International Review of Neurobiology Volume 53 Mitochondrial Function and Dysfunction

International R E V I E W O F Neurobiology Volume 53 Mitochondrial Function AND Dysfunction International REVIEW O...

Author: Anthony Schapira (Editor)

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International R E V I E W O F

Neurobiology Volume 53

Mitochondrial Function AND

Dysfunction

International REVIEW OF

Neurobiology Volume 53 SERIES EDITORS RONALD J. BRADLEY Department of Psychiatry, School of Medicine Louisiana State University Medical Center Shreveport, Louisiana, USA

R. ADRON HARRIS Waggoner Center for Alcohol and Drug Addiction Research The University of Texas at Austin Austin,Texas,USA

PETER JENNER Division of Pharmacology and Therapeutics GKT School of Biomedical Sciences King’s College, London,UK

EDITORIAL BOARD PHILIPPE ASCHER ROSS J. BALDESSARINI TAMAS BARTFAI COLIN BLAKEMORE FLOYD E. BLOOM DAVID A. BROWN MATTHEW J. DURING KJELL FUXE PAUL GREENGARD SUSAN D. IVERSEN

KINYA KURIYAMA BRUCE S. MCEWEN HERBERT Y. MELTZER NOBORU MIZUNO SALVADOR MONCADA TREVOR W. ROBBINS SOLOMON H. SNYDER STEPHEN G. WAXMAN CHIEN-PING WU RICHARD J. WYATT

Mitochondrial Function AND

Dysfunction EDITED BY

ANTHONY H. V. SCHAPIRA University Department of Clinical Neurosciences Royal Free and University College Medical School and Institute of Neurology London, United Kingdom

Amsterdam Boston London New York Oxford Paris San Diego San Francisco Singapore Sydney Tokyo

Cover Photo Credit: Cultured skin ﬁbroblast immunocytochemically stained for mitochondria DNA-encoded subunit I of cytochrome-c oxidase. Note that the subunit is present throughout the thread-like mitochondrial network. (Photo by J.-W. Taanman). ∞ This book is printed on acid-free paper.

C 2002, Elsevier Science (USA). Copyright

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To my daughter Sarah, a constant inspiration.

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CONTENTS

CONTRIBUTORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xv xix

SECTION I MITOCHONDRIAL STRUCTURE AND FUNCTION

Mitochondrial DNA Structure and Function CARLOS T. MORAES, SARIKA SRIVASTAVA, ILLIAS KIRKINEZOS, JOSE OCA-COSSIO, CORINA VANWAVEREN, MARKUS WOISCHNICK, AND FRANCISCA DIAZ I. II. III. IV. V. VI. VII. VIII. IX. X. XI.

Mammalian Mitochondrial Genomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Human mtDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure of the Human mtDNA D-Loop Region . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondrial DNA Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Initiation of L-Strand DNA Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alternative Mode of mtDNA Replication. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General Features of Factors Associated with mtDNA Replication . . . . . . . . . . . Regulation of mtDNA Replication. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondrial Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Translation of Mitochondrial Transcripts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3 5 7 7 9 9 10 11 12 15 17 17

Oxidative Phosphorylation: Structure, Function, and Intermediary Metabolism SIMON J. R. HEALES, MATTHEW E. GEGG, AND JOHN B. CLARK I. II. III. IV.

Historical Background. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Mitochondrial Electron Transport Chain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Intermediary Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

25 27 45 52 52

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Import of Mitochondrial Proteins MATTHIAS F. BAUER , SABINE HOFMANN, AND WALTER NEUPERT I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. The Pathways of Mitochondrial Preprotein Import . . . . . . . . . . . . . . . . . . . . . . . . . . III. Mitochondrial Biogenesis and Human Neurodegenerative Diseases . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

57 59 78 82

SECTION II PRIMARY RESPIRATORY CHAIN DISORDERS

Mitochondrial Disorders of the Nervous System: Clinical, Biochemical, and Molecular Genetic Features DOMINIC THYAGARAJAN AND EDWARD BYRNE I. II. III. IV. V. VI. VII. VIII. IX.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Historical Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetics and Pedigree Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Important Clinical Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Major Mitochondrial Syndromes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biochemical Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diagnostic Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Treatment of Mitochondrial Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

94 94 98 103 105 106 112 115 123 132

SECTION III SECONDARY RESPIRATORY CHAIN DISORDERS

Friedreich’s Ataxia J. M. COOPER AND J. L. BRADLEY I. II. III. IV.

Features of Friedreich’s Ataxia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effect of FRDA Gene Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Models of FRDA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FRDA Molecular Mechanisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

147 150 152 154

CONTENTS

V. Therapeutic Intervention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix 162 165 167

Wilson Disease C. A. DAVIE AND A. H. V. SCHAPIRA I. II. III. IV. V. VI. VII. VIII.

The Role and Transport of Copper in Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aceruloplasminemia and Menkes’ Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experimental Models of Wilson Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondrial Dysfunction in Animal Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondrial Dysfunction in Human Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular Basis for the Variation in Phenotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cranial Magnetic Resonance Imaging (MRI) and Spectroscopy in Wilson Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

175 177 178 179 180 182 183 183 185 187 187

Hereditary Spastic Paraplegia CHRISTOPHER J. MCDERMOTT AND PAMELA J. SHAW I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Clinical Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

191 192 193 201

Cytochrome c Oxidase Deﬁciency GIACOMO P. COMI, SANDRA STRAZZER, SARA GALBIATI, AND NEREO BRESOLIN I. II. III. IV.

Cytochrome c Oxidase Biogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nuclear Genes Affecting COX Assembly and Stability . . . . . . . . . . . . . . . . . . . . . . . Nuclear Genes Affecting mtDNA Level and/or Stability. . . . . . . . . . . . . . . . . . . . . mtDNA Defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

205 209 221 225 233

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SECTION IV TOXIN-INDUCED MITOCHONDRIAL DYSFUNCTION

Toxin-Induced Mitochondrial Dysfunction SUSAN E. BROWNE AND M. FLINT BEAL I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Inhibitors of Mitochondrial Complex I: NADH Ubiquinine Oxidoreductase III. Inhibitors of Mitochondrial Complex II: Succinate Ubiquinol Oxidoreductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Inhibitors of Mitochondrial Complex IV: Cytochrome c Oxidase . . . . . . . . . . V. Manganese . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. 3-Acetylpyridine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Myopathies and Myotoxic Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Discussion: What Determines the Regional and Cellular Speciﬁcity of Mitochondrial Toxins?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

243 244 254 258 260 262 264 265 267

SECTION V NEURODEGENERATIVE DISORDERS

Parkinson’s Disease L. V. P. KORLIPARA AND A. H. V. SCHAPIRA I. II. III. IV. V.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondrial Dysfunction in Parkinson’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . Etiology of Parkinson’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondrial Dysfunction and the Pathophysiology of Parkinson’s Disease Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

283 284 287 294 302 303

Huntington’s Disease: The Mystery Unfolds? A˚ SA PETERSE´ N AND PATRIK BRUNDIN I. II. III. IV. V.

Huntington’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epidemiology and Symptomatology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neuropathology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other Triplet Repeat Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

315 316 316 317 318

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VI. VII. VIII. IX. X. XI. XII.

Intracellular Localization of Normal and Mutant Huntingtin . . . . . . . . . . . . . . . Function of Normal and Mutant Huntingtin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell Death in Huntington’s Disease: Apoptosis and Authophagy . . . . . . . . . . . Oxidative Stress and Metabolic Dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dopamine Toxicity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transgenic Mouse Models of Huntington’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

319 321 323 324 325 326 328 329

Mitochondria in Alzheimer’s Disease RUSSELL H. SWERDLOW AND STEPHEN J. KISH I. II. III. IV. V. VI.

VII. VIII. IX. X. XI. XII. XIII. XIV.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Historical Overview and the Amyloid Cascade Hypothesis. . . . . . . . . . . . . . . . . . . Metabolic Dysfunction in Alzheimer’s Disease has been Reported. . . . . . . . . . Morphological Studies Demonstrate Mitochondrial Abnormalities in Alzheimer’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PDHC and KGDHC in Alzheimer’s Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Brain Biochemical Studies of Mitochondrial Enzymes in Alzheimer’s Disease: Is Cytochrome Oxidase Reduction Characteristic of Alzheimer’s Disease?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Is a Brain Cytochrome Oxidase Deﬁciency a Robust Feature of Alzheimer’s Disease? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Studies of Cytochrome Oxidase in Non-CNS Tissues in Alzheimer’s Disease: Clues to the Origin of the Enzyme Change? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cytochrome Oxidase Dysfunction in Alzheimer’s Disease: Possible Genetic Component? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cytochrome Oxidase Dysfunction in Alzheimer’s Disease: Genetic Studies are Still Inconclusive. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cybrid Data Suggest mtDNA Contributes to Alzheimer’s Disease Cytochrome Oxidase Dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Unresolved Issues in Alzheimer’s Disease Cybrid Studies: Where is the mtDNA “Mutation?” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Could a Cytochrome Oxidase Defect Cause Alzheimer’s Disease? . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

342 342 344 345 345

346 355 356 358 361 366 371 372 373 375

Contributions of Mitochondrial Alterations, Resulting from Bad Genes and a Hostile Environment, to the Pathogenesis of Alzheimer’s Disease MARK P. MATTSON I. Overview of Neurodegenerative Cascades in Alzheimer’s Disease . . . . . . . . . . II. Mitochondrial Alterations in Alzheimer’s Disease Patients and Experimental Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

387 389

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III. Genetic Factors and Mitochondrial Alterations in Alzheimer’s Disease. . . . . IV. Environmental Factors and Mitochondrial Alterations in Alzheimer’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

394 397 401 401

Mitochondria and Amyotrophic Lateral Sclerosis RICHARD W. ORRELL AND ANTHONY H. V. SCHAPIRA I. II. III. IV. V.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathogenic Hypotheses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transgenic Mouse Models of ALS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

411 413 414 420 423 424

SECTION VI MODELS OF MITOCHONDRIAL DISEASE

Models of Mitochondrial Disease DANAE LIOLITSA AND MICHAEL G. HANNA I. II. III. IV. V. VI. VII. VIII. IX. X.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Classiﬁcation of mtDNA Defects Causing Respiratory Chain Disease . . . . . . . Cell Models Employed to Study mtDNA Defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell Models of Respiratory Chain Disease Associated with Speciﬁc mtDNA Defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Classiﬁcation of Nuclear DNA Defects Causing Respiratory Chain Disease Cell Models of Respiratory Chain Disease Associated with Nuclear DNA Defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Application of Cell Models for the Development of Therapeutic Strategies in mtDNA Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Animal Models of Respiratory Chain Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other Models of Respiratory Chain Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

430 431 432 434 442 445 447 449 455 457 458

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SECTION VII DEFECTS OF β-OXIDATION INCLUDING CARNITINE DEFICIENCY Defects of β-Oxidation Including Carnitine Deﬁciency K. BARTLETT AND M. POURFARZAM I. II. III. IV.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Background Biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inherited Disorders of Mitochondrial β-Oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

469 470 480 505 505

SECTION VIII MITOCHONDRIAL INVOLVEMENT IN AGING

The Mitochondrial Theory of Aging: Involvement of Mitochondrial DNA Damage and Repair NADJA C. DE SOUZA-PINTO AND VILHELM A. BOHR I. II. III. IV. V. VI.

Mitochondria: The Biological Clock? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Damage to Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Accumulation of Oxidative Damage to mtDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Repair in Mammalian Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Changes in mtDNA Repair with Age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

519 520 522 525 528 530 530

INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTENTS OF RECENT VOLUMES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

535 549

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CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

K. Bartlett (469), Department of Child Health and Department of Clinical Biochemistry, Sir James Spence Institute of Child Health, Royal Victoria Inﬁrmary, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 4LP, United Kingdom Matthias F. Bauer (57), Institute of Clinical Chemistry, Molecular Diagnostics and Mitochondrial Genetics, and Diabetes Research Group, Academic Hospital Munich-Schwabing, D-8000 Munich, Germany M. Flint Beal (243), Department of Neurology, New York Presbyterian Hospital and Weill Medical College of Cornell University, New York, New York 10021 Vilhelm A. Bohr (519), Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224 J. L. Bradley (147), University Department of Clinical Neurosciences, Royal Free and University College Medical School, London NW3 2PF, United Kingdom Nereo Bresolin (205), Institute of Clinical Neurology, University of Milan, Milan 20122, Italy and IRCCS E. Medea, Associazione La Nostra Famiglia, Bosisio Parini, Italy Susan E. Browne (243), Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York, New York 10021 Patrik Brundin (315), Department of Physiological Sciences, Wallenberg Neuroscience Center, Lund University, Lund S-223 62, Sweden Edward Byrne (93), Center for Neuroscience, Department of Pathology, University of Melbourne, Victoria 3010, Australia John B. Clark (25), Departments of Neurochemistry and Clinical Biochemistry, Institute of Neurology, London, WC1N 3BG, United Kingdom Giacomo P. Comi (205), Institute of Clinical Neurology, University of Milan, Milan 20122, Italy J. M. Cooper (147), University Department of Clinical Neurosciences, Royal Free and University College Medical School, London NW3 2PF, United Kingdom xv

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C. A. Davie (175), University Department of Clinical Neurosciences, Royal Free and University College Medical School, London NW3 2PF, United Kingdom Nadja C. de Souza-Pinto (519), Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224 Francisca Diaz (3), Department of Neurology, University of Miami School of Medicine, Miami, Florida 33136 Sara Galbiati (205), Institute of Clinical Neurology, University of Milan, Milan 20122, Italy Matthew E. Gegg (25), Departments of Neurochemistry and Clinical Biochemistry, Institute of Neurology, London, WC1N 3BG, United Kingdom Michael G. Hanna (429), Clinical Neurogenetics, National Hospital for Neurology and Neurosurgeary, and Institute of Neurology, University College London, London WC1N 3BG, United Kingdom Simon J. R. Heales (25), Departments of Neurochemistry and Clinical Biochemistry, Institute of Neurology, London, WC1N 3BG, United Kingdom Sabine Hofmann (57), Institute of Clinical Chemistry, Molecular Diagnostics and Mitochondrial Genetics, and Diabetes Research Group, Academic Hospital Munich-Schwabing, D-8000 Munchen, Germany Ilias Kirkinezos (3), Department of Neurology, University of Miami School of Medicine, Miami, Florida 33136 Stephen J. Kish (341), Human Neurochemical Pathology Laboratory, Centre for Addiction and Mental Health, Toronto, Ontario M5T 1R8, Canada Danae Liolitsa (429), Centre for Neuromuscular Disease and Department of Molecular Pathogenesis, Institute of Neurology, Queen Square, London, WC1N 3BG, United Kingdom Mark P. Mattson (387), Laboratory of Neurosciences, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, and Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Christopher J. McDermott (191), Academic Neurology Unit, Division of Genomic Medicine, University of Shefﬁeld, Shefﬁeld S10 2RX, United Kingdom Carlos T. Moraes (3), Department of Neurology, University of Miami School of Medicine, Miami, Florida 33136 Walter Neupert (57), Adolf Butenandt Institute for Physiological Chemistry, Ludwig-Maximilans Universit¨at Mu¨ nchen, 81377 Munich, Germany Jose Oca-Cossio (3), Department of Neurology, University of Miami School of Medicine, Miami, Florida 33136

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Richard W. Orrell (411), University Department of Clinical Neurosciences, Royal Free and University College Medical School, London NW3 2PF, United Kingdom ˚ Peters´en (315), Section of Neuronal Survival, Wallenberg Neuroscience Asa Center, Lund University, Lund SE-221 84, Sweden M. Pourfarzam (469), Department of Child Health, Sir James Spence Institute of Child Health, Royal Victoria Inﬁrmary, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 4LP, United Kingdom Anthony H. V. Schapira (411), University Department of Clinical Neurosciences, and Institute of Neurology Royal Free and University College Medical School, London NW3 2PF, United Kingdom Sarika Srivastava (3), Department of Neurology, University of Miami School of Medicine, Miami, Florida 33136 Pamela J. Shaw (191), Academic Neurology Unit, Division of Genomic Medicine, University of Shefﬁeld, Shefﬁeld S10 2RX, United Kingdom Sandra Strazzer (205), Institute of Clinical Neurology, University of Milan, Milan 20122, Italy and IRCCS E. Medea, Associazione La Nostra Famiglia, Bosisio Parini, Italy Russell H. Swerdlow (341), Department of Neurology, Neurosciences Service Center, University of Virginia Health System, Charlottesville, Virginia 22908 Dominic Thyagarajan (93), Department of Neurology, Flinders Medical Centre, Bedford Park 5042, South Australia Corina vanWaveren (3), Department of Neurology, University of Miami School of Medicine, Miami, Florida 33136 Markus Woischnick (3), Department of Neurology, University of Miami School of Medicine, Miami, Florida 33136 L. V. P. Korlipara (283), University Department of Clinical Neurosciences Royal Free and University College Medical School London, United Kingdom, NW3 2PF A. H. V. Schapira (283), Institute of Neurology London, United Kingdom, WC1N 3BG

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PREFACE

Since their ﬁrst recognition in 1840, their naming in 1898, and structural analysis in the 1950’s, mitochondria have been an object of considerable attention to the cell biologist. Their normal function is critical to the survival of the cell. They house numerous crucial biochemical pathways, and in particular are responsible for the generation of ATP by oxidative phosphorylation. More recently mitochondria have been shown to have a crucial role in mediating pathways to apoptotic cell death. Of particular interest has been the identiﬁcation and characterization of mitochondrial DNA and the use of this molecule in migration studies. The identiﬁcation of maternally inherited human diseases predicted the discovery of mitochondrial DNA mutations causing human pathology. Since the ﬁrst description of mitochondrial DNA mutations causing chronic progressive external opthalmaplegia and Kearns Sayre syndrome in 1988, over 100 different mutations of mitochondrial DNA are now associated with various human diseases. These represent a huge clinical spectrum, and epidemiological studies have suggested that mutations of mitochondrial DNA may be one of the most common causes of inherited neurological disease. This text serves to form a bridge between basic mitochondrial science and mitochondrial pathology. An adequate knowledge of mitochondrial structure and function is fundamental to an understanding of the contribution of mitochondrial dysfunction to etiology and pathogenesis of diseases. The foundation of this book therefore lies upon excellent contributions on mitochondrial DNA oxidative phosphorylation, intermediary metabolism, and mitochondrial transport. Our appreciation of the breadth of mitochondrial pathology now encompasses both the primary and secondary respiratory chain disorders. The primary disorders include all the archetypal mitochondrial encephalomyopathies caused by mutations of mitochondrial DNA, or mutations of nuclear genes encoding respiratory chain sub-units. The subject of secondary respiratory chain disorders is complex, but includes important diseases such as Friedreich’s ataxia, Wilson disease, hereditary spastic paraplegia and cytochrome c oxidase deﬁciency caused by nuclear gene mutations affecting assembly and stability of mitochondrial DNA or the holoprotein. These diseases represent examples of mutations affecting non-respiratory chain

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mitochondrial or non-mitochondrial proteins that result in secondary mitochondrial dysfunction. There are increasing examples of environmental toxins that cause mitochondrial abnormalities. These are attracting particular attention, as environmental agents may contribute to certain human diseases via a mitochondrial pathway. Several mitochondrial toxins are used as tools to model human diseases in vitro and in vivo. These have been particularly important and relevant to certain neurodegenerative disease, including Parkinson’s disease and Huntington’s disease. Here the contribution of mitochondrial dysfunction to etiology and pathogenesis remains uncertain. This is particularly so for Alzheimer’s disease and amyotrophic lateral sclerosis. The separate sections on these topics reﬂect the evidence for mitochondrial abnormalities, but also discuss in a balanced approach how these defects might arise. Several genetic models of mitochondrial disease are now available and these include both mitochondrial and nuclear genomic defects. Reﬁnements in transgenic technology will undoubtedly improve our ability to model mitochondrial genome mutations. Finally, although much of the focus of this text is on the respiratory chain, there have been several important advances in the pathology of beta oxidation, and these are reﬂected in an outstanding section on this topic. Aging has been suggested to be the most common mitochondrial disease of all! There is abundant evidence that mitochondrial DNA mutations and mitochondrial dysfunction are seen more commonly in senescence. The relevance of these observations to cell dysfunction or aging itself is an important topic and is covered in an excellent section that completes this text. With kind regards Tony

SECTION I MITOCHONDRIAL STRUCTURE AND FUNCTION

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MITOCHONDRIAL DNA STRUCTURE AND FUNCTION

Carlos T. Moraes,1 Sarika Srivastava, Ilias Kirkinezos, Jose Oca-Cossio, Corina vanWaveren, Markus Woischnick, and Francisca Diaz Department of Neurology University of Miami School of Medicine Miami, Florida 33136

I. II. III. IV. V. VI. VII.

VIII. IX.

X. XI.

Mammalian Mitochondrial Genomes The Human mtDNA Structure of the Human mtDNA D-Loop Region Mitochondrial DNA Replication Initiation of L-Strand DNA Replication Alternative Mode of mtDNA Replication General Features of Factors Associated with mtDNA Replication A. DNA Polymerase γ B. Mitochondrial Single-Strand Binding Protein Regulation of mtDNA Replication Mitochondrial Transcription A. Transcription Initiation B. Transcription Elongation and Termination C. Posttranscriptional Modifications Translation of Mitochondrial Transcripts Concluding Remarks References

I. Mammalian Mitochondrial Genomes

In 1963, DNA was ﬁrst detected within mitochondria (N. M. K. Nass and S. Nass, 1963). In the next 30 years, the complete mitochondrial DNA (mtDNA) sequence [approximately 17,000 base pairs (bp)] was determined in more than a dozen species, including humans (Anderson et al., 1981). Most vertebrate cells in culture appear to have approximately 1000–5000 molecules of the circular mitochondrial genome (Bogenhagen and Clayton, 1974; Shmookler Reis and Goldstein, 1983). The mtDNA localizes to the mitochondrial matrix and seems to be associated with proteins and lipids (Hillar et al., 1979). In yeast, the larger [∼80 kilobases (kb)] mitochondrial genomes are organized in 10–20 distinct nucleoids (i.e., protein–DNA 1

To whom correspondence should be addressed.

INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 53

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Copyright 2002, Elsevier Science (USA). All rights reserved. 0074-7742/02 $35.00

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FIG. 1. Structure of the human mitochondrial DNA. Panel A depicts the 16,569-bp human mtDNA showing 13 protein coding genes as well as 2 rRNA- and 22 tRNA-coding genes. Genes coding for subunits of complex I (ND1–ND6), complex III (Cyt b), complex IV (COX I– COX III), and complex V (A8 and A6) are shown by different hatches. The insert in panel A illustrates the mechanisms associated with mtDNA replication and transcription, including the approximately binding sites for the mitochondrial RNA polymerase, the mitochondrial

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complexes), which are spherical or ovoid, measuring 0.3–0.6 μ in diameter. Nucleoids contain between 3 and 4 mitochondrial genomes and as many as 20 different polypeptides (Miyakawa et al., 1987; Kaufman et al., 2000). It is unclear if animal mtDNA is also organized as DNA–protein complexes (nucleoids), although this possibility has been suggested as a system to maintain genetic stability ( Jacobs et al., 2000). One of the most striking differences between the yeast and the animal systems can be observed during development of animal cells. Mitochondrial DNA copy number seems to be strictly controlled during development (Piko and Taylor, 1987; Lefai et al., 2000b), and speciﬁc mechanism may have evolved because of these needs. The recent identiﬁcation of a mitochondrial helicase termed Twinkle, which shows a punctate localization compatible with a nucleoid structure, gives support for this model (Spelbrink et al., 2001). Electron microscopy analyses showed that mammalian mtDNA can be arranged as unicircular monomers, but also as unicircular dimers or catenated forms (Clayton, 1982). These early studies also showed what has been termed the displacement loop or “D-loop” as a separation of strands in a speciﬁc region of the mtDNA. It is now known that most sequences associated with initiation of mtDNA replication or transcription are in the proximity of the D-loop region (Clayton, 1982). Both transcription and replication of one strand and transcription of the complementary strand initiate in the proximity of the D-loop. This 1123-bp stretch of DNA is often in a singlestranded conﬁguration, and contains sites for DNA-binding proteins that control mtDNA replication and transcription. Mutations in this region have been observed to accumulate during aging (Michikawa et al., 1999; Wang et al., 2001), but it is still unclear if these alterations affect mtDNA replication or gene expression.

II. The Human mtDNA

The human mtDNA is representative of mammalian mitochondrial genomes. It is a 16,569-bp, double-stranded, circular molecule encoding 13 polypeptides (Fig. 1). All mtDNA-encoded polypeptides are members of the oxidative phosphorylation complexes (OXPHOS). These include seven transcription factor mtTFA, the RNA processing enzyme RNAse MRP, and the transcription termination factor mTERF. The origins of replication for the H- and L- (OH and OL) strands are also shown. Panel B shows the structure of the regulatory D-loop region in more detail, including the approximate position of the conserved sequence boxes believed to play a role in replication and RNA primer processing. It also shows the location of the two hypervariable regions (HSV1 and HSV2) commonly used for evolutionary studies.

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subunits of complex I, one subunit of complex III, three subunits of complex IV, and two of complex V. Besides protein coding genes, mtDNA also codes for 22 transfer RNAs (tRNAs) and two ribosomal RNAs (12S and 16S rRNAs). The expression and maintenance of mtDNA depends on a large number of nuclear-coded factors that are synthesized in the cytosolic ribosomes as precursor polypeptides and imported into the mitochondria via specialized import pores (Attardi and Schatz, 1988). Although the catalytic subunits of the OXPHOS system are encoded by the mtDNA, these enzyme complexes also contain a large number of nuclear-coded subunits that are necessary for their function. The asymmetric distribution of guanine and cytosine permits separation of mtDNA into “heavy” (H-strand) and “light” (L-strand) strands in alkaline density gradient centrifugation. The rRNAs, all but one polypeptide, and 14 of the 22 tRNAs are encoded in the heavy-strand genes. In contrast to Saccharomyces cerevisiae mtDNA, vertebrate mtDNA are devoid of introns (Anderson et al., 1981). There are very few noncoding intergenic regions, with the exception of the regulatory region containing the promoters and origin of heavy-strand replication. The genetic information is so condensed that there is an overlap in some coding sequences, and termination codons can be generated by the addition of adenines to the transcript during polyadenylation of mRNAs (Anderson et al., 1981). The genetic code of vertebrates’ mtDNA differs from the nuclear-cytoplasmic code. Instead of being a termination codon, TGA codes for tryptophan in vertebrate’s TABLE I MAMMALIAN MITOCHONDRIAL GENETIC CODEa UUU UUC UUA UUG CUU CUC CUA CUG AUU AUC AUA AUG GUU GUC GUA GUG a

Phe Phe Leu Leu Leu Leu Leu Leu Ile Ile Met(Ile) Met Val Val Val Val

UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG

Ser Ser Ser Ser Pro Pro Pro Pro Thr Thr Thr Thr Ala Ala Ala Ala

UAU UAC UAA UAG CAU CAC CAA CAG AAU AAC AAA AAG GAU GAC GAA GAG

Tyr Tyr Ter Ter His His Gln Gln Asn Asn Lys Lys Asp Asp Glu Glu

UGU UGC UGA UGG CGU CGC CGA CGG AGU AGC AGA AGG GGU GGC GGA GGG

Cys Cys Trp(Ter) Trp Arg Arg Arg Arg Ser Ser Ter(Arg) Ter(Arg) Gly Gly Gly Gly

Amino acids in parentheses correspond to universal genetic code.

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mitochondria. ATA codes for methionine in mitochondria but isoleucine in the cytosol. Finally, AGA or AGG in mitochondria code for a stop codon instead of arginine (Table I) (Anderson et al., 1981).

III. Structure of the Human mtDNA D-Loop Region

Comparison of the nucleotide sequences of mammals’ mtDNA revealed some degree of conservation in the promoter regions as well as in three other regions (termed Conserved Sequence Blocks, or CSB I, CSB II, and CSB III) (Walberg and Clayton, 1981). These sequences are conserved in the D-loop regions of many vertebrates, suggesting important roles for these motifs. The CSBs are hypothesized to be involved in some aspect of mtDNA replication because they are located in the D-loop region, and in the case of CSB I, almost always near the initiation site for H-strand DNA synthesis. However, the absence of certain CSBs in some vertebrates suggests that either the function of these elements can be obviated by speciﬁc D-loop region conﬁgurations or that other novel nucleotide sequences (or protein factors) can provide the same function in these organisms. The majority of the D-loop region contain noncoding sequences and include hypervariable regions (Greenberg et al., 1983). Although the overall rate of mutations in these hypervariable regions are signiﬁcantly higher than in the rest of mtDNA (Greenberg et al., 1983), some nucleotide positions seem to be hot spots for changes (Stoneking, 2000). The two hypervariable segments (HV1 and HV2; positions 16024–16383 and 57–372, respectively) have been very useful in studying evolution of eukaryotes (Lang et al., 1999), and more speciﬁcally, of human populations ( Jorde et al., 2000).

IV. Mitochondrial DNA Replication

In most cases, mtDNA replication in mammals is an asynchronous process, beginning at the origin of the H-strand replication (OH) and proceeding around two thirds of the mitochondrial genome, until the origin of the L-strand replication (OL) (Fig. 1) is forced into a single-strand conﬁguration by the extending daughter H-strand. At this point, the displaced H-strand starts to be copied into the daughter L-strand. The precise mapping of RNA and DNA species in the D-loop region provided evidence that RNA derived from the L-strand promoter (LSP) serves as a primer for H-strand DNA replication (Chang and Clayton, 1985; Chang et al., 1985). There is also

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evidence suggesting that the CSBs are involved in formation of a properly conﬁgured RNA primer. This RNA synthesized from the L-strand promoter (7S RNA) is correctly processed by a mitochondrial RNA processing (MRP, see below) activity. The existence of an RNA–DNA hybrid downstream of human LSP has been demonstrated (Xu and Clayton, 1996). The human hybrid is conﬁned to a speciﬁc region of the origin (spanning CSB II and CSB I), and hybrid formation is virtually abolished by mutations in the upstream CSB III element. A detailed study of the mammalian RNA–DNA hybrid using mouse OH revealed an unusual structure, where a molecule containing an extremely stable R-loop consisting of two DNA strands and one RNA strand (with one of the DNA strands displaced by the hybridized RNA molecule) (Lee and Clayton, 1996). Because this was also observed using a plasmid construct containing the isolated OH region, the nucleic acid sequence of mouse OH appears to contain all of the information required for formation of a stable RNA–DNA hybrid. To provide an appropriate primer for replication, a site-speciﬁc mitochondrial RNA processing endoribonuclease (RNase MRP) processes 7S RNA substrates at sites that match some of the RNA–DNA transition sites (i.e., potential DNA replication priming sites) that have been mapped at the H-strand origin in vivo (Dairaghi and Clayton, 1993). The MRP enzyme contains, in addition to protein components, an RNA essential for activity (Chang and Clayton, 1987; Chang and Clayton, 1989). The pattern of RNA cleavage by RNase MRP is consistent with a role for the enzyme in providing primers for mtDNA replication with the substrate being probably the triple-stranded RNA-DNA hybrid, rather than single-stranded RNA. These cleavages seem to be dependent on the presence of CSB I, suggesting that RNase MRP is necessary for the processing that produces the RNA primers in mammalian mitochondria. These ﬁndings also implicate the RNA–DNA hybrid as the substrate for the RNA processing that leads to formation of the primers for H-strand replication. RNase MRP activity is also found in the cell nucleus (Chang and Clayton, 1987; Gold et al., 1989). Several lines of evidence demonstrated that nuclear RNase MRP is involved in late stages of 5.8S rRNA processing in the nucleus (Lygerou et al., 1996). However, a small amount of MRP RNA has been localized to the mitochondrion in mouse cells (Li et al., 1994) and Xenopus laevis oocytes (Davis et al., 1995) by in situ hybridization. In addition, mutations in the genes encoding the RNA component of Saccharomyces cerevisiae and S. pombe RNase MRP RNA have been isolated that result in a mitochondrial phenotype (Paluh and Clayton, 1996). Taken together, these observations suggest the existence of a larger pool of RNAse MRP in the cell nucleus that is responsible for rRNA processing, and a smaller pool in mitochondria that appears to be involved in mitochondrial RNA primer processing.

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V. Initiation of L-Strand DNA Replication

In vertebrates, the origin of light-strand replication (OL) is located a large distance from OH on the mtDNA molecule (Figure 1). Initiation of L-strand DNA replication has been studied in mammals, where it occurs within a small [30 base pair(bp)] noncoding region that is ﬂanked by tRNA genes. The DNA sequence in this region has the potential to assume a stable stem-loop structure (Tapper and Clayton, 1981) that is thought to form after the replication fork from initiation of H-strand synthesis passes OL and exposes the parental H-strand in this region as a single strand. Most of our understanding of OL function comes from studies that utilized an in vitro replication system for the human OL. These studies showed that OL is capable of initiating L-strand DNA synthesis at sites that match those mapped from nucleic acids isolated from mitochondria in vivo (Wong and Clayton, 1985). Initiation of L-strand DNA synthesis requires a DNA primase responsible for generating short RNA molecules with 5 -ends that map to the T-rich portion of the loop in the predicted OL stem-loop structure. In most vertebrates, a noncoding region with conserved predicted secondary structure is found within the sequence in the mtDNA molecule that encodes a cluster of tRNAs for the amino acids Trp, Ala, Asn, Cys, and Tyr (Anderson et al., 1981).

VI. Alternative Mode of mtDNA Replication

Holt and colleagues (2000) proposed that the mammalian mitochondrial genome has two modes of replication. The ﬁrst one, described above (Clayton, 1982), involves the asymmetric replication of the leading and lagging strands. The second one, based on the observation of replication intermediates in two-dimensional (2D) gels suggested that replication, in a certain number of mtDNA molecules, involves coupled leading- and lagging-strand synthesis. Interestingly, they found a higher percentage of the latter mechanism in cells that were transiently depleted of their mtDNA. Although there are questions on whether these observations actually reﬂect alternative modes of mtDNA replication, different modes could have an active role in controlling mtDNA copy number, as speciﬁc factors may be involved in different modes of replication. As a possible control mechanism, factors necessary for the coupled leading- and lagging-strand synthesis may be limiting, and once mtDNA levels are close to normal they can no longer participate in the replication of most molecules, thereby decreasing the overall mtDNA replication in the cell.

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VII. General Features of Factors Associated with mtDNA Replication

Many factors involved in mammalian mtDNA replication have been characterized. These include subunits A (catalytic, also referred as subunit α) and B (accessory, also known as subunit β) of DNA polymerase γ , mitochondrial RNA polymerase, mitochondrial single-stranded binding protein (mtSSB), mitochondrial transcription factor A (mtTFA), and RNA processing enzymes (reviewed in Lecrenier and Foury, 2000; Shadel and Clayton, 1997). A mitochondrial DNA ligase, apparently related to the nuclear DNA ligase III, that is likely to participate in the resolution of replicated strands also has been characterized (Pinz and Bogenhagen, 1998; Lakshmipathy and Campbell, 1999). Even though the primary functions of these factors are understood, their role in the regulation of mtDNA copy number is not. In addition, mitochondrial helicases (Spelbrink et al., 2001) and topoisomerases (Topcu and Castora, 1995) are also likely to have a role in this process. Experimental evidence also exists suggesting that the cell nucleus may exert a negative control on the mitochondrial genome through some short-lived nuclear substance(s) (Rinaldi et al., 1979).

A. DNA POLYMERASE γ Overexpression of the catalytic subunit (subunit A) of the mtDNA polymerase γ in cultured insect or human cells did not alter mtDNA levels (Lefai et al., 2000a; Spelbrink et al., 2000). However, in transgenic ﬂies overexpressing pol γ -A, the number of mitochondrial genomes was reduced drastically, indicating that although cells can tolerate a variable amount of the pol γ catalytic subunit under some conditions, the levels of subunit A could be critical in the context of the whole organism (Lefai et al., 2000b). Flies with mutations in pol γ -A show problems with the visual system and altered behavior in the wandering stage, both of which seemed to be a consequence of defects in locomotion (Iyengar et al., 1999). The expression of the accessory subunit of pol γ (subunit B) seems to correlate better with mtDNA replication activity. The steady-state level of pol γ -B mRNA increases during the ﬁrst hours of development, reaching its maximum value at the start of mtDNA replication in Drosophila embryos. This pattern of expression was not observed with pol γ -A mRNA (Lefai et al., 2000b). A potential link between nuclear and mitochondrial DNA replication also has been described in Drosophila. The pol γ -B promoter contains a DNA replication-related site (DRE), previously identiﬁed in genes involved in nuclear DNA replication, which is essential for its transcription, suggesting a common regulatory

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mechanism controlling nuclear and mitochondrial DNA replication (Lefai et al., 2000b). Recently, a mutation in the DNA pol γ subunit A was associated with multiple mtDNA deletions in patients with progressive external ophthalmoplegia (Goethem et al., 2001).

B. MITOCHONDRIAL SINGLE-STRAND BINDING PROTEIN The mtSSB also has an important role in mtDNA replication. The rate of DNA synthesis by Drosophila DNA polymerase γ was increased nearly 40-fold upon addition of mtSSB. The stimulation of both 5 –3 DNA polymerase and 3 –5 exonuclease activities of Drosophila pol γ by mtSSB results from increased primer recognition, binding, and rate of initiation (Farr et al., 1999). Similar to the pol γ -B promoter, putative transcription factor binding sites clustered within the promoter region of the mtSSB gene include two Drosophila DREs. Deletion and base substitution mutagenesis of the DRE sites demonstrated that they are required for efﬁcient promoter activity, and gel electrophoretic mobility shift analyses showed that DRE binding factor (DREF) binds to these sites (Ruiz De Mena et al., 2000). The link between mitochondrial and nuclear DNA replication is probably very complex and regulated by additional factors as mRNA levels for mtSSB varies independently of cell proliferating activity (Ruiz De Mena et al., 2000). Flies with a disruption in the mtSSB show a marked mtDNA depletion, defective mitochondrial respiration, and a “low-power” phenotype, similar to the one observed in mutants of pol γ -A (Maier et al., 2001).

VIII. Regulation of mtDNA Replication

The available information on the regulation of mtDNA replication factors in adult animal cells is compatible with the concept that the levels of these factors do not increase signiﬁcantly when mtDNA levels decrease. Schultz et al. (1998) found that the DNA pol γ subunit A is expressed at similar levels in different tissues and does not seem to be regulated by physiological changes. Davis et al. (1996) showed that DNA pol γ -A transcripts and protein levels in human cells devoid of mtDNA were comparable with those of controls. Larsson et al. (1994) and Moraes et al. (1999) did not ﬁnd a signiﬁcant alteration in mRNA levels of genes coding for factors involved in mtDNA replication when cells were depleted of mtDNA. Therefore, the available information suggests that mtDNA levels do not seem to inﬂuence transcriptional expression of the known mtDNA replication-related genes.

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Because of their observations showing that cells maintain a certain mass of mitochondrial genomes, Tang et al. (2000) suggested that the regulation of mtDNA copy number could be provided by the control of the organellar nucleoside pools. Although cellular nucleotide pools are tightly regulated, defects in nucleotide metabolism are associated with human diseases and mtDNA stability (Nishino et al., 1999; Kaukonen et al., 2000). Control of the available dNTP pools inside mitochondria is known to be important for replication ﬁdelity (Kunkel and Alexander, 1986; Wernette et al., 1988) and depends on factors that regulate nucleotide metabolism such as mitochondrial deoxyribonucleotidases (Rampazzo et al., 2000), which can also offer another level of regulation. Evidence suggesting that mtDNA maintenance and copy-number control depend on factors other than nucleotide pools or housekeeping replication factors comes from the observation of Shoubridge and colleagues ( Jenuth et al., 1997) that in some mouse tissues there is a tissue-speciﬁc and age-related directional selection for different mtDNA genotypes. This suggests the presence of tissue-speciﬁc nuclear genes important for mtDNA maintenance. Moraes et al., (1999) found that ape mtDNA (gorilla or chimpanzee mtDNA) could repopulate human cells devoid of mtDNA (ρ ◦ cells) at a rate similar to wild-type human mtDNAs. However, ape mtDNA was not maintained in human cells harboring wild-type or defective human mtDNA (either with a large deletion or a point mutation). These observations suggested that competition between the two haplotypes prevented the maintenance of ape genomes, underscoring the importance of recognition of the mtDNA primary sequence by cognate replication factors.

IX. Mitochondrial Transcription

A. TRANSCRIPTION INITIATION There are two major transcription initiation sites in the human mtDNA D-loop region (termed OH and OL) situated within 150 bp of one another (Fig. 1). A promoter element with a 15-bp sequence motif, 5 -CANACC(G) CC(A)AAAGAN, surrounds the transcription initiation sites and is necessary for transcription (Chang and Clayton, 1984). Heavy-strand transcription starts at nucleotide position 561, located within the H-strand promoter (HSP) and ﬂanked by the tRNAPhe gene, whereas L-strand transcription starts at nucleotide position 407, within the LSP (Fig. 1). Despite the close proximity of the HSP and LSP, the initial in vitro transcription studies demonstrated that these elements are functionally independent (Chang and Clayton, 1984; Hixson and Clayton, 1985; Topper

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and Clayton, 1989). This functional autonomy was conﬁrmed in patients with progressive external ophthalmoplegia, with mutated muscle mtDNA harboring large-scale deletions including the HSP. In affected muscle cells, LSP was fully active (Moraes et al., 1991). A second initiation site for H-strand transcription has been described around nucleotide position 638, adjacent to the gene for 12S rRNA. Its promoter region only shows limited similarity with the 15-bp consensus sequence and is used less frequently for transcription of the H-strand (Montoya et al., 1983). Fractionation of human mitochondrial transcription extracts showed the requirement for at least two factors for transcriptional activity: (1) a relatively nonselective core RNA polymerase and (2) a dissociable transcription factor that confers promoter selectivity to the polymerase (Fisher et al., 1987). A human cDNA specifying the mitochondrial RNA polymerase was identiﬁed by screening of an expressed sequence tags (EST) database with the yeast sequence (Tiranti et al., 1997). It was found that the C-terminal half of the predicted polypeptide shares signiﬁcant amino acid sequence identity with the single subunit RNA polymerases of T3, T7, and SP6 bacteriophages. A mitochondrial transcription factor was identiﬁed by Clayton and colleagues (Fisher and Clayton, 1985). This factor, currently known as mtTFA (or Tfam) is a 25-kDa mitochondrial protein that contains two high mobility group (HMG) domains separated by a 27-amino acid residue linker and followed by a 25-amino acid residue basic C-terminal tail. HMG domains are involved in DNA binding, and are found in a diverse family of proteins whose members have been implicated in processes such as transcription enhancement and chromatin packaging. Mutation analysis of the human mtTFA has demonstrated that its C-terminal tail is important for speciﬁc DNA recognition and is essential for efﬁcient transcription (Dairaghi et al., 1995a). The mechanism of transcription stimulation by mtTFA seems to be related to its ability to bend DNA upon binding, thereby facilitating DNA strands unwinding (Fisher et al., 1992). Scanning transmission electron microscopy revealed that the Xenopus homologue also causes sharp bending of the DNA duplex at the promoter activation site (Antoshechkin et al., 1997). These mtTFA-induced conformational changes of mtDNA may be required to allow the core RNA polymerase access to the template for initiation of the transcription process. As described above, both major transcription promoters in human mitochondria can function bidirectionally, in vitro as well as in vivo (Chang et al., 1986). The asymmetric binding of mtTFA relative to the transcription start site may ensure that transcription proceeds primarily in a unidirectional fashion (Fig. 1). The existing 10-bp spacing between the mtTFA binding site and the start site of transcription seem to be necessary for efﬁcient transcription (Dairaghi et al., 1995b).

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An additional protein, which is necessary for mitochondrial RNA polymerase activity, has been identiﬁed in yeast and Xenopus laevis (Antoshechkin and Bogenhagen, 1995; Lisowsky and Michaelis, 1988). This factor, designated mtTFB, exhibits sequence homology to the dissociable subunit of bacterial RNA polymerases which is responsible for promoter recognition. More recently, McCulloch and colleagues (2002) and Falkemberg and colleagues (2002) identiﬁed this second mitochondrial transcription factor in human cells (mtTFB or TFBM). The latter study identiﬁed two forms of this factor (TFB1M and TFB2M) with TFB2M having the strongest activity. These proteins have homology to rRNA dimethyltransferases and RNA adenine methyltransferases suggesting that they are part of a family of nucleic acid binding proteins. Although they seem to interact with the mitochondrial RNA polymerase, their mode of action remains to be determined.

B. TRANSCRIPTION ELONGATION AND TERMINATION The mtDNA L-strand is transcribed as a single polycistronic precursor RNA, encompassing most of the genetic information encoded on the strand (Murphy et al., 1975; Montoya et al., 1981). The HSP seems to direct transcription of the entire H-strand in a similar fashion. In all studied cells, the rRNAs are synthesized at a much higher rate than the individual mRNAs encoded on the H-strand (Gelfand and Attardi, 1981). This difference has been explained by two possible mechanisms: (1) the existence of two distinct initiation sites for H-strand transcription. According to this model, transcription starts relatively frequent at the ﬁrst HSP and then terminates at the downstream end of the 16S rRNA gene. This transcription process would be responsible for synthesis of the vast majority of the two rRNA species. In contrast, transcription starting at a second HSP would be less frequent but would result in polycistronic molecules corresponding to almost the entire H-strand, yielding all the mRNAs and most of the tRNAs encoded on the H-strand (Montoya et al., 1982). (2) the difference in synthesis rate of rRNA/mRNA could also be explained by transcription attenuation at the junction of the 16S rRNA and tRNALeu(UUR) genes by a factor named mTERF (Kruse et al., 1989) (or mtTERM; Hess et al., 1991). In addition to an attenuation function for H-strand transcription, mTERF may also stop L-strand transcription at a site where no L-strand-encoded genes are found downstream (Hess et al., 1991). Because mTERF also mediates termination of transcription by heterologous RNA polymerases, it probably stops elongation of transcription by constituting a physical barrier, rather than by speciﬁc interactions with the RNA polymerase, (Shang and Clayton, 1994). These two mechanisms are not mutually exclusive, and may work in a coordinated manner.

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A polypeptide of around 34 kDa has been associated with mTERF function. The cDNA coding for the human polypeptide was cloned and sequenced. The polypeptide contains two widely separated basic regions and three leucine zipper motifs that were necessary for its DNA-binding capacity (Fernandez-Silva et al., 1997). However, the recombinant protein was unable to promote transcription termination in an in vitro system, suggesting that an additional component may be required for the termination activity.

C. POSTTRANSCRIPTIONAL MODIFICATIONS No intron sequences are present in vertebrate mtDNA and intergenetic sequences are minimal. The processing of the long polycistronic H- and L-strand messengers is thought to require only a few enzymes. Genes for tRNAs ﬂank the two rRNA genes and nearly every protein coding gene (Fig. 1). This unique genetic organization has suggested that the secondary structure of the tRNA sequences provide “cleavable tags” (Ojala et al., 1981). Precise endonucleolytic excision of the tRNAs from the nascent transcripts will concomitantly yield correctly processed rRNAs, and in most cases, correctly processed mRNAs (Ojala et al., 1981; Montoya et al., 1982). In cases in which the mRNA termini cannot be accounted for by tRNA excision, the processing enzyme possibly recognizes a secondary structure that shares features with the cloverleaf structures of tRNAs. Mitochondrial mRNAs are polyadenylated by a mitochondrial poly(A) polymerase during or immediately after cleavage (Rose et al., 1975). Maturation of mitochondrial tRNAs involves three enzymatic activities: (1) cleavage at the 5 end. This activity is performed by a mitochondrial RNase P (mtRNase P). In contrast to vertebrates’ mtRNase P, yeast mtRNase P has been characterized in detail. The enzyme of S. cerevisiae is composed of a nuclear-encoded protein and a mtDNA-encoded RNA species (Dang and Martin, 1993). The RNA moiety of the ribonucleoprotein complex is AU-rich and forms the catalytic core of the enzyme. (2) Cleavage at the 3 end. The endonuclease responsible for 3 end cleavage of tRNAs has not been characterized. (3) Maturation of the excised tRNAs. This process is completed by addition of the sequence CCA to their 3 end catalyzed by ATP(CTP):tRNA nucleotidyltransferase (Rossmanith et al., 1995).

X. Translation of Mitochondrial Transcripts

There are close to 100 ribosomes per mitochondrion (Cantatore et al., 1987). Mammalian mitochondrial ribosomes have an unusually low RNA

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content, and consequently, a low sedimentation coefﬁcient (approximately 55S (Hamilton and O’Brien, 1974)). The 39S and 28S ribosomal subunits contain the 16S and 12S rRNA species, respectively, which are encoded by the mtDNA (Attardi and Ojala, 1971; Brega and Baglioni, 1971). Twodimensional gel electrophoresis has allowed the identiﬁcation of 85 protein spots from bovine and 86 from rodent mitochondrial ribosomes (Matthews et al., 1982; Cahill et al., 1995). It is possible that some of these mitochondrial ribosomal proteins have adopted structural and functional roles of rRNA sequences. Although mammalian mitochondrial ribosomes differ in many aspects from both eukaryote (cytosolic) and prokaryote ribosomes, they retain some properties of the putative prokaryote ancestral, such as sensitivity to chloramphenicol and insensitivity to cyclohexemide (Lamb et al., 1968). The mitochondrial rRNA and tRNA species are relatively small when compared to other systems. Mammalian mitochondrial mRNAs have no 5 untranslated region (5 UTR) and are devoid of a cap structure (Grohmann et al., 1978). Because of this lack of 5 UTR, coding sequences start at or very near the 5 end with the codon for the initiating N-formylmethionine (Montoya et al., 1981). Approximately 400 nucleotides are required for efﬁcient binding of mRNAs to the small ribosomal subunit, even though a smaller region actually interacts with the ribosome (Denslow et al., 1989; Liao and Spremulli, 1989; Liao and Spremulli, 1990). After binding of the small ribosomal subunit to the messenger, the subunit appears to move to the 5 end of the mRNA mediated by yet unknown auxiliary initiation factors (Denslow et al., 1989). The only initiation factor identiﬁed in mammalian mitochondria to date is mtIF-2 (Liao and Spremulli, 1991). The cDNA for the human mtIF-2 has been cloned and sequenced (Ma and Spremulli, 1995). This monomeric protein belongs to the family of GTPases and promotes fMet-tRNA binding to the small ribosomal subunit in the presence of GTP and a template. Detailed in vitro characterization of bovine mtIF-2 suggested that mtIF-2 may bind to the small ribosomal subunit prior to its interaction with GTP. GTP would enhances the afﬁnity between mtIF-2 and the small subunit and allow fMettRNA to join the complex (Ma and Spremulli, 1996). Hydrolysis of GTP seems to facilitate the release of mtIF-2 and the association of the large (39S) ribosomal subunit to form the 55S initiation complex. Nonhydrolysable analogues of GTP can still promote formation of the initiation complex, indicating that GTP hydrolysis is not required for subunit joining (Liao and Spremulli, 1991). The mitochondrial elongation factors, mtEF-Tu, mtEF-Ts, and mtEF-G, have been puriﬁed from bovine liver (Schwartzbach and Spremulli, 1989; Chung and Spremulli, 1990). The human cDNAs for all three factors have

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been cloned and sequenced (Ma and Spremulli, 1995; Woriax et al., 1995; Xin et al., 1995). The in vitro characterization of the puriﬁed factors and the cDNA sequence information have revealed similarities with the corresponding prokaryotic factors, suggesting that elongation of the nascent mitochondrial polypeptide in mitochondria proceeds in a similar fashion as in bacterial systems (Nierhaus, 1996).

XI. Concluding Remarks

Because of its probable prokaryote origin, in many aspects, the mitochondrion behaves as an independent entity living inside an eukaryotic cell. All basic processes associated with life (DNA maintenance, transcription, and translation) occur inside the organelle. However, the vast majority of the factors involved in promoting and controlling these processes are borrowed from the cytoplasm, where nuclear-coded proteins are synthesized. Our understanding of the intricate relationships between mitochondrial and nuclear genomes is still limited, due mainly to the fact that genetic manipulation and in vitro systems are difﬁcult to develop for organelles. Nevertheless, a picture is emerging that shows the mitochondrial genetic system has many features in common with the putative prokaryote ancestral, but yet has developed a number of unique mechanisms as it evolved as an endosymbiont taking advantage of what a complex eukaryote nuclear genome has to offer.

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Rampazzo, C., Gallinaro, L., Milanesi, E., Frigimelica, E., Reichard, P., and Bianchi, V. (2000). A deoxyribonucleotidase in mitochondria: Involvement in regulation of dNTP pools and possible link to genetic disease. Proc. Natl. Acad. Sci. USA 97, 8239–8244. Rinaldi, A. M., De Leo, G., Arzone, A., Salcher, I., Storace, A., and Mutolo, V. (1979). Biochemical and electron microscopic evidence that cell nucleus negatively controls mitochondrial genomic activity in early sea urchin development. Proc. Natl. Acad. Sci. USA 76, 1916–1920. Rose, K. M., Morris, H. P., and Jacob, S. T. (1975). Mitochondrial poly(A) polymerase from a poorly differentiated hepatoma: puriﬁcation and characteristics. Biochemistry 14, 1025– 1032. Rossmanith, W., Tullo, A., Potuschak, T., Karwan, R., and Sbisa, E. (1995). Human mitochondrial tRNA processing. J. Biol. Chem. 270, 12885–12891. Ruiz De Mena, I., Lefai, E., Garesse, R., and Kaguni, L. S. (2000). Regulation of mitochondrial single-stranded DNA-binding protein gene expression links nuclear and mitochondrial DNA replication in drosophila. J. Biol. Chem. 275, 13628–13636. Schultz, R. A., Swoap, S. J., McDaniel, L. D., Zhang, B., Koon, E. C., Garry, D. J., Li, K., and Williams, R. S. (1998). Differential expression of mitochondrial DNA replication factors in mammalian tissues. J. Biol. Chem. 273, 3447–3451. Schwartzbach, C. J., and Spremulli, L. L. (1989). Bovine mitochondrial protein synthesis elongation factors. Identiﬁcation and initial characterization of an elongation factor Tu-elongation factor Ts complex. J. Biol. Chem. 264, 19125–19131. Shadel, G. S., and Clayton, D. A. (1997). Mitochondrial DNA maintenance in vertebrates. Annu. Rev. Biochem. 66, 409–435. Shang, J., and Clayton, D. A. (1994). Human mitochondrial transcription termination exhibits RNA polymerase independence and biased bipolarity in vitro. J. Biol. Chem. 269, 29112– 29120. Shmookler Reis, R. J., and Goldstein, S. (1983). Mitochondrial DNA in mortal and immortal human cells. Genome number, integrity, and methylation. J. Biol. Chem. 258, 9078–9085. Spelbrink, J. N., Toivonen, J. M., Hakkaart, G. A., Kurkela, J. M., Cooper, H. M., Lehtinen, S. K., Lecrenier, N., Back, J. W., Speijer, D., Foury, F., and Jacobs, H. T. (2000). In vivo functional analysis of the human mitochondrial DNA polymerase POLG expressed in cultured human cells. J. Biol. Chem. 275(32), 24818–24828. Spelbrink, J. N., Li, F. Y., Tiranti, V., Nikali, K., Yuan, Q. P., Tariq, M., Wanrooij, S., Garrido, N., Comi, G., Morandi, L., Santoro, L., Toscano, A., Fabrizi, G. M., Somer, H., Croxen, R., Beeson, D., Poulton, J., Suomalainen, A., Jacobs, H. T., and Zeviani, M. (2001). Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria. Nat. Genet. 28(3), 223–231. Stoneking, M. (2000). Hypervariable sites in the mtDNA control region are mutational hotspots. Am. J. Hum. Genet. 67, 1029–1032. Tang, Y., Schon, E. A., Wilichowski, E., Vazquez-Memije, M. E., Davidson, E., and King, M. P. (2000). Rearrangements of human mitochondrial DNA (mtDNA): New insights into the regulation of mtDNA copy number and gene expression. Mol. Biol. Cell 11, 1471–1485. Tapper, D. P., and Clayton, D. A. (1981). Mechanism of replication of human mitochondrial DNA. Localization of the 5 ends of nascent daughter strands. J. Biol. Chem. 256, 5109–5115. Tiranti, V., Savoia, A., Forti, F., D’Apolito, M. F., Centra, M., Rocchi, M., and Zeviani, M. (1997). Identiﬁcation of the gene encoding the human mitochondrial RNA polymerase (h-mtRPOL) by cyberscreening of the Expressed Sequence Tags database. Hum. Mol. Genet. 6, 615–625. Topcu, Z., and Castora, F. J. (1995). Mammalian mitochondrial DNA topoisomerase I preferentially relaxes supercoils in plasmids containing speciﬁc mitochondrial DNA sequences. Biochim. Biophys. Acta 1264, 377–387.

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OXIDATIVE PHOSPHORYLATION: STRUCTURE, FUNCTION, AND INTERMEDIARY METABOLISM

Simon J. R. Heales,1 Matthew E. Gegg, and John B. Clark Departments of Neurochemistry and Clinical Biochemistry (Neurometabolic Unit) Institute of Neurology and National Hospital London, WC1N 3BG, United Kingdom

I. Historical Background II. The Mitochondrial Electron Transport Chain A. Complex I B. Complex II C. Complex III D. Complex IV E. Complex V F. ADP–ATP Translocator III. Intermediary Metabolism A. Pyruvate Dehydrogenase B. The TCA Cycle C. Mitochondrial Fatty Acid Oxidation D. Ketone Body Metabolism IV. Concluding Remarks References

I. Historical Background

Although K¨olliker (1856) had described granules in striated muscle in the middle nineteenth century, it was not until the turn of the twentieth century that the name mitochondrion came into use. Altman (1894), in his “Die Elementar Organismen und ihre Beziehungen zu den Zellen,” spoke of primitive self-replicating bodies or bioblasts that he stained speciﬁcally and referred to as “elementary particles.” However, it was the cytologist Benda who in 1898 coined the name mitochondrion from the Greek for thread (mitos) and grain (chondros) from his studies on the thread-like granules he observed in sperm and ova. Two years later Michaelis, using a variety of dyestuffs including Janus Green, demonstrated that these granules had oxidoreduction activities. 1

To whom correspondence should be addressed.

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We then enter what can only be described as the golden era of the German school in which the likes of Warburg, Wieland, and more later Krebs studied the respiration and metabolism of various cellular preparations. Warburg in 1913 described oxygen respiratory granules in liver cells and Wieland in 1932 published on the mechanism of oxidation. These studies were complemented by those of Keilin and Hartree, on cytochromes during the same period. However, the importance of these phenomena both in terms of their cellular localization to the mitochondria and their relevance to cellular energetics and ATP production were largely unappreciated until the late 1930s. In the same way, it is interesting to note that Meeves in 1918 suggested that mitochondria have hereditary characteristics, a suggestion which was largely ignored until the controversies of the mid-1960s (Lehninger, 1965). The advent of the electron microscope and the high-speed refrigerated centrifuge during the 1940s and 1950s allowed a quantum leap in our understanding of both the structure and function of mitochondria. The development of the technique of differential centrifugation by Claude and others in the 1940s allowed the isolation of relatively pure preparations of mitochondria, permitting detailed studies of the main metabolic activities of these organelles in the early 1950’s by Kennedy, Lehninger, Hogeboom, and others. This was complemented by the high-resolution electron micrograph (EM) studies by Palade and Sj¨ostrand, thus providing the basis for a ﬁrm and a detailed understanding of the structure and function of mitochondria. Following on from this period, the next two decades or more were taken up with the sometimes heated controversies relating to the mechanism of the process of oxidative phosphorylation. Contributors to this were many but include Boyer, Chance, Green, Mitchell, Slater, and Williams, resulting in a consensus at this time that although basically chemiosmotic in nature nevertheless has aspects drawn from the other so-called chemical and conformational theories. The mid-1960s also brought a renewal of the controversy of whether mitochondria contained DNA. The work of Roodyn, Wilkie, and Work (Roodyn and Wilkie, 1968) was central to this, providing the evidence that this was not due to bacterial contamination, and Nass concluded in 1965 that “DNA is an integral part of most and probably all mitochondria (Roodyn and Wilkie, 1968).” This also provided support for the concept that the mitochondrion has evolved from a symbiotic bacterium and had its own capability of coding for and synthesizing its own proteins. This was of course proved beyond doubt, when in the 1980s the complete sequence of mitochondrial DNA (mtDNA) was sequenced by the laboratories of Sanger and Attardi (Anderson et al., 1981; Chomyn et al., 1985). This coincided with a growing recognition of mitochondrial diseases, pioneered by the work of Clark, Morgan-Hughes, Land (Morgan-Hughes et al., 1977),

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and others in which the biochemical defects at the level of the mitochondrial electron transport chain had been described in certain neuromuscular disorders. In the 21st century we are now grappling with relating the clinical phenotype with genotype in these diseases, together with attempting to understand the mechanisms whereby mitochondrial dysfunction is caused, e.g., oxidative stress and how this relates to cell death (apoptosis/necrosis) in neurodegenerative disease.

II. The Mitochondrial Electron Transport Chain

Each human cell contains hundreds of mitochondria that are approximately 1 μm in length. The shape of these organelles varies from spherical to rod-like, and on occasion, they appear to form a network. Each mitochondrion has a double membrane structure, i.e., the outer mitochondrial membrane surrounds the inner membrane. This inner membrane is invaginated and forms cristae (Schefﬂer, 1999). The space between the two membranes is known as the intermembrane space while the inner membrane encloses that matrix where a number of metabolic processes occur, e.g., the tricarboxylic acid (TCA) cycle, heme synthesis, part of the urea cycle and fatty acid oxidation. The inner mitochondria membrane is the site of the electron transport chain (ETC) and is where the process of oxidative phosphorylation occurs that facilitates ATP synthesis. The ETC is composed of more than 80 polypeptides components that are grouped together into four enzymatic complexes (Fig. 1). The polypeptides that constitute complex I (NADH: ubiquinone oxidoreductase), III (ubiquinol cytochrome c reductase), and IV (cytochrome c oxidase) are coded for by both nuclear and mitochondrial DNA. In contrast, complex II (succinate:ubiquinone oxidoreductase) is coded exclusively by the nuclear genome. In general terms, transfer of reducing equivalents from NADH or FADH2 (generated, e.g., from carbohydrate or fatty acid metabolism, see below) to molecular oxygen is coupled to the pumping of protons across the inner mitochondrial membrane, i.e, from the matrix into the intermembrane space. This transport of protons generates an electrochemical gradient that has two components: (a) a pH gradient resulting in a pH difference (pH) across the inner membrane of approximately 1.4, and (b) a membrane potential (ψ), due to charge separation, of about 150 mV. The resulting proton motive force is then dissipated, when there is a need to synthesise ATP, i.e, when the cellular ADP concentration increases. Dissipation of this gradient through the membrane

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C

Q

Q

FIG. 1. Schematic of the mitochondrial ETC. Details of this system can be found in the text. C and Q represent the mobile electron carriers, cytochrome c and ubiquinone, respectively.

sector of the ATP synthase leads to the phosphorylation of ADP (Mitchell, 1961). The rate of ATP synthesis, by the above system, is under tight control and is regulated via ADP. The cellular concentration of ADP is approximately 0.14 mmol/L, which is about 10-fold lower than that of ATP. Thus, a small decrease in ATP concentration, due to an increase in metabolic demand, is accompanied by a relatively large percentage increase in cellular ADP. Regulation of this system by ADP is known as respiratory control and ensures that oxidative phophorylation occurs only when there is a need to replenish ATP. In view of the key role the ETC plays in energy metabolism, damage to one or more of the respiratory chain complexes could lead to an impairment of cellular ATP formation. However, each of the complexes of the ETC appears to exert varying degrees of control over respiration. Furthermore, in vitro, studies suggest that substantial loss of activity of an individual complex may be required before ATP synthesis is compromised. However, the degree of control a particular complex exerts over respiration may differ between cell types. Within the brain, mitochondria appear to be heterogeneous. Thus, complex I, of nonsynaptic mitochondria, has to be inhibited by approximately 70% before inhibition of ATP synthesis occurs. However, for synaptic mitochondria, impairment of ATP synthesis occurs when complex I is inhibited by 25% (Davey et al., 1997).

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A. COMPLEX I NADH:ubiquinone oxidoreductase (complex I; EC 1.6.5.3) is the ﬁrst and largest enzyme in the electron transport chain. Complex I catalyzes the transfer of two electrons from NADH to ubiquinone. These are transferred through the enzyme by bound prosthetic groups. This transfer is coupled to the translocation of four to ﬁve protons from the matrix, across the inner membrane, to the intermembrane space. The three-dimensional structures of complex I from Escherichia coli, Neurospora crassa, and bovine heart have been determined by electron microscopy (Guenebaut et al., 1997; Grigorieff, 1998; Guenebaut et al., 1998). All the structures show a characteristic L shape, with one arm embedded in the membrane and the other projecting into the matrix (Fig. 2). The matrix domain has a globular structure, and it is connected to the elongated membrane domain by a narrow stalk. A constriction in the membrane domain is present in both the N. crassa and bovine enzymes. Bovine complex I has 43 different subunits with a molecular mass of approximately 900 kDa. The molecular masses of the matrix domain, including the stalk, and the membrane domain were determined as 520 and 370 kDa, respectively (Grigorieff, 1999).

FIG. 2. Structure of complex I. Characteristic L-shaped structure of complex I. The NADH oxidation occurs in the peripheral matrix domain, while subunits in the membrane domain are thought to be responsible for proton pumping. Treatment of complex I with detergent yields the subcomplexes Iα and I β. Harsher treatment divides the membrane domain into Iβ and Iγ (denoted by dashed line). Transfer of electrons to ubiquinone (Q) is thought to be mediated by subunits located in Iγ .

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Complex I has one noncovalently bound ﬂavin mononucleotide molecule (FMN) and at least six iron–sulfur clusters and two ubiquinone binding sites. Only four or ﬁve iron–sulfur clusters have been resolved and characterized by electron paramagnetic resonance spectroscopy. The location of the remaining iron–sulfur clusters (Ohnishi, 1998) and the ubiquinone binding sites (Tormo and Estornall, 2000) is still highly contentious. Consequently, the mechanism of electron transfer, and how this is coupled to proton transfer, remains unresolved. In the absence of crystal structures and genetic approaches, treatment of bovine complex I with the chaotrope, percholate, and the detergent, N,N-dimethyldodecylamine N-oxide, have contributed to the understanding of both the location, organization, and properties of the 43 subunits. Treatment of the bovine enzyme with percholate releases three fractions, a water-soluble fragment known as the ﬂavoprotein (FP) fraction, the iron– sulfur protein, and a hydrophobic complex. The FP fraction retains the ability to transfer electrons from NADH to ferricyanide, and it consists of three subunits, the 51-, 24-, and 10-kDa subunits. The 51-kDa subunit is the site for binding of both NADH and the primary electron acceptor, FMN. The 51- and 24-kDa subunits also both contain iron–sulfur clusters. The nondenaturing detergent N,N-dimethyldodecylamine N-oxide dissociates complex I differently, yielding two subcomplexes termed Iα and Iβ. The Iα retains the biochemical activity of the complex and primarily contains the soluble peptides that reside in the matrix domain. The membrane domain with no biochemical activity is therefore the Iβ complex. Complex I from E. coli and other bacteria are made up of at least 14 polypeptides and are all present as homologues in both N. crassa and mammalian mitochondria. These proteins are considered to be the “minimal” subunits required for electron transfer and proton translocation. Seven bovine homologues from the bovine Iα fraction are found in E. coli: 75, 51, 49, 30, 24, TYKY, and PSST. The polypeptides in Iα that are not minimal subunits have been termed as “accessory” proteins, although the majority of subunits have yet to be assigned a particular function. Many of the polypeptides have no relation to other proteins. The 18-kDa subunit contains a cAMP-dependent kinase phosphorylation site motif (Sardanelli et al., 1995). Phosphorylation of this subunit activates complex I, and it is proposed to be an additional mechanism whereby overall respiratory chain activity is regulated (Papa et al., 2001). Subunit SDAP is an acyl-carrier protein and may be involved in lipid biosynthesis and/or repair. The stalk between the matrix and membrane domains has a diameter of 30 A˚ and is postulated to be part of the electron transfer pathway linking

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the NADH binding domain in the matrix to the ubiquinone binding sites of the membrane domain. The iron–sulfur cluster N2 is considered to play an important role in complex I. It has the highest reduction potential of all the clusters in complex I and its one electron reduction/oxidation is coupled to the binding and release of one proton. This cluster has been located to the stalk region. Both PSST and TYKY have been advocated to be the subunit that binds N2 (Ohnishi et al., 1998). The two candidates are both amphipathic and in direct interaction with the membrane domain. The N2 cluster transfers electrons to ubiquinone; the distance between N2 ˚ The N2 cluster is and one of the ubiquinone binding sites is only 8–11 A. most likely located inside the membrane. Seven subunits of mammalian complex I are coded for by mitochondrial DNA: ND 1, 2, 3, 4, 4L, 5, and 6. They are all located in the membrane domain and constitute the remaining seven minimal subunits found in bacterial complex I. The ND subunits are similar to bacterial cation/H+ antiporters, and they are thought responsible for proton translocation. The constriction of the membrane arm divides the domain into one-third and two-thirds portions. Relatively harsh N,N-dimethyldodecylamine N-oxide treatment produces, in addition to subcomplexes Iα and Iβ, the small subcomplex known as Iγ (Fig. 2). The smaller Iγ fraction contains subunits from the smaller part of the membrane arm, while Iβ constitutes the larger part of the arm (Sazanov et al., 2000). The ND1, 2, 3, 4L, and the nuclear-encoded KFY1, are found in Iγ , while ND4 and ND5 and 11 nuclear subunits reside in Iβ. The ND6 could not be located. ND1 and ND2 form a subcomplex within Iγ . The ND1 binds rotenone and ubiquinone, and it is probably intimately involved with ubiquinone binding and reduction. The location of ND1 in Iγ locates the subunit close to the redox centers of Iα and the stalk. At least two functional and spatially distinct ubiquinone reaction centers are thought to exist in complex I. A wide inhibitor binding domain between the two ubiquinone reaction centers has been proposed (Tormo and Estornell, 2000). Experiments in N. crassa have indicated that the matrix and membrane domains undergo independent assembly (Videira, 1998). Whether this phenomena is analogous to mammals is uncertain. In fungi, the nuclear- and mitochondrial-coded genes are exclusive to the matrix and membrane domains respectively. This is not the case in mammalian mitochondria. Frame shift mutations in ND4 and ND6 (in human and mouse) results in defective assembly of the mitochondrial-encoded subunits with loss of complex I activity. However, NADH:ferricyanide oxidoreductase activity is unaffected, indicating that the ﬂavoprotein fragment is present (Bai and Attardi, 1998).

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B. COMPLEX II The ﬂavoprotein succinate:ubiquinone oxidoreductase (complex II; EC 1.3.5.1) oxidizes succinate to fumarate, transferring the electrons to ubiquinone. Complex II is the only enzyme that serves as a direct link between the citric acid cycle (succinate dehydrogenase) and the electron transport chain. The enzyme is both structurally and catalytically closely related to the fumarate reductases. Fumarate reductases are synthesized in anaerobic organisms that utilize fumarate as the terminal electron acceptor. The elucidation of complex II structure and function has been achieved using both the mammalian enzyme and prokaryotic fumarate reductases (reviewed in Ackrell, 2000, and Hagerhall, 1997). Bovine complex II is comprised of a hydrophilic domain that projects into the matrix and a hydrophobic membrane anchor (Fig. 3). The hydrophilic domain contains a ﬂavoprotein subunit (70 kDa) intimately associated with an iron–sulfur subunit (30 kDa). This domain functions as a succinate dehydrogenase in the presence of an artiﬁcial electron acceptor such as ferricyanide, but does not interact directly with ubiquinone. The anchor domain contains the two polypeptides QPs-1 and QPs-3 (15 and 13 kDa, respectively). The anchor domain needs to be present for the reduction of ubiquinone to occur. Ubisemiquinone has been detected bound to intact or reconstituted complex II formed from QPs and succinate dehydrogenase, but not succinate dehydrogenase alone. The primary sequences of both the

FIG. 3. Structure of mammalian complex II. The matrix domain responsible for catalytic activity contains the ﬂavoprotein (FP), the capping domain (C), and the iron-sulfur protein (IP). The matrix domain is attached to the membrane by QPS-1 and QPS-3. The membrane spanning domain contains the ubiquinone binding sites (Q).

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ﬂavoprotein and the iron–sulfur subunits are highly homologous between species, while the anchor domain illustrates greater diversity. Unlike the other complexes in the electron transport chain, the four polypeptides of mammalian complex II are all coded for by nuclear genes (Hirawake et al., 1999). The ﬂavoprotein subunit polypeptide is folded into four domains [a large ﬂavin adenine dinucleotide (FAD) binding domain, a mobile capping domain, a helical domain, and a C-terminal consisting of an antiparallel β-sheet] and contains the dicarboxylate binding site (Hagerhall, 1997; Lancaster et al., 1999; Ackrell, 2000). The FAD binding domain has a Rossmann-type fold and is very similar to other FAD binding domains such as thioredoxin reductase. The FAD prosthetic group is covalently bound to the protein by a histidine residue (several H-bonds further hold the FAD in place). Flavin adenine dinucleotide is the primary electron acceptor in complex II. To aid electron transfer, the dicarboxylate binding site is predominantly formed by the FAD isoalloxazine ring (Lancaster et al., 1999). The iron–sulfur subunit has an N-terminal “plant ferredoxin” domain and a C-terminal “bacterial ferredoxin” domain, and binds three iron–sulfur centers. The N-terminal domain contains the [2Fe–2S] iron–sulfur center, while the [4Fe–4S] and [3Fe–4S] iron–sulfur centers are located in the C-terminal. Three groups of highly conserved cysteine residues serve as ligands to the centers. X-ray crystallography has indicated that the [2Fe–2S] iron–sulfur center is closest to the FAD moiety (12.3 A˚ in W. succinogenes and E.coli (Ackrell, 2000)). The [4Fe–4S] center connects the [2Fe–2S] center with the [3Fe–4S] center. Electrons are passed singly from the [3Fe–4S] center to ubiquinone forming semiubiquinone before becoming fully reduced and exchanging with the ubiquinone pool in the membrane. The [3Fe–4S] center also appears to have an important structural role. Puriﬁed ﬂavoprotein + iron–sulfur protein fractions can only rebind to the anchor domain when the [3Fe–4S] center is intact (Hagerhall, 1997). The cysteine residues that ligate this center are within segments that are in contact with the anchor domain (Lancaster et al., 1999). The structure of the anchor domain varies greatly between species. The anchors are classiﬁed into four types, and differ in topology, number of polypeptides, and cytochrome b content (Hagerhall, 1997; Hirawake et al., 1999). Mammalian complex II consists of two membrane subunits, QPs-1 and QPs-3, and one cytochrome b prosthetic group. Each subunit has three helices that span the membrane (Yu et al., 1992; Hagerhall and Hederstedt, 1996). Structural, EPR, and inhibitor studies indicate that there are probably two ubiquinone binding sites in the mammalian membrane anchor, with

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both polypeptides providing a site each (Lee et al., 1995; Shenoy et al., 1999). The QPs-1 site is located close to the negative (matrix) side of the membrane. This site appears to be bordered by both the iron–sulfur and anchor domains, and it is close to both the [3Fe–4S] center and the b-type heme. The QPs-3 site is located on the positive side of the membrane. Isolated QPs contains 27 nmol of cytochrome b/mg of protein. The function of the heme in complex II is still unknown. Studies of Bacillus subtilis and E. coli succinate:ubiquinone oxidoreductase have implicated the heme in playing an important role in the assembly of the enzyme. Absence of heme leads to the synthesis of apocytochrome, and to the accumulation of both the ﬂavoprotein and iron–sulfur domains in the cytoplasm (Hagerhall, 1997). The ligand for the b-type heme in complex II has been identiﬁed as being a bishistidine. Expression of both polypeptide anchors in E. coli is necessary for heme insertion and enzyme activity, indicating that one ligand is provided from each polypeptide (Shenoy et al., 1999). The core of the membrane anchor in mammalian complex II is proposed to be a four-helix antiparallel bundle (two helices each from QPs-1 and QPs-3) with the heme group oriented approximately perpendicular to the membrane plane (Hagerhall and Hederstedt, 1996).

C. COMPLEX III Ubiquinol:cytochrome c reductase (complex III; EC 1.10.2.2) is also known, because of the two cytochromes found within it, as the bc1 complex. This component of the ETC transfers electrons from reduced ubiquinone (ubiquinol) to cytochrome c. This electron transfer is coupled to proton pumping from the matrix to the inner membrane space, contributing to the proton gradient required for ATP synthesis. The structure of complex III in a variety of mammalian species has been elucidated (Iwata et al., 1998; Kim et al., 1998; Zhang et al., 1998). The protein exists as a homodimer with each monomer consisting of 11 different subunits with a total molecular mass of approximately 240 kDa (see Table I). The two monomers of the complex have a twofold axis of symmetry in the plane of the membrane (Fig. 4). Chicken complex III is 150 A˚ in length, spanning the membrane, and projecting into both the intermembrane space and matrix by 31 and 79 A˚ respectively (Zhang et al., 1998). Functionally, the most important subunits in complex III are cytochrome b (containing both a low and high potential b-type heme, bL and bH), cytochrome c (containing one c1-type heme), and the Rieske protein (bound to a [2Fe–2S] iron–sulfur center). This observation is supported by the fact that in purple bacteria, the complex is comprised of just three or four subunits containing the redox centers above. The functions of the eight

TABLE I SUBUNITS OF BOVINE HEART COMPLEX III Subunit

Prosthetic group

1. Core 1 2. Core 2 3. Cytochrome b 4. Cytochrome c1

Hemes bH, bL Heme c1

5. Rieske protein

[2Fe–2S]

6. 13.4 K 7. Q binding 8. c1 hinge 9. Presequence of Rieske protein 10. c1 associated 11. 6.4 K

Location

Mr(kDa)

Matrix Matrix Membrane Membrane and intermembrane space Membrane and intermembrane space Matrix Membrane Intermembrane space Matrix

49.1 46.5 42.6 27.3

Membrane Membrane

21.6 13.3 9.6 9.2 8 7.2 6.4

FIG. 4. Structure of complex III. Complex III exists as a dimer with the monomers related by a twofold axis in the plane of the paper (dashed line). The intermembrane domain of the Rieske protein (shaded grey with a star denoting the Fe–S center) is mobile. The domain can be close to the transmembrane domain, which is also the location of the two ubiquinone binding sites, Q o (black triangle) and Q i (white triangle), and the high (bH) and low potential (bL) b-type hemes (white squares). In the other conformation, the Reiske protein is located close to cytochrome c1 and soluble cytochrome c.

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additional polypeptides present in mammalian complex III are still largely unknown. Genetic studies in yeast have indicated that, with the exception of subunit 6, mutations inserted into subunits containing no prosthetic groups are respiration deﬁcient. Therefore these polypeptides are still necessary for complex III activity. The intermembrane side of bc1 contains the functional domains of cytochrome c1 (including the heme), the Rieske iron–sulfur protein and subunit 8. The transmembrane domain is comprised of 13 transmembrane helices, one each from cytochrome c1, the Rieske protein, and subunits 7, 10, and 11 and eight from cytochrome b. Cytochrome b also has four horizontal helices on the intermembrane side. The intermembrane domains of cytochrome c1, the iron–sulfur protein and subunit 8 are positioned on top of these helices (Iwata et al., 1998). Hemes bH and bL are close to the matrix and intermembrane sides, respectively, and are in the middle of a four-helix bundle. More than half of the molecular mass of the complex is located in the matrix domain. The two large core proteins, subunits 1 and 2, subunit 6 and subunit 9, reside in this domain. These subunits are thought to have more of a structural role, with subunit 2 implicated in the stabilization of the dimer. Subunits 1 and 2 have homology with the two subunits of mitochondrial matrix processing peptidase. Evidence for the possible protease activity of subunits 1 and 2 is provided by the location of subunit 9. Subunit 9 is the presequence of the nuclear-encoded Rieske protein. In mammals, subunit 9 is cleaved from the iron sulfur protein following import into the mitochondria and resides between subunits 1 and 2 (Iwata et al., 1998; Schefﬂer, 1999). The mechanism by which electrons are transferred through complex III has been termed the Q cycle. Complex III has two ubiquinone sites, Q o and Q i, that are located near the membrane surface facing the intermembrane space and matrix, respectively. Electron transfer from ubiquinol bound at the Q o site is bifurcated. One electron is sequentially transferred to the Rieske iron–sulfur protein, cytochrome c1 and ﬁnally to soluble cytochrome c. The oxidation of ubiquinol by the Rieske protein results in the release of two protons into the intermembrane space and the generation of ubisemiquinone at the Q o site. The electron from semiubiquinone bound at Q o is transferred consecutively to heme bL, bH, and ﬁnally to ubiquinone bound at the Q i site, thus forming semiubiquinone. The sequential oxidation of a second ubiquinol at Q o will reduce semiubiquinone to ubiquinol at the Q i site. The two protons required for this are taken up from the matrix. Ubiquinol is then free to bind to Q o, thus completing the cycle (Crofts et al., 1999; Snyder et al., 2000). The X-ray crystal structures of complex III from chicken, cow, and rabbit in the absence and the presence of inhibitors of quinone oxidation have

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shown that the extrinsic domain of the Rieske iron–sulfur protein assumes one of two conformations (Zhang et al., 1998). Crystals in the presence of stigmatellin, a Q o site inhibitor, show the extrinsic domain of the iron sulfur center close to the heme groups of cytochrome b and the Q o site. Histidine 161, a ligand for the iron–sulfur center, is in an H-bond distance of the Q o site (Zhang et al., 1998). This is termed the proximal conformation. However, crystals in the native form show the extrinsic domain of the iron– sulfur center is close to the electron acceptor, the heme of cytochrome c1 (distal conformation). The relative position of the iron–sulfur center in chicken crystals in the presence of inhibitor is 16 A˚ from that of the native structure. When the Rieske protein is in the distal conformation (close to cytochrome c1), the distance from the [2Fe–2S] center to the expected center of the substrate (in this case stigmatellin, ubiquinol in vivo) is approximately ˚ Rapid electron transfer is possible over this distance given a proper 22 A. protein matrix. However, when the Rieske protein is in the distal conformation, the iron–sulfur center is separated from the Q o site by a cleft, which is likely to be aqueous (Crofts et al., 1999). Given the differing conformations observed, and the inherent physical obstacles preventing efﬁcient electron transfer between donor and acceptor sites, it has been suggested that the reaction mechanism of complex III involves movement of the extrinsic domain of the Rieske iron–sulfur protein. Both the transmembrane helix and matrix side are unaltered in the presence of stigmatellin. The coil consisting of residues 68–73 is stretched in the presence of stigmatellin, implying that this region is responsible for the movement of the extrinsic domain (Zhang et al., 1998). In the proximal conformation, the Q o binding pocket is buried between the [2Fe–2S] center and the heme of cyt bL. The binding pocket is bifurcated, with a lobe to both cyt bL and the iron–sulfur protein docking interface. The Q i site is thought to either bind the inhibitor antimycin or at least overlap with the inhibitor’s binding site (Kim et al., 1998). X-ray crystals indicate that antimycin is bound in a cavity surrounded by heme bH, three transmembrane helices and the amphipathic surface helix of cytochrome b (Zhang et al., 1998). The same face of the iron–sulfur protein interacts with both the Q o site and cyt c1. A loop present in cytochrome c and c2 is absent in cyt c1, exposing heme propionates to the surface. This is within the electron transfer distance of the iron–sulfur center in the distal conformation, and it could be the route by which cyt c1 is reduced (Zhang et al., 1998). Reduction of cytochrome c by c1 is thought to require subunit 8, also termed the “hinge protein.” The protein has eight glutamate residues at the N-terminal that may form part of the cytochrome c docking site together with helix α1 of cytochrome c1 (Iwata et al., 1998).

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D. COMPLEX IV Cytochrome c oxidase (complex IV; EC 1.9.3.1) is the terminus for electron transfer in the respiratory chain. The enzyme couples the reduction of oxygen to water, using electrons from cytochrome c, to the pumping of protons from the matrix. Cytochrome c oxidase has the distinction of being the ﬁrst complex of the ETC to be crystallised. Crystallization of bovine heart complex IV by Tsukihara et al. (1996) revealed that the mammalian enzyme has 13 different subunits. Biochemical and spectroscopic analysis had previously alluded to the presence of two cytochromes (hemes a and a3) and two copper sites. Crystallization of the complex not only pinpointed their location but also revealed the location of two additional metal centers (one magnesium, one zinc), two cholates, and eight phospholipids (ﬁve phosphatidyl ethanolamine and three phosphatidyl glycerols) associated with it. The protein exists in the inner membrane as a dimer with each monomer having a molecular mass of 204 kDa (211 kDa including constituents). Viewed from the cytosolic side, the monomers face each other around a twofold axis of symmetry. The surface of each monomer facing the other is concave, forming a large opening between them (Fig. 5b). The X-ray structure failed to reveal any association between the phospholipid, cardiolipin, and complex IV. Cardiolipin is essential for complex IV activity and Tsukihara et al. (1996) suggest that there is space for two cardiolipin molecules within the intermonomer space. Subunits I–III are mitochondrially encoded and form the core of the protein. Subunit I binds heme a and heme a3 and also forms the CuB redox center, while subunit II binds the CuA center. Elucidation of the bacterial cytochrome c oxidase in Paracoccus dentriﬁcans (Iwata et al., 1995; Michel et al., 1998) illustrates that the protein contains only four subunits, the core of which, subunits I–III, are virtually identical at an atomic level to their mammalian counterpart. Only subunits I and II are required for a functionally active protein. This suggests that subunits I–III form the functional core of the protein. Viewed perpendicularly to the membrane, the core of cytochrome c oxidase looks like a trapezoid with an extension on the smaller side (Fig. 5a). The trapezoid forms the transmembrane domain, while the extension is a globular domain of subunit II that projects into the intermembrane space. Subunit I is a membranous protein with 12 transmembrane helices. Viewed from the intermembrane side, the helices are arranged in an anticlockwise fashion into three semicircles, each containing four helices bundles. This arrangement forms a “whirlpool” conformation (Tsukihara et al., 1996) with a threefold axis of symmetry. This structure forms three pores,

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FIG. 5. Schematic representation of complex IV. The trapezoid topology of subunits I–III perpendicular to the membrane plane is shown in (a). Cytochrome c binds at the corner formed by subunits I and II on the intermembrane side. The complex IV dimer as a cross section at the membrane surface when viewed from the cytosolic side is shown in (b). The three 4 helices bundles of subunit I, which form pores A, B, and C (open circles), are shown as dashed curves. Heme in pore C is represented by a dashed diagonal line. The heme a3–CuB center in pore B is denoted by a diagonal line (a3) and a diamond (Cu).

A, B and C (Fig. 5b). Subunit I contains the two hemes, heme a is located in pore C, while heme a3 is found in pore B. Heme a3, together with the copper atom CuB, forms the binuclear site involved in the reduction of oxygen to water. Both hemes are arranged perpendicularly to the membrane plane. Pore A is mainly ﬁlled with conserved aromatic residues. The helices of subunit I are not completely perpendicular to the membrane

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surface, with the helices sloping toward convergence on the intermembrane side. Subunits II and III associate with subunit I without any direct contact between each other. Subunit II has two transmembrane helices that interact with subunit I and an extramembranous globular domain in the intermembrane space. The globular domain has a ten-stranded β-barrel and sits upon part of the intermembrane face of subunit I (Tsukihara et al., 1996). This domain also contains the CuA site (comprised of two copper atoms) and is only 7 A˚ from the surface of the protein. The CuA site is the primary electron acceptor from cytochrome c. The corner formed by the extramembrane domain of subunit II and the ﬂat cytosolic surface of subunit I is thought to be the most likely cytochrome c binding site (Fig. 5a, Michel et al., 1998). This region contains ten exposed acidic residues that could bind the lysine residues at the heme edge of cytochrome c. The electrons are then transferred to heme a and then ﬁnally on to the heme a3–Cub binuclear site for the reduction of oxygen. The two heme edges are only 4.5 A˚ apart in subunit I. A hydrophilic cleft between subunits I and II proceeds from the binuclear site to the intermembrane surface of the enzyme and is thought to be a water channel. The channel has highly conserved hydrophilic residues and the magnesium binding site. Subunit III is almost entirely housed within the membrane and consists of seven transmembrane helices. These helices are divided into two bundles (helices I–II and III–VII) by a V-shaped cleft. In the mitochondria, the cleft contains two phosphatidylethanolamine and one phosphatidylglycerol molecule. The V-shaped cleft has been proposed to be the oxygen channel. The channel starts at the center of the lipid bilayer, where oxygen solubility is greater than in the aqueous phase, above a tightly bound lipid molecule, and leads directly to the binuclear site in subunit I. The mechanisms of proton transfer to the oxygen reduction site and proton pumping are still highly contentious (Michel, 1998; Michel et al., 1998; Riistama et al., 2000; Yoshikawa et al., 2000). Putative pathways for the transfer of protons in a protein moiety via a network of hydrogen bonds have been identiﬁed. Coupled proton pumping may occur either via a direct conformational change at the binuclear site or a structural change distant from the active site. The remaining ten subunits of mitochondrial cytochrome c oxidase are nuclear encoded. The function of these subunits is still largely unknown. They may play a role in insulation, regulation, stabilization, or assembly. No cytochrome c oxidase activity is observed in yeast in the absence of either subunit IV, VI, VII, or VIIa. In mammals, the nuclear-encoded subunits IV, VIa, VIIa, and VIII exist as two tissue-speciﬁc isoforms (Grossman and Lomax, 1997; Huttemann et al., 2001). The isoforms vary in the N-terminus of the protein (termed heart and liver type), and they are coded for by separate genes. The heart-type isoforms are expressed in heart and skeletal

OXIDATIVE PHOSPHORY LATION

41

muscle, while the liver-type isoform appears to be ubiquitously expressed. In humans, only the liver-type isoform of VIII is present in all tissues. These isoforms may provide a method by which cytochrome c oxidase can be differentially regulated depending on the tissue’s requirements. Seven subunits each possess one transmembrane helix, forming an irregular cluster surrounding the metal sites. The packing of the transmembrane subunits with one another is thought to aid the stability of the enzyme and increase the solubility of the core subunits within the membrane. Many areas of the core remain uncovered, especially on the cytosolic side. The remaining three subunits have extramembrane domains. Subunits Va and Vb are located on the matrix side, while VIb, which binds zinc, is on the cytosolic side.

E. COMPLEX V The ATP synthase (F1F0-ATP synthase) uses the proton motive force generated across the inner mitochondrial membrane by electron transfer through the ETC to drive ATP synthesis. Bovine heart ATP synthase is comprised of 16 different subunits and is divided into three domains (Abrahams et al., 1994). The matrix globular domain, F1, containing the catalytic site is linked to the intrinsic membrane domain, F0, by a central stalk (Fig. 6a) (Abrahams et al., 1994; Karrasch and Walker, 1999). Proton ﬂux through F0 is coupled to ATP synthesis in the F1 domain by rotation of the central stalk. The F1 catalytic domain is a ﬂattened sphere 80 A˚ high and 100 A˚ in diameter, and contains three α subunits and three β subunits [(αβ)3 ]. The subunits are arranged alternately like segments of an orange about the central stalk that contains the γ , δ, and ε subunits (Fig. 6a) (Gibbons et al., 2000). The α and β subunits are homologous (20% identical), and have a very similar fold. Both subunits bind nucleotides, however, only the β subunits show catalytic activity. The nucleotide binding sites are located at the interfaces between the α and β subunits. The catalytic sites are predominantly in the β subunits with some residues from the α-subunits contributing. The structures of the three β-subunit catalytic sites are always different and cycle through “open,” “loose,” and “tight” states (Fig. 6b). This cycle is known as the “binding-change mechanism,” and was originally proposed by Paul Boyer and colleagues (1997). When the catalytic site is in the tight state, there is a high afﬁnity for ADP and inorganic phosphate resulting in ATP forming spontaneously. The open state has very low afﬁnity for substrate/product, while the loose state binds substrate reversibly. Release of ATP from the open state depends on binding of ADP and Pi to the loose state (Boyer, 1997), indicating cooperative binding between sites. Structural, biochemical, and spectroscopic studies have suggested that the γ subunit of the stalk rotates, coupling the proton motive force at the

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FIG. 6. Structure and mechanism of action of ATP synthase (complex V). A representation of the proposed structure of ATP synthase is shown in (a). The stalk rotates in an anticlockwise direction when viewed from the membrane. The (αβ)3 domain is prevented from rotating by the stator. The stalk rotation occurs in 120◦ steps, this movement results in the three β subunits cycling through the three states proposed by the binding-change mechanism (b). In the absence of an input of energy (rotation of the stalk), the tight state (T) is occupied by ATP and the loose state (L) is able to bind ADP and Pi. A 120◦ rotation of the stalk changes the conformations of the β subunits, trapping bound ADP and Pi in the tight state and allowing ATP to escape from the open state (O). A second ATP is formed in the tight state and a new set of substates (ADP and Pi) is free to bind to the β subunit currently in loose state. And so the cycle repeats.

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membrane to ATP synthesis over 100 A˚ away. The C-terminal of the γ subunit is a 90 A˚ α-helix and ﬁts into the central cavity formed by the (αβ)3 domain. The C-terminal emerges to form a dimple, 15 A˚ below the top of the hexameric domain. The lower half of the helix forms an asymmetric antiparallel coiled coil leading into a single α-helix at the N-terminal. This helix extends 47 A˚ below the (αβ)3 domain and forms part of the stalk domain between the F1 and F0 domains (Abrahams et al., 1994; Gibbons et al., 2000). Reversible disulﬁde crosslinks between a mixture of radioactive and unlabeled β subunits and the γ subunit conﬁrmed that the γ subunit can bind each β subunit freely, regardless of which state it is in. Furthermore, the (αβ)3 domain loses most of its catalytic activity and shows little cooperative binding of nucleotides when the γ subunit is disassociated. Several speciﬁc polar interactions and hydrophobic loops between the (αβ)3 domain and γ subunit have also been observed. Attachment of a ﬂuorescent actin ﬁlament to the γ subunit showed directly that the γ subunit rotates counterclockwise in ATP synthase when viewed from the F0 domain (Noji et al., 1997). Neither the δ or ε subunits (the two remaining components of the stalk) are necessary for rotation. The γ subunit rotates in 120◦ steps with a frequency of 100–200 Hz. This rotation changes the nucleotide binding afﬁnities of each β subunit, cycling them through the open, loose, and tight states (Fig. 6b). This is because each β subunit is sequentially exposed to a different surface of the γ subunit as it rotates. For example, in the open state, the position of the γ subunit, relative to the β-subunit, prevents the β subunit from adopting a nucleotide binding formation. Crystallization of the F1 domain bound to the inhibitor dicyclohexylcarbodiimide resolved the structure of the stalk. A hitherto unseen Rossmann fold toward the bottom of the γ subunit at the base of the stalk (adjacent to the F0 domain) was identiﬁed. The δ and ε subunits interact extensively with this fold, forming a foot (Gibbons et al., 2000). This foot interacts with the c ring of the Fo domain. Electron microscopy of bovine ATP synthase also has revealed a peripheral stalk connecting the (αβ)3 domain to a collar (possibly the foot) at the top of the Fo domain (Karrasch and Walker, 1999). This is postulated to be a stator, preventing the (αβ)3 domain from following the rotation of the γ subunit. Subunits b, d, F6 and oligomycin-sensitivityconferring protein (OSCP) of the Fo domain have been proposed to be part of this peripheral stalk. The peripheral stalk (stator) in bacterial ATP synthases is comprised of just two b subunits from F0 and the bacterial homologue of OSCP. The two copies of the b subunit extend to the top of F1 where they interact with the OSCP homologue that is associated with the F1 domain. The F0 domain spans the membrane and is the site of proton translocation required to drive ATP synthesis. Unfortunately, no high-resolution crystal structures are available for this domain. The F0 domain of bovine

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heart ATP synthase contains 9 different subunits, a, b, c, d, e, f, g, A6L, and F6. Subunits a, b, d, and F6 are present in the complex with one copy each. In bacteria, two copies of b are observed. One of the subunits present in eukaryoate, but absent in bacterial ATP synthase, probably substitutes for the second copy of b required in the stator. The stoichiometry of subunit c is unclear, 9–12 copies have been suggested to form a ring. Subunit a in conjunction with the ring of c subunits is thought to provide the pathway for proton translocation. Subunit a is believed to act as a proton inlet channel. At the interface between the a and c subunits, a proton that has passed through subunit a, is thought to bind to Asp61 of the c subunit. The c-subunit ring of E. coli ATP synthase has been shown to rotate (Tsunoda et al., 2001). Therefore, upon protonation, the c-subunit site leaves the interface with the a subunit and rotates into the lipid phase. The c subunit rotates nearly 360◦ , releasing the proton to the outlet channel in subunit a as it reenters the subunit a–subunit c interface. The presence of one mutant c subunit blocks proton translocation, indicating that there is cooperativity between the c subunits. The inhibition of ATP synthase exerted by dicyclohexylcarbodiimide is achieved by a unique reaction with Asp61. If the mammalian ATP synthase has 12 c subunits, one full turn of the rotor will yield three ATP molecules (four protons translocated per ATP). The γ , δ, and ε subunits of the central stalk are intimately attached to the ring of c subunits. The rotation of the stalk conferred to it by the movement of the c ring provides a mechanism by which proton translocation across the membrane is coupled to ATP synthesis in the matrix over 100 A˚ away.

F. ADP–ATP TRANSLOCATOR ATP generated in the mitochondrial matrix is transported to the cytosol via the ATP–ADP translocator. For every ATP molecule exported, an ADP molecule from the cytosol is imported. The exchange of ATP for ADP is driven by the membrane potential since ATP has one more negative charge than ADP. The ATP–ADP translocator is an integral protein with six transmembrane helices and a molecular mass of 32 kDa. Dimerization of the translocator subunits is thought to form the channel through which ATP and ADP are transported (Klingenberg, 1992; Schefﬂer, 1999). It is estimated that the translocator accounts for up to 15% of the total protein content of mitochondria. The use of two speciﬁc ATP–ADP translocator inhibitors, atractyloside and bongkrekic acid, have shed light on the mechanism of translocation. Atractyloside only binds to the cytoplasmic side of the translocator since it is unable to cross the inner membrane, while bongkrekic acid can enter

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the mitochondria and binds exclusively to the matrix side. The presence of inhibitors prevents the binding of both ATP and ADP. However, both inhibitors cannot bind at the same time, despite occupying opposite sides of the translocator. This indicates that the ATP–ADP translocator is only open for one substrate at a time (e.g., ATP on the matrix side). The postulated transition between the two conformational states (open on the matrix side to open on the cytosolic side) results in the translocation of the substrate across the membrane (Schefﬂer, 1999). Studies have suggested that the ATP–ADP translocator is one of the components of the mitochondrial permeability transition pore. Formation of this pore is postulated to be a factor in the initiation of apoptosis (Tatton and Olanow, 1999).

III. Intermediary Metabolism

Reducing equivalents, for utilization by the ETC, are generated via a number of integrated metabolic pathways. Below are brief descriptions of the predominant metabolic pathways, located to mitochondria, that are responsible for NADH and FADH2 generation. Details of other metabolic pathways that occur within mitochondria but are not directly related to energy transduction, e.g., heme synthesis and the urea cycle, are not covered, but can be found elsewhere (e.g., Schefﬂer, 1999).

A. PYRUVATE DEHYDROGENASE Cytosolic pyruvate, under aerobic conditions, is metabolized further by the TCA cycle. The transport of pyruvate into mitochondria is via the monocarboxylate translocator, and entry of pyruvate into the TCA cycle (see below) is regulated by pyruvate dehydrogenase (PDH). This enzyme complex catalyzes the conversion of pyruvate to acetyl CoA and NADH. The PDH complex consists of 132 subunits and is composed of three main enzymes: (a) pyruvate decarboxylase (E1) which is a tetramer, encoded by two genes on the X chromosome and composed of 2α and 2β subunits; (b) a transacetylase (E2) of 52 kDA, which exists as a monomer with lipoic acid; and (c) dihydrolipoyl dehydrogenase (E3), a 55-kDA dimer that also functions in the branched chain ketoacid dehyrogenases and the α-ketoglutarate dehydrogenase complex. A lipoic acid containing moiety known as the “X” protein is also present in the complex and is believed to have an acyl transfer function (Patel and Roche, 1990). As PDH catalyzes a key regulatory step of aerobic glucose oxidation, activity is tightly regulated. The mechanism for this regulation is phosphorylation (inactive) and dephosphorylation

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(active) of PDH by a kinase and a phosphatase, respectively (Linn et al., 1969; Schefﬂer, 1999). B. THE TCA CYCLE The TCA cycle, also known as the Kreb’s cycle or the citric acid cycle, was elucidated in 1937. A major function of this cycle is generation of reduced NADH and FADH2 that can be utilized by the ETC for ATP synthesis. This cycle of eight enzyme catalyzed reactions is located to the mitochondrial matrix and links a number of metabolic pathways that generate acetyl CoA (Fig. 7). Furthermore, intermediates generated in the cycle are utilized in a number of anaplerotic pathways. The enzymes of the TCA cycle are all

FIG. 7. Integration of energy metabolism within the mitochondria. Acetyl CoA, generated via PDH and fatty acid β oxidation, is metabolized by TCA (Kreb’s cycle). Reducing equivalents (NADH and FADH2) generated by this cycle, PDH activity, and β oxidation are oxidized by the electron transport chain resulting in the generation of ATP. The enzymes of the TCA cycle are as follows: (1) citrate synthase, (2) aconitase, (3) isocitrate dehydrogenase, (4) α-ketoglutarate dehydrogenase, (5) succinyl-CoA synthase, (6) succinate dehydrogenase, (7) fumarase, (8) malate dehydrogenase. The splitting of the cycle into “mini cycles” is depicted by the dotted line and requires aspartate amino transferase, 9.

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encoded by nuclear genes and are constitutively expressed. Further details relating to the cycle and disorders affecting this pathway can be found in Rustin et al. (1997) and Schefﬂer (1999). Metabolically related enzymes of the cycle appear to be associated together within the matrix in order to allow for substrate channeling (Robinson and Srere, 1985). Regulation of the cycle occurs at the level of citrate synthase, isocitrate dehydrogenase, and α-ketoglutyrate dehydrogenase. Thus, alterations in the NADH:NAD+ ratio, the energy charge, and calcium can act to regulate the TCA cycle. Functional splitting of the TCA cycle into complementary “mini Krebs cycles” has been proposed (Yudkoff et al., 1994). It is suggested that two independent segments of the cycle exist, i.e., from α-ketoglutarate to oxaloacetate and from oxaloacetate to α-ketoglutarate. For these two cycles to function, aspartate amino transferase needs to be present (Fig. 7). The ﬁnding of normal respiration rates in cells derived from patients with TCA cycle defects is suggested to arise as a result of an upregulation of the minicycle with the full complement of enzymes (Rustin et al., 1997).

C. MITOCHONDRIAL FATTY ACID OXIDATION Fatty acids are a major energy source, particularly during periods of fasting. While most tissues exhibit an ability to oxidize fatty acids, this process appears particularly important in muscle where approximately 70% of energy demands, under resting conditions, are met by fatty acid oxidation (Di Donato, 1997). Fatty acids, depending on carbon chain length and degree of unsaturation, can be oxidized, via a number of reactions (α, β, or ω oxidation), which utilize enzyme systems found within peroxisomes and mitochondria. However, we focus here only upon the mitochondrial β oxidation of saturated straight chain fatty acids. Further details relating to peroxisomal fatty acid metabolism, oxidation branch chain, and unsaturated fatty acids can be found in Moser (1997) and Wanders et al. (1999). Following liberation from adipose tissue, fatty acids are transported to tissues bound primarily to albumin. The cellular uptake and transport of fatty acids from the cell membrane to the mitochondrion is poorly understood, but may involve speciﬁc membrane transporters and cytosolic binding proteins. The initial step in the process of harnessing energy from fatty acids is the generation of an acyl-CoA thioester from free coenzyme A and the corresponding free fatty acid. For long chain fatty acids (greater than 12 carbons) this reaction is catalyzed by a long-chain acyl-CoA synthetase located on the outer mitochondrial membrane (Roe and Coates, 1995).

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The series of reactions that are involved in fatty acid β oxidation are catalyzed by group of enzymes located on the matrix side of the inner mitochondrial membrane and within the mitochondrial matrix. However, the inner mitochondrial membrane is not permeable to long-chain (>12 carbon units) fatty acyl-CoA esters. In order to traverse this membrane, a transport system involving carnitine has evolved (Brivet et al., 1999). 1. Carnitine Transport of Long Chain Fatty Acids Carnitine palmitoyl transferase I (CPT I), found on the outer mitochondrial membrane, transfers the fatty acyl moiety from acyl CoA to carnitine, leading to the formation of an acyl carnitine. This acyl carnitine is then “shuttled,” by the carnitine–acylcarnitine translocase, across the inner mitochondrial membrane, in exchange for free carnitine. Carnitine palmitoyl transferase II (CPT II) then transfers the acyl group back to CoA and the liberation of free carnitine. The regenerated fatty acyl CoA can then enter the β-oxidation spiral (Fig. 7). CPT I and CPT II have different mitochondrial locations, are distinct proteins, and display different biochemical properties, e.g., CPT I, in contrast to CPT II, can be inhibited by malonyl CoA. Furthermore, CPT I exists as tissue-speciﬁc isoforms, i.e, liver and muscle type that are encoded by genes that are located on chromosomes 11 and 22, respectively. The two isoforms are of similar size (liver: 773 amino acids, 88.1 kDa; Muscle: 772 amino acids, 88.2 kDa), but they differ in their kinetic properties. Tissue-speciﬁc isoforms of CPT II have not been reported. This enzyme is encoded on chromosome 1, and a 658 amino acid proenzyme is synthesized that is imported into the mitochondrion. Following import, a 25 amino acid leader sequence is removed. The active protein has an approximate molecular mass of 71 kDa. Further details relating to the carnitine transport system can be found in Brivet et al. (1999). The gene for the carnitine–acylcarnitine translocase has been assigned to chromosome 3, and encodes for a protein comprising of 301 amino acids. In common with other mitochondrial carrier proteins, the translocase contains a three-fold repeat sequence of approximately 100 amino acids. Furthermore, there are six transmembrane α-helices that are connected by hydrophilic loops (Indiveri et al., 1997). 2. β-Oxidation of Fatty Acids The complete oxidation of unsaturated fatty acyl-CoA molecules to acetyl CoA is achieved by a series of four enzyme reactions, i.e., dehydrogenation (oxidation), hydration, further dehydrogenation (oxidation), and thiolysis (Fig. 8).

OXIDATIVE PHOSPHORY LATION

FIG. 8. Mitochondrial β oxidation of fatty acids.

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The initial step of β oxidation is catalyzed by a group of enzymes known as the acyl-CoA dehydrogenases. At least four enzymes have been identiﬁed that catalyze essentially the same reaction but display speciﬁcity toward acylCoA molecules of differing carbon chain lengths. These enzymes, referred to as the short-chain (SCAD), medium-chain (MCAD), long-chain (LCAD), and very long-chain (VLCAD) acyl-CoA dehydrogenases, insert a double bond between the α and β carbons of the acyl-CoA molecule. An enoyl-CoA molecule is the product of this reaction, and the electrons removed from the acyl CoA are donated to an electron transfer ﬂavoprotein (ETF). This ETF is then oxidized by ETF dehydrogenase, leading to formation of ubiquinol, which is oxidized by the ETC (Wanders et al., 1999). The true role of LCAD in the oxidation of fatty acids, in vivo, is not clear. Studies, in vitro, suggest considerable overlap in speciﬁcity for LCAD and VLCAD. Furthermore, cell culture studies suggest that VLCAD is exclusively required for palmitate (C16) oxidation. Current data now suggest that the major role of LCAD is in the oxidation of branched chain fatty acids and it is proposed that LCAD be renamed as long-branch chain acyl-CoA dehydrogenase (Wanders et al., 1998). Considerable data are available relating to SCAD, MCAD, and LCAD. The active forms of these enzymes are to be found in the mitochondrial matrix and are each composed of four identical subunits that bind FAD. These enzyme subunits are synthesized in the cytosol as precursor proteins that contain leader sequences that direct them to the mitochondrion. Following mitochondrial import, the enzyme subunits are processed into the active enzymes, i.e., leader sequences are removed followed by tetramerization and incorporation of FAD. VLCAD is bound, in contrast to the other acyl CoA dehyrogenases, to the inner mitochondrial membrane and is ideally situated to receive long-chain substrates that have been transported by the carnitine system (Wanders et al., 1999). The second step in fatty acid β oxidation is hydration of enoyl CoA to form 3-hydroxyacyl CoA. Current evidence suggests that there are at least two mitochondrial enzymes that catalyze this reaction. Short-chain enoyl CoA hydratase, also known as crotonase, is found in the mitochondrial matrix and is active, with decreasing efﬁciency, on enoyl-CoA molecules of chain length between 4 and 16 carbon units. Crotonase is comprised of six identical subunits that are synthesized in the cytosol as precursors containing mitochondrial targeting signals. Following transport into the mitochondria assembly of the hexamer can occur. The long-chain enoyl CoA hydratase is part of the membrane-bound mitochondrial trifunctional protein (see below) (Wanders et al., 1999). The next step in β oxidation is a dehydrogenation reaction catalyzed by the 3-hydroxyacyl CoA dehydrogenases. At least two enzymes have been

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identiﬁed that have speciﬁcity for short- and long-chain hydroxyacyl CoA molecules. The NADH generated by these enzymes is utilized by the ETC for ATP synthesis. Short-chain hydroxyacyl-CoA dehydrogenase (SCHAD) is a dimer comprised of identical subunits (33 kDa). Precursor proteins are synthesized in the cytosol and are transported into the mitochondrial matrix where assembly of the active enzyme occurs. The SCHAD appears to have a broad speciﬁcity, i.e., is capable of oxidising hydroxyacyl CoA molecules of between 4 and 16 carbon units. However, maximal activity is toward substrates having between 4 and 10 carbon units. Long-chain hydroxyacyl-CoA dehydrogenase (LCHAD) is membrane bound and is a constituent of the mitochondrial trifunctional protein (see below). The enzyme has broad substrate speciﬁcity and displays maximal activity toward hydroxylacyl-CoA molecules having between 12 and 16 carbons (Wanders et al., 1999). The ﬁnal stage in mitochondrial β oxidation is thiolytic cleavage. In this step, the 3-oxoacyl CoA generated by SCHAD or LCHAD is split into acetylCoA and a shortened acyl-CoA ester that can reenter the β oxidation spiral. The acetyl CoA generated at this stage can then be metabolized further by the TCA cycle. Two mitochondrial thiolases have been identiﬁed that are involved in β oxidation; a general (medium-chain) thiolase and a thiolase associated with the mitochondrial trifunctional protein (see below). The general thiolase is active toward 3-oxoacyl CoA molecules, located in the mitochondrial matrix, a homotetramer, and it is active toward 3-oxoacyl CoA molecules with between 4 and 12 carbons (Wanders et al., 1999). The mitochondrial trifunctional protein (MTP), as the name suggests, displays enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and thiolase activity. This inner mitochondrial membrane complex has an approximate molecular mass of 460 kDa, and is an heteroctomer comprised of four α and four β subunits. The α subunits are associated with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activity, while the β units contain the thiolase (Uchida et al., 1992).

D. KETONE BODY METABOLISM Plasma levels of the ketone bodies, acetoacetate and 3-hydroxybutyrate, signiﬁcantly rise during periods of starvation as a result of accelerated catabolism of fatty acids (Girard et al., 1992). Under such conditions, entry of acetyl CoA into the TCA cycle is limited as oxaloacetate is also being used for gluconeogenesis. Three mitochondrially located enzymes are involved in the formation of acetoacetate. Thus, in the presence of acetoacetyl-CoA thiolase, two molecules of acetyl CoA are utilized to form acetoacetyl CoA. A third

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molecule of acetyl CoA is then utilized to form 3-hydroxy-3-methylglutaryl CoA (HMG CoA) a reaction catalyzed by HMG-CoA synthase. The HMG CoA so formed is then further metabolized by a HMG-CoA lyase to form acetoacetate and acetyl CoA. In the presence of NADH, the acetoacetate is reduced to 3-hydroxybutyrate by 3-hydroxybutyrate dehydrogenase. The liver is traditionally considered to be a major site of ketogenesis, while brain muscle and heart are referred to as nonketogenic. However, studies have provided evidence to suggest that the brain may have the full complement of enzymes required for ketone body production (Cullingford et al., 1998). During periods of starvation, ketone bodies become an increasingly important metabolic fuel for the brain. Acetoacetate and 3-hydroxybutyrate, generated by the liver, cross the blood–brain barrier and are subsequently metabolized. 3-Hydroxybutyrate dehydrogenase, located on the inner mitochondrial membrane, forms acetoacetate and NADH from 3-hydroxybutyrate. In the presence of 3-ketoacyl-CoA transferase, CoA is transferred from succinyl CoA to acetoacetate, thereby forming succinate and acetoacetyl CoA. Finally, in the presence of free CoA and acetoacetyl-CoA thiolase, two molecules of acetyl CoA are formed. This acetyl CoA can then be oxidized via the TCA cycle (Mitchell et al., 1995).

IV. Concluding Remarks

Optimal mitochondrial function, as discussed above, is clearly essential for cell survival. In view of this critical role, it is perhaps not surprising that inherited deﬁciencies affecting mitochondrial metabolism are often associated with a striking clinical picture. Furthermore, there is an increasing body to evidence to suggest that mitochondrial dysfunction occurs in a number of neurodegenerative disorders. Subsequent chapters in this book consider potential mechanisms and the metabolic consequences of impaired mitochondrial function. References

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IMPORT OF MITOCHONDRIAL PROTEINS

Matthias F. Bauer1 and Sabine Hofmann Institute of Clinical Chemistry Molecular Diagnostics and Mitochondrial Genetics and Diabetes Research Group Academic Hospital Munich-Schwabing K¨ olner Platz, D-80804 M¨unchen, Germany

Walter Neupert Institute of Physiological Chemistry University of Munich Butenandtstrasse 5 81377 M¨unchen, Germany

I. Introduction II. The Pathways of Mitochondrial Preprotein Import A. Targeting and Sorting of Preproteins to Mitochondria is Mediated by Specific Signals B. The Translocation System of the Outer Mitochondrial Membrane—The TOM Complex C. The Presequence Translocase of the Inner Membrane—The TIM23 Complex D. The Translocase for Import of Carrier Proteins into the Mitochondrial Inner Membrane—The TIM22 Complex E. Mitochondrial Translocases in Mammals III. Mitochondrial Biogenesis and Human Neurodegenerative Diseases A. Dysfunction of Mitochondrial Preprotein Import as a Cause of Progressive Neurodegeneration—Mohr-Tranebjaerg Syndrome B. Defects of Quality Control of Mitochondrial Inner Membrane Proteins—Hereditary Spastic Paraplegia References

I. Introduction

Mitochondria are present in virtually all eukaryotic cells, and they arise by growth and division of preexisting mitochondria. This growth occurs by insertion of newly synthesized components leading to the expansion of 1

Author to whom correspondence should be addressed.

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the four compartments of the mitochondria. Two of these mitochondrial compartments are formed by the outer and the inner membrane, which delimitate two aqueous compartments, the intermembrane space, and the matrix. There has been considerable advance in the ﬁeld of transport of mitochondrial constituents, in particular of protein components, to these mitochondrial compartments. Almost all mitochondrial proteins are encoded as precursors by the nuclear genome. A major aspect of mitochondrial biogenesis is, therefore, the transfer of nuclear encoded, cytosplasmically synthesized precursor proteins across and into the mitochondrial membranes and their assembly to the supramolecular complexes of the various mitochondrial compartments. The number of different proteins undergoing these processes may amount to roughly 1000. In contrast, only a few protein components are encoded by the mitochondrial DNA (mtDNA). In mammals the mitochondrial genome contains the genes for RNA species [two ribosomal (rRNAs) and 22 transfer (tRNAs)] required for mitochondrial protein biosynthesis and for 13 polypeptides that represent components of the various complexes of oxidative phosphorylation. All of these latter proteins are synthesized on mitochondrial ribosomes and they are inserted from the matrix side into the mitochondrial inner membrane (Stuart and Neupert, 1996). Together with the imported preproteins encoded by nuclear genes, these mitochondrial gene products are assembled into the hetero-oligomeric respiratory chain complexes I, III, and IV, and the ATP synthase. The use of simple model organisms, such as the yeast Saccharomyces cerevisiae and Neurosporacrassa, has helped considerably to investigate the structure and function of a rather large number of components involved in targeting and sorting of nuclear-encoded preproteins to mitochondria. Several pathways that guide mitochondrial preproteins to their sites of function have been characterized and the energetics of the various steps of import have been studied in some detail (Ryan and Jensen, 1995; Schatz, 1996; Bauer et al., 2000; Herrmann and Neupert, 2000; Pfanner and Geissler, 2001). As demonstrated by these investigations, uptake of protein components into mitochondria is a multistep process facilitated by the coordinated action of specialized translocation systems, so-called preprotein translocases. These translocases decode the signal sequences of the precursor proteins and mediate translocation, insertion, and intramitochondrial sorting to their correct destination. Hydrophilic precursor proteins destined for the matrix must cross both membranes as well as the intermembrane space before reaching their ﬁnal location. The precursors of the membrane-integrated components of the outer membrane are bound on the surface and are sorted directly into the lipid bilayer, whereas inner membranes have to cross the outer membrane without getting arrested in it and have to pass through the aqueous intermembrane space. Notably, all subunits of the mitochondrial

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translocation systems are themselves nuclear encoded, and the precursors of these components have to be sorted and inserted into the membranes by preexisting translocases. Recent research on mitochondrial protein translocation has focused mainly on the molecular nature of the translocation machineries. These translocation systems are of considerably higher complexity and higher versatility than may have been expected. In this chapter we provide an overview on the structural organization and the function of the import systems that mediate protein targeting to mitochondria. We will also discuss how genetic alterations of these systems contribute to the development of neurodegenerative disorders in humans.

II. The Pathways of Mitochondrial Preprotein Import

Nuclear-encoded mitochondrial proteins are synthesized on ribosomes in the cytosol as precursor proteins (or preproteins) that are directed to the submitochondrial compartments by means of speciﬁc mitochondrial targeting signals. Newly synthesized mitochondrial preproteins in the cytosol are believed to be maintained in a translocation competent state by speciﬁc binding proteins. A number of cytosolic components were reported to interact with nascent polypeptide chains, i.e., even before they are released from the ribosome to mediate stabilization and (partial) folding (for review, see Hartl, 1996). In particular, cytosolic Hsp70s, members of the heat shock protein family of 70 kDa as well as binding factors speciﬁc for presequences appear to be involved in these processes. Moreover, proteins in the cytosol may exert a more speciﬁc function, namely guiding preproteins to the surface of mitochondria, in a similar way as has been discovered for secretory proteins in bacteria and eukaryotes (“targeting function”) (Rapoport et al., 1996; Schatz and Dobberstein, 1996). At the outer surface of the mitochondrial outer membrane, speciﬁc receptors are exposed that recognize and bind the precursors prior to their translocation. The transfer across the membranes is then mediated by the distinct import systems embedded in the outer and the inner membranes (Fig. 1). Upon translocation of a precursor into the mitochondrial matrix, these machineries interact dynamically, thereby bringing the two membranes into close proximity. In eukaryotes, three distinct preprotein import systems have been described (Ryan and Jensen, 1995; Schatz, 1996; Bauer et al., 2000; Herrmann and Neupert, 2000; Pfanner and Geissler, 2001). The TOM complex (“t ranslocase of the o uter mitochondrial m embrane”) mediates the initial recognition of preproteins, their transfer through the outer membrane, and the insertion of resident outer membrane

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FIG. 1. Mitochondrial preprotein import and export pathways. Nuclear-encoded preproteins are imported and distributed into the four mitochondrial compartments along distinct pathways (schematically depicted as arrows). Cytosolic preproteins are recognized by specialized import receptors of the TOM complex of the outer membrane, and then, depending on their ﬁnal destination, sorted into the outer membrane (OM) and the intermembrane space (IMS), or are handed over to the TIM translocases of the inner membrane (IM). Preproteins carrying a presequence (matrix-targeting signal) are imported via the TIM23 complex into the inner membrane or the matrix space. Hydrophobic proteins are shuttled by the help of soluble chaperones across the intermembrane space to the TIM22 complex, which mediates their insertion into the inner membrane. Mitochondrial protein components encoded by the mtDNA are exported into the inner membrane via the OXA translocase and by the help of Pnt1.

proteins (Fig. 1). This complex is most likely used by all nuclear-encoded precursors. The TOM complex contains speciﬁc hydrophilic receptors recognizing newly synthesized precursors in the cytosol (Kiebler et al., 1990; Pfaller et al., 1988). The bound precursor proteins are then transferred to a protein conducting channel, also referred to as the “general import/insertion pore” (GIP), which translocates preproteins across the outer membrane into the intermembrane space (Pfaller et al., 1988; Hill et al., 1998; Kunkele et al., 1998a). Preproteins cross the membranes in an unfolded conformation, and folded domains of preproteins present on the surface of mitochondria are unfolded during this translocation process. Further movement of the translocation intermediates into and across the inner membrane is mediated by two distinct translocases in the inner membrane, the TIM23 and the TIM22 complex (Fig. 1). Both TIM complexes cooperate with the TOM complex upon transfer of a preprotein into and across the mitochondrial

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inner membrane thereby forming so-called translocation contact sites. The two different TIM complexes differ in their speciﬁcity for preprotein substrates and direct preproteins to different destinations (Ryan and Jensen, 1995; Sirrenberg et al., 1996; Kerscher et al., 1997; Koehler et al., 1998a,b; Sirrenberg et al., 1998). The transfer of preproteins via the TIM23 complex across the inner membrane strictly requires both an electrochemical potential () across the inner membrane and ATP in the matrix as energy sources. The TIM22mediated insertion of hydrophobic proteins into the inner membrane depends on the presence of a but does not require ATP (Ryan and Jensen, 1995; Schatz, 1996; Bauer et al., 2000; Herrmann and Neupert, 2000; Pfanner and Geissler, 2001). At least one further translocase, called OXA, exists in the inner membrane (Fig. 1). This translocase contains the Oxa1 protein and mediates insertion of distinct classes of preprotein substrates from the matrix side into the inner membrane. These substrates include mitochondrially encoded subunits of the respiratory chain complexes and certain nuclear-encoded inner membrane proteins that are ﬁrst imported into the matrix space via the TIM23 complex and from there into the inner membrane (Hell et al., 1998). This insertion pathway also requires, at least in many cases, a membrane potential across the inner membrane, and it resembles the Secindependent, pH-dependent insertion of polytopic proteins into the bacterial plasma membrane (Herrmann et al., 1997). Recently, a second export component, Pnt1, has been identiﬁed in a genetic approach screening for yeast mutants defective for the export of mitochondrially encoded proteins (Fig. 1) (He and Fox, 1999). Pnt1 is involved in the export of the C-terminus of subunit 2 of the cytochrome c oxidase (Cox2). Its precise role in export, however, has not been determined, and there is experimental evidence that Pnt1 and Oxa1p exhibit overlapping functions in yeast. It has become clear that a variety of additional steps exist that act on precursors during the import and allow them to reach their ﬁnal destinations. In particular, molecular chaperones support folding of precursors, and facilitate their assembly into functional complexes or sorting to the correct compartment. In addition, maturation steps can occur during import, which include covalent and noncovalent modiﬁcations.

A. TARGETING AND SORTING OF PREPROTEINS TO MITOCHONDRIA IS MEDIATED BY SPECIFIC SIGNALS All proteins of an eukaryotic cell with the exception of the few mitochondrially encoded ones are translated on cytosolic polysomes and are

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eventually sorted to the various different cellular compartments. The information necessary for targeting of preproteins to mitochondria is contained in relatively minor portions of the nascent or newly completed polypeptide chains. These portions were designated as signal or targeting sequences. Most targeting signals are contained within N-terminal extensions, the majority of which are cleaved upon import into the mitochondria. As they direct preproteins, at least partially, into the matrix space they are referred to as “matrix targeting signals.” Many preproteins, on the other hand, do not carry N-terminal presequences but internal targeting signals. Additional signals exist in preproteins that mediate sorting and insertion into the mitochondrial membrane. 1. Matrix-Targeting Signals (Presequences) The majority of precursor proteins carry the targeting sequence in an N-terminal extension of about 10–80 amino acid residues. In particular, most soluble matrix proteins carry such presequences, which are proteolytically cleaved off upon reaching their ﬁnal destination. When fused to a cytosolic protein presequences could be shown to speciﬁcally direct “passenger” proteins across both membranes into the matrix (Hurt et al., 1984; Horwich et al., 1985). In addition, N-terminal presequences or presequence-like signals are also sufﬁcient to target many preproteins to the other three compartments, the outer membrane, the intermembrane space, and the inner membrane (see below). The presequences of different precursors do not share sequence similarities. They share, however, distinct structural features. They contain abundant positively charged, and quite frequently hydrophobic and hydroxylated, amino acid residues (von Heijne et al., 1989). As a rule, they are predicted to form amphipathic α-helices presenting a positively charged face on one side and a hydrophobic face on the opposite side of the helix (Roise et al., 1986; Roise and Schatz, 1988). The potential to form a polar α-helical structure is thought to prevail in a hydrophobic environment and is considered an essential prerequisite for the function of the presequences (Gavel et al., 1988). It has been proposed that this helical structure is responsible for the initial interaction with the lipid bilayer of the outer membrane mainly on the basis of experiments with artiﬁcial lipid vesicles (de Kroon et al., 1991; de Kruijff, 1994; Tamm, 1991). The signiﬁcance of such a reaction in vivo, however, is not clear. The prevailing concept includes the idea that the speciﬁc recognition of the targeting signals occurs via proteinaceous receptor components of the complex on the mitochondrial surface. These receptors have been identiﬁed and their role in translocation could be demonstrated (Fig. 2) (see below). The structural features of the presequences recognized by the receptors of the TOM complex are partly known. Recent studies have indicated

FIG. 2. Composition and speciﬁcity of the translocation systems of the mitochondrial membranes. The majority of mitochondrial preproteins carry positively charged matrix-targeting signals at their N-termini (presequences), which are recognized by receptor components Tom22/Tom20. From this so-called cis site, the presequence is transferred through the GIP consisting of Tom40, Tom22, and the small Tom proteins. Upon reaching the intermembrane face of the outer membrane, the presequence binds to a trans site, which is constituted by Tom22, and possibly Tom40. A subset of preproteins, including the ADP/ATP carrier (AAC) and related proteins carrying internal targeting signals, are ﬁrst bound to the specialized receptor Tom70. For further translocation, the TOM complex cooperates with the TIM23 complex and the TIM22 complex in the inner membrane. The transfer of preproteins via the TIM23 complex across the inner membrane strictly requires both, an electrochemical potential () across the inner membrane and ATP in the matrix as energy sources. Insertion of the presequence into the TIM23 complex is thought to be driven electrophoretically by the membrane potential and complete transport of the precursor into the matrix is mediated by an ATPpowered import motor consisting of mtHsp70 and the nucleotide exchange factor Mge1p, which are attached to the inner outlet of the TIM23 complex. A number of preproteins with internal signals are guided by hetero-oligomeric complexes of small Tim proteins from the TOM complex across the aqueous intermembrane space to the TIM22 complex. The composition of these hetero-oligomeric complexes differs depending on whether they are soluble in the intermembrane space or are attached to the membrane integral portion of the TIM22 complex. The TIM22-mediated insertion of hydrophobic proteins into the inner membrane depends on the presence of a but does not require ATP. Abbreviations: Tom20 (20), Tom22 (22), Tom40 (40), Tom70 (70), Tom5 (5), Tom6 (6), Tom7 (7), mt-Hsp70 (70), Tim44 (44), Tim17 (17), Tim23 (23), Mge1p (E), Tim22 (22), Tim54 (54), Tim18 (18), Tim9 (9), Tim10 (10), Tim12 (12), Tim8 (8), and Tim13 (13).

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that hydrophobic interactions rather than the positive charges appear to mediate binding of the presequence to Tom20, one of the major two TOM receptors (Fig. 2) (Abe et al., 2000). As the binding of the presequences is labile particularly under conditions of high ionic strength, it was assumed that weak electrostatic forces may act between the presequences and the receptor components (Mayer et al., 1995a). The cytoplasmic domain of Tom22, on the other hand, carries a cluster of 18 negatively charged residues. This region was proposed to interact with the positive charges of the presequences (Kiebler et al., 1993). The presequences are recognized not only on the mitochondrial surface but also on the inner face of the outer membrane, and in a further step, by components located at the surface of the inner membrane. In particular, binding by the TIM23 complex and translocation across the inner membrane appears to involve the positively charged amino acid residues of the presequence (Fig. 2) (Bauer et al., 1996). Whereas the presequences of the vast majority of matrix-targeted proteins are located at the amino-terminus, recently a mitochondrial matrix protein was identiﬁed that carries a cleavable targeting signal at its C-terminus. This C-terminal signal of the precursor of the yeast DNA helicase Hmi1p is similar to classical N-terminal presequences and consists of a stretch of positively charged amino acids that has the potential to form an amphipathic α-helix (Lee et al., 1999). In contrast to the precursors carrying Nterminal presequences, this preprotein is imported in a reverse orientation with a C- to N-terminal direction, demonstrating that the import systems are able recognize the targeting signals irrespective of their position within the precursor protein. The presequences of most of the precursor proteins, including that of Hmi1p, are cleaved off by the mitochondrial processing peptidase in the matrix (MPP) during or after their translocation across the inner membrane (Wang and Weiner, 1993, 1994; Arretz et al., 1991, 1994; Glaser and Dessi, 1999). In a number of cases the initial proteolytical processing, performed by MPP, is followed by an additional proteolytic maturation step in the matrix. A second portion is removed either by MPP, as with the precursor of F0-ATPase subunit 9 (Schmidt et al., 1984) or by the monomeric metalloprotease, MIP (m itochondrial i ntermediate p eptidase), which removes an octapeptide from the N-termini generated by MPP (Kalousek et al., 1988; Isaya et al., 1992). 2. Variations on Targeting Signals for Sorting to Mitochondrial Subcompartments Many preproteins destined for the inner and outer membrane and the intermembrane space carry N-terminal presequences or presequence-like

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signals in their mature parts. In addition to the positively charged targeting signals, more hydrophobic segments exist that mediate the sorting and insertion in the membrane compartments. Precursors destined for the outer membrane do not carry cleavable presequences, but they do contain targeting signals in their mature parts that are only partially characterized so far. The targeting signals of outer membrane proteins, like Tom70, which carries a single N-terminal anchor, has been analyzed in some detail. Yeast Tom70 exposes approximately 10 N-terminal amino acid residues into the intermembrane space, which are followed by a 20-residue membrane anchor and a large 60 kDa domain in the cytosol (Riezman et al., 1983; Millar and Shore, 1994). Both the information for targeting and for membrane integration are located in the ﬁrst 30 residues (McBride et al., 1992). The structural features that target these preproteins to the outer membrane are not known, although they are bound via the receptor components, which also bind presequence-carrying preproteins. Insertion of these precursors into the outer membrane is mediated by hydrophobic stretches. As classical presequences, these stretches do not share distinct sequence motifs. Other signals for targeting to the outer membrane appear to present in C-terminal segments of proteins that are anchored to the membrane by hydrophobic segments located close to the C-terminus (Mitoma and Ito, 1992; Nguyen et al., 1993; Shore et al., 1995). The speciﬁcity of recognition and the mechanism of their insertion are not understood. Many intermembrane space proteins are initially synthesized without an N-terminal targeting signal. The internal signals are not known or only partially characterized, like for the intermembrane space protein cytochrome c heme lyase (CCHL) (Steiner et al., 1996). In the case of cytochrome b2 arrest at the level of the inner membrane by a stop-transfer sequence has been suggested. Other models imply partial or complete passing of such preproteins through the matrix space. Most inner membrane proteins and some intermembrane space proteins have positively charged matrix targeting signals at their N-termini that are complemented by more hydrophobic sorting signals either within the mature part of the protein or in tandem with the presequences. Three different kinds of such signals are known: ﬁrst, sorting signals, which consist of hydrophobic segments with charged ﬂanking regions that become arrested when they cross the inner membrane; second, precursors with a hydrophobic segment preceeding a hydrophobic transmembrane segment that becomes inserted in a kind of loop structure; and third, a hydrophobic segment in a preprotein that has a matrix targeting signal and becomes completely or partially imported into the matrix. Subsequently, the hydrophobic sorting signal then gets inserted into the inner membrane and adjacent segments

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get exported into the intermembrane space. Apparently, it is the combination of the topologies of the charged sequences and the ﬂanking hydrophobic membrane segments that determine targeting and membrane insertion. How this information is decoded by the components of the translocation systems of both membranes remains obscure. 3. Multiple Internal Targeting Signals An exceptional case with respect to the structure of the targeting signals is the large family of mitochondrial carrier proteins of the inner membrane with the ATP/ADP carrier (AAC) as the most prominent member. The carrier proteins do not carry cleavable presequences, but are targeted by means of internal signals that are repeated three or in one case even six times within these carriers (Fig. 2). The ATP/ADP carrier is characterized by three domains each of about 100 amino acid residues (Saraste and Walker, 1982). Stretches of about 20 amino acids are present in the carboxy-terminal half of each domain predicted to form α-helices (Aquila et al., 1985) and resemble the classical mitochondrial presequences (Ito et al., 1985; Von Heijne, 1986; Smagula and Douglas, 1988). The internal signals appear to exert a cooperative effect in recruiting several receptors to one precursor molecule (Endres et al., 1999; Wiedemann et al., 2001). Only little is known, however, about the structural characteristics and the mode of action of these internal targeting signals. Studies have just begun to address the questions of how such precursors use the TOM complex and how they become inserted into the inner membrane by using the TIM22 machinery (Fig. 2).

B. THE TRANSLOCATION SYSTEM OF THE OUTER MITOCHONDRIAL MEMBRANE—THE TOM COMPLEX The TOM complex is composed of seven to eight protein subunits with different and distinct functions in the recogniton and the translocation of preproteins. All of them are integral membrane proteins (Fig. 2). They can be classiﬁed into receptor components, channel-forming components, and small membrane-spanning proteins with not yet clearly deﬁned accessory functions. The preprotein receptors Tom70 and Tom20 expose hydrophilic domains of approximately 65 and 17 kDa, respectively, at the surface of the mitochondria, which recognize and bind the targeting signals of newly synthesized precursors present in the cytosol (S¨ollner et al., 1989, 1990; Hines et al., 1990; Kiebler et al., 1993; Ramage et al., 1993; Lithgow et al., 1994; Nakai and Endo, 1995; Honlinger et al., 1996). The Tom40 is the key structural component of the protein conduction channel, the GIP in the outer

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membrane that guides the precursors across the outer membrane in their unfolded conformation. It is essential for viability in yeast and Neurospora (Vestweber et al., 1989; Kiebler et al., 1990). Multiple copies of Tom40 are organized in the TOM core complex together with up to three small, membrane-embedded components, Tom5, Tom6, and Tom7 (Kassenbrock et al., 1993; Alconada et al., 1995; Cao and Douglas, 1995; Honlinger et al., 1996; Dietmeier et al., 1997) and Tom22, a subunit with hydrophilic domains exposed to both sides of the outer membrane (Kiebler et al., 1993; Lithgow et al., 1994; Nakai and Endo, 1995). The Tom22 fulﬁlls two functions. It acts as receptor together with Tom20 and is a constituent of the GIP complex (Fig. 2) (Court et al., 1996; Kunkele et al., 1998b). Two further proteins Tom71, (Schlossmann et al., 1996) and Tom37 (Gratzer et al., 1995) were found in the yeast S. cerevisiae. Tom70 and Tom71 are structurally closely related (53% sequence identity, 70% similarity) (Bomer et al., 1996a; Schlossmann et al., 1996). So far, no protein with homology to Tom71 and Tom37 could be detected in any other higher eukaryotic organism. The recent isolation and puriﬁcation of the TOM holo complex of Neurospora crassa has provided further insight into the composition, structure, and function of the TOM complex (Kunkele et al., 1998b; Ahting et al., 1999; Stan et al., 2000; Ahting et al., 2001). The isolated holo complex contained the established import receptors (Tom70 and Tom20) as well as the TOM core complex, consisting of Tom40, Tom22, Tom6, and Tom7 (Kunkele et al., 1998b; Ahting et al., 1999). The Tom6 and Tom7 were found to be in direct contact with the major component of the pore, Tom40. In addition, Tom6 was observed to interact with Tom22 in a manner that depends on the presence of preproteins in transit (Dembowski et al., 2001). The TOM core complex has the characteristics of the general insertion pore GIP; it contains high-conductance channels and binds preprotein in a targeting sequence-dependent manner (Stan et al., 2000). Electron microscopic (EM) analysis and tomographic studies revealed single particles with one, two, and three putative channels. The majority of these complexes seem to contain two protein-conducting channels (Ahting et al., 1999). As estimated from three-dimensional reconstruction by electron tomography and from electrophysiological measurements, the size of the two open pores traversing the complex is roughly 2.1 nm and has a height of approximately 7 nm, which is large enough to allow translocation of a polypeptide chain (Ahting et al., 1999). A TOM subcomplex consisting exclusively of Tom40 of N. crassa has been isolated (Ahting et al., 2001). Structural analyses as determined by circular dichroism measurements and Fourier transform infrared spectroscopy revealed 31% β-sheet topology and 22% α-helix (Ahting et al., 2001).

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Isolated Tom40 was functional and forms pores with channel-forming activities very similar to those found with both TOM core complex and TOM holo complex, supporting the view that Tom40 is the central constituent of the protein-conducting channel of the TOM complex. Electron microscopy of puriﬁed Tom40 revealed particles primarily with one center of stain accumulation. They presumably represent an open pore with a diameter of 2.5 nm, similar to the pores found in the TOM complex. Thus, Tom40 is the core element of the TOM translocase and it forms the protein-conducting channel in an oligomeric assembly. Early studies have provided insights into the speciﬁcity of the mitochondrial preprotein receptors (S¨ollner et al., 1989; Ramage et al., 1993). The Tom22 was shown to act in concert with Tom20, thereby forming a receptor assembly that preferentially binds preproteins with positively charged presequences and precursors destined to be inserted into the outer membrane (Fig. 2) (Mayer et al., 1995a; Brix et al., 1997; Abe et al., 2000). The so-called cis site of this receptor is involved in the recognition of precursor proteins on the surface of the outer membrane and provides an extended binding area on which the various targeting signals can dock, and thereby are guided into the outer membrane translocation pore (Lill et al., 1996). The cytosolic domain of Tom20 contains a hydrophobic groove that accommodates a positively charged amphipathic α-helical matrix-targeting sequence. Although the positive charges are necessary for translocation of the presequence across the inner membrane, binding of the presequence to Tom20 is mediated by hydrophobic interactions (Abe et al., 2000). The molecular basis of binding of a presequence to Tom22 are not known. However, binding to the cis site is readily reversible and weakened in its stability at increasing salt concentrations. This may indicate the involvement of weak electrostatic forces acting between the presequences and the receptor components (Haucke et al., 1995; Mayer et al., 1995b). Hydrophobic precursor proteins that carry internal targeting information, such as the members of the family of the mitochondrial carriers, are preferentially bound by the receptor components Tom70 and Tom71. As these signals occur repeatedly within one carrier preprotein, it was assumed that several Tom70 molecules simultaneously bind, thereby stabilizing a hydrophobic preprotein on the mitochondrial surface (Pfanner et al., 1987; Wiedemann et al., 2001; Schlossmann et al., 1994). The high tendency of hydrophobic precursors to aggregate is not only a problem at the surface of mitochondria but occurs also in the cytosol. This problem is overcome by the action of several components in the cytosol that maintain precursor proteins in an import-competent state and perhaps protect them from rapid proteolytic degradation. The Tom70 may also act as a docking site for

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these cytosolic targeting factors, which comprise members of the cytosolic Hsp70 family and probably additional binding factors. MSF (mitochondrial import stimulating factor) is the best characterized factor so far. It belongs to the 14–3–3 protein family (Hachiya et al., 1994; Mihara and Omura, 1996). MSF was proposed to recognize the mitochondrial precursor proteins in the cytosol, forms a complex with them and targets them to the receptors on the outer surface of mitochondria in an ATP-dependent manner (Hines et al., 1990; Hines and Schatz, 1993; Hachiya et al., 1995; Komiya et al., 1997). ATP hydrolysis is likely required to facilitate release of the preproteins from factors such as cytosolic Hsp70 and MSF. This release may promote binding of the preprotein by receptors of the TOM complex. Binding of preproteins to the cis site is followed by the transfer through the translocation channel or GIP, which allows interaction of the N-terminal targeting sequence with a second speciﬁc binding site located at the inner face of the outer membrane called trans site (Fig. 2). Insertion of the N-terminal part of the mature protein into GIP is accompanied by the unfolding of the following segments of the preprotein (Mayer et al., 1995b). The molecular nature of the trans site is not entirely clear. The Tom40 is considered to be the main component generating the trans site. In addition, Tom22 may contribute to this binding site. The presence of speciﬁc presequences binding sites were proposed for the intermembrane space portions of both proteins (Mayer et al., 1995a; Hill et al., 1998; Athing et al., 1999). What drives the translocation of the presequence across the outer membrane? Apparently, neither a membrane potential nor ATP are necessary for directing the presequence to the trans site. The energy derived from presequence binding could constitute the driving force for transfer across the outer membrane. The much higher afﬁnity of the presequences to the trans site as compared to the cis site could provide the driving force for movement and determine its directionality (Mayer et al., 1995b). A related concept is the “acid chain hypothesis,” which proposes that the positively charged presequences are recognized by the negatively charged clusters of the TOM components via ionic interactions. This concept is supported by the observation that puriﬁed cytosolic and intermembrane space domains of several Tom proteins and Tim23 interacted with mitochondrial precursors in a sequential manner. Other noncovalent forces, like hydrophobic forces also appear to play an important role in the interaction between matrix-targeted preproteins and TOM components. A modiﬁed model has been proposed in which preproteins are transferred in a stepwise manner along a chain of binding sites that guides the precursor across the outer membrane into the intermembrane space (binding chain hypothesis) (Pfanner and Geissler, 2001).

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C. THE PRESEQUENCE TRANSLOCASE OF THE INNER MEMBRANE—THE TIM23 COMPLEX At the inner outlet of the TOM channel presequence-containing preproteins are bound at the trans site before they are sorted to the TIM23 complex, which mediates further translocation into the matrix and into the inner membrane (Figs. 1 and 2). During transport, the TOM complex and the TIM23 complex are transiently linked by the translocating polypeptide chain, thereby forming so-called translocation contact sites (Fig. 3) (Donzeau et al., 2000). Insertion of the presequence into the TIM23 complex is thought to be driven electrophoretically by the membrane potential (Martin et al., 1991; Bauer et al., 1996), and complete transport of the precursor into the matrix is mediated by an ATP-powered import motor attached to the inner outlet of the TIM23 complex (Scherer et al., 1992; Kronidou et al., 1994; Schneider et al., 1994). The TIM23 complex consists of a membrane-integrated section that is composed by the subunits Tim23 and Tim17; and by a section attached to it at the matrix side, which is composed by the components Tim44, mtHsp70, and Mge1 (Fig. 3) (Maarse et al., 1992; Dekker et al., 1993; Emtage and Jensen, 1993; Maarse et al., 1994; Ryan et al., 1994). The Tim23 forms a receptor for the presequence in the intermembrane space and together with Tim17 a preprotein conducting channel across the inner membrane (Berthold et al., 1995; Bauer et al., 1996). The Tim23 can be divided into a hydrophilic N-terminal and a hydrophobic C-terminal half. It is anchored in the inner membrane by its C-terminal portion (Emtage and Jensen, 1993; Donzeau et al., 2000), resulting in an N-out and C-out topology. In the N-terminal half of Tim23 an intermediate domain can be discriminated from an N-terminal domain. The intermediate domain is exposed in the intermembrane space, whereas the N-terminal domain is penetrating the outer membrane so that a small segment is exposed on the mitochondrial surface where it is accessible to added protease (Donzeau et al., 2000). Thus, Tim23 is the ﬁrst mitochondrial protein with a two-membrane-spanning topology. The Tim17 is structurally related to Tim23 in its membrane-integrated portion but lacks a hydrophilic N-terminal portion (Maarse et al., 1994; Ryan et al., 1994). The Tim17 and Tim23 are organized as a dimeric complex (Bauer et al., 1996; Moro et al., 1999). How these components generate the protein conducting channel is not clear. The section of the TIM23 translocase at the inner face of the inner membrane forms the import motor. The Tim44 is a hydrophilic peripheral membrane protein associated with the inner face of the inner membrane and forms a dimer (Maarse et al., 1992; Blom et al., 1993; Milisav et al., 2001). In contrast to Tim17 and Tim23, Tim44 is not accessible to added proteases from the outer side of the inner

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FIG. 3. Dynamic interaction between the TOM complex and the TIM23 complex during import of preproteins with matrix-targeting signals into the mitochondrial matrix. Schematic outline of the formation of translocation contact sites. (1) The dimeric TIM23 complex contains two molecules of Tim23, Tim17, and Tim44, which recruit two molecules of mtHsp70 to the outlet of the translocation channel. Tim23 is integrated into both mitochondrial membranes. The N-terminal domain of Tim23 is embedded into the outer membrane (OM), the intermediate domain dimerizes and forms a negatively charged presequence receptor in the IMS, and the C-terminal half is integrated in the inner membrane (IM). A precursor in association with the TOM complex is shown. The positively charged matrix targeting signal (zigzag) is bound to the trans site (hatched) at the inner side of the outer membrane. The TIM23 complex, tethered to the outer membrane via its N-terminal domain, screens by lateral diffusion the inner side of the outer membrane. (2) The presequence receptor domain of Tim23 encounters the TOM complex and triggers the release of the presequence from the trans site. (3) Binding of the presequence destabilizes the interaction of the dimerized intermediate domains, leading to the -dependent opening of the protein conducting channel of the TIM23 complex. The presequence is translocated across the inner membrane. Upon entering the matrix, further translocation is driven by ATP-dependent reaction cycles of the import motor consisting of mtHsp70 (70), Tim44 (44), and Mge1p (E).

membrane. The mtHsp70 is a matrix-localized mitochondria-speciﬁc member of the large Hsp70 protein family. It associates with Tim44 in an ATPdependent manner and this is regulated by the nucleotide exchange factor Mge1. How can one envision the function of this molecular machine in the translocation of preproteins? The TIM23 complex comprises four distinct functional elements (Fig. 3) (Bauer et al., 1996; Donzeau et al., 2000): (a) By virtue of its simultaneous integration into two membranes, Tim23 forms contacts between the outer and inner mitochondrial membranes. Tethering

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the TIM23 complex to the outer membrane facilitates the transfer of preproteins from the TOM complex to the inner membrane translocase, thereby increasing the efﬁciency of protein import. (b) The TIM23 complex receives the preprotein from the TOM complex and binds the N-terminal matrix-targeting signals (presequences) through the presequence receptor domain of Tim23. The receptor is formed upon dimerization of the intermediate domain of Tim23 in a membrane potential-dependent manner. The receptor domains are negatively charged and may interact with the positively charged amphipathic matrix-targeting signals. Upon interaction with the presequence, the dimer of the intermediate domain dissociates, thereby triggering the opening of the protein-conducting import channel across the inner membrane. (c) The channel is formed by the membrane-integrated portion of Tim17 and Tim23. Opening of the channel allows translocation of the presequence across the inner membrane in a membrane-potentialdependent manner. The components forming the channel interact with the unfolded preprotein in transit, but they do not tightly bind the precursor and thus allow oscillation of the presequence in the channel (Ungermann et al., 1994; Berthold et al., 1995; Dekker et al., 1997). (d) for further inward movement a molecular motor is attached at the inner side of the inner membrane. Two models for the action of this molecular motor are currently under debate. In the ﬁrst model, a Tim44 dimer recruits two molecules of mtHsp70 to the outlet of the protein-conducting channel (Kronidou et al., 1994; Rassow et al., 1994; Schneider et al., 1994; Schneider et al., 1996). The Tim44 and the mtHsp70, in cooperation with its cochaperone Mge1p, may constitute a molecular ratchet that drives complete translocation of the polypeptide chains into the matrix with ATP as an energy source. Within this ratchet, mtHsp70 appears to trap incoming segments of unfolded precursor proteins. Thus retrograde movements of the translocating polypeptide chain in the channel are prevented. Repeated cycles of mtHsp70 binding and release, in a kind of “hand-over-hand” mechanism, may facilitate vectorial translocation into the matrix in a stepwise manner (Moro et al., 1999; Schneider et al., 1994). In the second model, Tim44-bound mtHsp70 undergoes signiﬁcant conformational changes, thereby pulling the polypeptide chain of a precursor through the translocation channel. It is not known, however, what the strength of a pulling force exerted by small conformational changes could be and so it is not clear whether such a mechanism would be sufﬁcient to drive the import of an entire polypeptide chain and in particular to provide the energy for unfolding of folded domains of precursor proteins in transit (Voos et al., 1996; Huang et al., 1999; Voisine et al., 1999). In addition to the energy derived from ATP hydrolysis, a second driving force is required for protein translocation into the matrix. This energy is

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present in the form of a total proton-motive force across the inner membrane, which must be sealed against pronounced leakage of ions. Energization of the inner membrane is not only required for protein translocation but an absolute requirement for the mitochondria to perform oxidative phosphorylation. In contrast to the translocase of the outer membrane, the TIM23 complex and as described below the TIM22 complex require a tight regulation of opening and closing. A permanently opened TIM23 channel would otherwise impair the electrochemical gradient across the inner membrane and cause a breakdown of the oxidative phosphorylation. It is the electrical component of the total proton-motive force that promotes the dimerization of the intermediate domain of the Tim23, thereby presumably sealing the channel (Bauer et al., 1996). On the other hand, is required for the transfer of the targeting sequence of a preprotein across the inner membrane (Martin et al., 1991; Pfanner and Neupert, 1985); it is, however, not necessary for the movement of the mature part of the preprotein through the import channel of the inner membrane (Schleyer and Neupert, 1985). As discussed above, the membrane potential may exert an electrophoretic effect on the positively charged presequences in such a manner that translocation of the targeting signal is triggered and a gating effect is exerted (Martin et al., 1991).

D. THE TRANSLOCASE FOR IMPORT OF CARRIER PROTEINS INTO THE MITOCHONDRIAL INNER MEMBRANE—THE TIM22 COMPLEX Integral inner membrane proteins that carry a classical matrix targeting signal use the TIM23 complex for insertion. This can occur either in a “translocation arrest” pathway or by the transfer into the matrix and insertion from the inner face with the help of the OXA1 translocase (Hell et al., 1997, 1998). However, a number of inner membrane proteins carrying internal targeting signals do not use the TIM23 complex but rather are transferred from the TOM complex to the TIM22 complex for insertion into the inner membrane (Sirrenberg et al., 1996) (Fig. 1). Mitochondrial carrier proteins constitute the major class of precursors that are imported via this pathway (Sirrenberg et al., 1996, 1998; Koehler et al., 1998a,b; Endres et al., 1999). In addition, the TIM22 complex appears to mediate the import of precursors of other hydrophobic membrane proteins such as Tim23, Tim17, and Tim22, which do not belong to the class of mitochondrial carriers (Adam et al., 1999; Leuenberger et al., 1999; Paschen et al., 2000). The Tim22 is the central component of the TIM22 complex; it is structurally related to Tim17 and Tim23, suggesting that both TIM complexes

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have evolved from a common ancestor by gene duplication events (Sirrenberg et al., 1996; Bauer et al., 1999b). Two further membrane-integrated components of the TIM22 complex, Tim54 and Tim18, are known (Fig. 2) (Kerscher et al., 1997, 2000). Their functions are not clear so far. The Tim54 seems to inﬂuence the stability of Tim22 in the inner membrane, but may not directly interact with the preproteins during import (Kerscher et al., 1997). The Tim18 shows structural similarity to the subunit IV of complex II (succinate dehydrogenase) of the respiratory chain (Kerscher et al., 2000). During import, the membrane-integral portion of the TIM22 complex interacts with a set of small, structurally related proteins of the mitochondrial intermembrane space (Koehler et al., 1998a,b; Sirrenberg et al., 1998; Adam et al., 1999). The interaction of these small Tim proteins with the translocating preproteins is metal dependent (Sirrenberg et al., 1998). In yeast, ﬁve small intermembrane space proteins, Tim8, Tim9, Tim10, Tim12, and Tim13 have been identiﬁed. All of them contain a Cys4 motif that binds Zn2+ ions proposed to be required for the formation of typical zinc ﬁnger structures (Sirrenberg et al., 1998; Adam et al., 1999). In yeast, Tim9, Tim10, and Tim12 are essential for the cell viability, whereas Tim8 and Tim13 have no obvious deletion phenotype. In particular, Tim9, Tim10, and Tim12 were shown to mediate the insertion of members of the mitochondrial carrier family into the inner membrane; the insertion of a subclass of hydrophobic inner membrane proteins, such as Tim23, involves the assistance of Tim8 and Tim13 (Leuenberger et al., 1999; Paschen et al., 2000). 1. Import of Carrier Proteins The Tim9, Tim10, and Tim12 are organized in two distinct heterooligomeric 70 kDa complexes (Fig. 4) (Koehler et al., 1998a,b; Sirrenberg et al., 1998; Adam et al., 1999). The TIM9·10 complex appears to contain three molecules of Tim9 and three molecules of Tim10. The TIM9·10·12 complex is probably composed of three molecules of Tim9, two molecules of Tim10, and one molecule of Tim12. The TIM9·10·12 complex is ﬁrmly associated with the membrane integrated components of the TIM22 complex, whereas the TIM9·10 complex is mobile in the intermembrane space (Sirrenberg et al., 1998). The TIM22 complex cooperates with both the TIM9·10 and the TIM9·10·12 complex, which sequentially interact with hydrophobic precursors and maintain them in an insertion-competent conformation. Most recently, Luciano and co-worker (Luciano et al., 2001) were able to reconstitute the TIM9·10 complex by co-importing recombinantly expressed Tim9 and Tim10. Moreover, import of recombinant Tim10 into an AAC import-deﬁcient strain lacking the endogenous TIM9·10 complex

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FIG. 4. The TIM22 complex mediates insertion of the members of the carrier family into the inner membrane. Precursors of substrate carrier proteins such as the ADP/ATP carrier (AAC) contain internal targeting information. They are released from cytosolic ribosomes (stage I) and preferentially bind to the Tom70 receptor on the surface of the mitochondria (stage II). The precursor is then transferred to the general insertion pore of the TOM complex. Segments of the precursor that are translocated across the TOM complex are trapped by the TIM9·10 complex in the intermembrane space, resulting in partial translocation of the AAC across the outer membrane The precursor remains at this stage ﬁrmly bound to the TOM complex (stage IIIa). The precursor is then transferred to the TIM9·10·12 complex (stage IIIb) at the outer face of the inner membrane. Insertion of carrier proteins into the inner membrane is mediated by Tim22 in a -dependent manner (stage IV). Finally, the inserted AAC assembles into a functional dimer (Stage V; homodimerization).

was able to restore import and insertion of AAC to almost wild-type levels (Luciano et al., 2001). It was shown that the precursors of the carrier proteins interact with the hetero-oligomeric 70 kDa zinc ﬁnger protein complexes in the intermembrane space in a Zn2+-dependent manner (Sirrenberg et al., 1998). Thus, the interaction of the zinc ﬁngers of the small Tim proteins with the internal signals could be the molecular basis for the recognition of the mitochondrial carrier proteins by the import machinery. Translocation and membrane insertion of the carrier proteins involves the coordinated action of both the TOM complex and the TIM22 complex (Fig. 4) (Pfanner and Neupert, 1987; Ryan et al., 1999). The following pathway of import is proposed on the basis of the available experimental data: the cytosolic precursor of a carrier is initially recognized by the outer membrane receptor Tom70 of the TOM complex. The precursor is then transferred to

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the GIP and partially translocated across the outer membrane. It interacts with the TIM9·10 complex in the intermembrane space but remains ﬁrmly bound to the TOM complex (Adam et al., 1999). Still in contact with the TOM complex, the precursor is then handed over to the TIM9·10·12 complex at the inner membrane (Adam et al., 1999). Subsequently, the TIM22 complex triggers the release of the carrier from the TOM complex and mediates the insertion into the inner membrane (Adam et al., 1999). In contrast to the TIM23-mediated import into the matrix, the insertion of the hydrophobic preproteins into the inner membrane depends on the presence of a membrane potential but does not require ATP. Finally, the inserted carrier assembles into a functional dimer (Nelson et al., 1998). It is not clear whether hydrophobic preproteins are imported at translocation contact sites of the TOM complex and the TIM22 complex in a manner similar to the import of hydrophilic matrix-targeted precursors via the TIM23 complex (Donzeau et al., 2000). It appears likely that carrier proteins cross the outer membrane in a partially folded form, exposing loops at the inner outlet of the TOM channel into the intermembrane space. The translocation of carriers in a loop formation may lead to a cooperative effect of the internal import signals which are subsequently recognized by the small Tim proteins of the intermembrane space (Wiedemann et al., 2001). The complexes of small Tim proteins may act like molecular chaperones that stabilize the precursors of hydrophobic inner membrane proteins in the aqueous environment of the intermembrane space in that particular conformation and guide them to the TIM22 complex. Thus, translocation of carrier proteins does not involve a soluble translocation intermediate in the intermembrane space (Adam et al., 1999). 2. Import of Tim23 into the Inner Membrane The Tim8 and Tim13 also form a hetero-oligomeric 70 kDa complex in the intermembrane space. This complex is supposed to contain three molecules of each, Tim8 and Tim13, but none of the other Tim proteins (Koehler et al., 1999). The Tim8·13 complex is not required for the biogenesis of the mitochondrial carrier protein but rather affects import of noncarrier proteins of the inner membrane such as Tim23 (Kerscher et al., 1997; Leuenberger et al., 1999; Paschen et al., 2000). As most other inner membrane proteins, the precursors of Tim23 and Tim17 contain internal signals, which mediate insertion of the precursors into the inner membrane in the presence of a membrane potential, (Davis et al., 1998; Kaldi et al., 1998). The hydrophilic N-terminal domain of Tim23 contains, in addition, a targeting signal that mediates its import independent of (Kaldi et al., 1998). The TIM8·13 complex is proposed to stabilize the Tim23 precursor in a translocation-competent conformation in the intermembrane space,

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thereby facilitating its -dependent insertion into the inner membrane mediated by the TIM22 complex. Thus, the TIM8·13 complex and the related TIM9·10 complex have a different substrate speciﬁcity but appear to function in a similar manner. In yeast, the assistance of the TIM8·13 complex is not required, however, for import of Tim23 under all conditions (Paschen et al., 2000). Under normal growth conditions, the membrane potential is sufﬁcient to drive import and membrane insertion of Tim23 even without the assistance of the TIM8·13 complex. Only when the membrane potential is low, was the TIM8·13 complex found to be necessary to accumulate Tim23 precursor at the inner face of the outer membrane where it can contact the TIM22 complex to facilitate membrane insertion. This situation differs from that in humans (see below).

E. MITOCHONDRIAL TRANSLOCASES IN MAMMALS In contrast to the rather comprehensive knowledge on fungal systems, relatively little is known about the import components in mammalian mitochondria. On the other hand, it was to be expected that the proteinimport systems of mammalian mitochondria are basically similar to that of S. cerevisiae or N. crassa. Precursors from fungi were observed to be imported into isolated mammalian mitochondria, and precursor protein from mammalian cells could be imported into fungal mitochondria. Furthermore, similar requirements for import in vitro were seen with mitochondria from both types of organisms. Mitochondrial preprotein imports depends on similar energy requirements. Several mammalian homologs of components of the yeast import system have been identiﬁed. These are the mammalian homologues of the TOM receptor components Tom20, Tom22, and Tom70 (Goping et al., 1995; Hanson et al., 1996; Alvarez-Dolado et al., 1999; Saeki et al., 2000), and of the central core component Tom40 (Suzuki et al., 2000). Most notably, some of the newly identiﬁed mammalian proteins, such as human Tom20, can act as functional homologues to the yeast components and complement the respective null phenotype. Furthermore, human Tom34 (Nuttall et al., 1997; Young et al., 1998; Chewawiwat et al., 1999), and metaxin (Armstrong et al., 1997; Abdul et al., 2000) have been desribed as components of the mitochondrial import machinery in the outer membrane. Both mammalian components have no apparent counterpart in fungi but appear to be involved in mitochondrial import pathways in mammalians. Recently, also human components of the inner membrane translocases, TIM23 and TIM22, have been identiﬁed and characterized in more detail (B¨omer et al., 1996b; Ishihara and Mihara, 1998; Wada and

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Kanwar, 1998; Bauer et al., 1999a,b; Rothbauer et al., 2001). The structural composition of the TIM23 complex appears to be conserved from lower to higher eukaryotes; on the other hand, signiﬁcant differences have been observed. In contrast to yeast, two Tim17 homologues are expressed in mammalians, giving rise to two distinct functional TIM23 complexes in the inner mitochondrial membranes: Tim17a–Tim23 and Tim17b–Tim23 (Bauer et al., 1999a). The preservation of the gene indicates that the human TIM17 genes originated by duplication and subsequent translocation to another chromosome. The functional relevance of these structural differences is not clear. The expression of two distinct functional TIM23 translocases in higher eukaryotes might reﬂect the development of a higher complexity in the mitochondrial composition during evolution, and therefore the development of different requirements of preprotein import. Differences in the structural composition of the second inner membrane translocase, TIM22, have also been described. Whereas the human homolog of yeast Tim22 was recently identiﬁed (Bauer et al., 1999b), a Tim54 homologue appears not to be expressed in mammalian mitochondria. Most recently, the structural and functional analysis of the human TIM22 import pathway, in particular of the small zinc ﬁnger proteins of the Tim10 family, has allowed to elucidate the pathomechansim underlying a complex neurodegenerative syndrome.

III. Mitochondrial Biogenesis and Human Neurodegenerative Diseases

A. DYSFUNCTION OF MITOCHONDRIAL PREPROTEIN IMPORT AS A CAUSE OF PROGRESSIVE NEURODEGENERATION—MOHR-TRANEBJAERG SYNDROME The small Tim components of the intermembrane space belong to an evolutionary conserved protein family from which more than 50 ORFs have been identiﬁed throughout the eukaryotic kingdom (Bauer et al., 1999b). Six members of this protein family were shown to be expressed in humans (Bauer et al., 1999b; Jin et al., 1999). Based on the sequence alignments, humans contain two Tim8 homologues (hTim8a, hTim8b), one Tim13 homologue, one Tim9 homologue, and two Tim10 homologues (hTim10a, hTim10b), but no obvious Tim12 homologue. All human homologues appear to be expressed in a wide range of adult and fetal human tissues (Bauer et al., 1999b). Similar to yeast, the human small Tim proteins form distinct oligomeric complexes in the intermembrane space of mitochondria (Rothbauer et al., 2001). Human Tim8a is identical to DDP1, the deafness–dystonia peptide encoded on chromosome Xq22 ( Jin et al., 1996; Koehler et al., 1999). Mutations

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in the DDP1 gene are associated with a severe X-linked neurodegenerative disorder, the Mohr-Tranebjaerg syndrome (MTS) (McKusick, no. 304700) (Tranebjaerg et al., 1995). The phenotype of MTS includes progressive postlingual sensorineural hearing loss, often in combination with a variety of neurological symptoms including dystonia, muscle weakness, dementia, and blindness. Most of the DDP1 mutations are loss-of-function mutations predicted to lead to an absent or a truncated gene product. So far, only one missense mutation was found, causing a cysteine to tryptophan exchange (C66W) within the Cys4 motif (Tranebjaerg et al., 2000). By analogy to the function of Tim8 and Tim13 in yeast, it was suggested that the Mohr-Tranebjaerg syndrome is a new type of mitochondrial disease caused by a defect in the biogenesis of the human TIM23 complex (Paschen et al., 2000). However, the TIM8·13 complex in yeast is not strictly required for the import of Tim23 (see above). A requirement of the TIM8·13 complex was only observed when membrane insertion of Tim23 was compromised (Paschen et al., 2000). If this is true also for the human DDP1·Tim13 complex, how can loss of DDP1 function in MTS patients lead to such a severe neurodegenerative phenotype? Recent data suggest, that the human DDP1·hTim13 complex is functional in yeast. It rescues the growth defect observed at low temperature in the 8/13 yeast deletion mutant (Paschen et al., 2000; Rothbauer et al., 2001). In contrast, expression of a mutant DDP1 carrying a C66W amino acid exchange (the only missense mutation observed in MTS patients) does not complement the yeast deletion phenotype (Hofmann et al., 2002). The C66W mutations presumably leads to a nonfunctional zinc ﬁnger (Hofmann et al., 2002). Studies on the mutant DDP1C66W revealed that it does not accumulate in the intermembrane space of mitochondria from patient cell lines (C. K¨ohler, personal communication). This suggests that the mutant DDP1 protein is not able to fold properly and is rapidly degraded; this also explains the full-blown clinical phenotype observed in a patient harboring the mutant C66W allele on the X chromosome. The human DDP1·hTim13 complex facilitates the import of Tim23 precursor across the outer membrane at low in a manner similar to yeast (Fig. 5). However, import of human Tim23 into isolated yeast mitochondria required the assistance of the DDP1·hTim13 complex even when was high (Rothbauer et al., 2001). Under these conditions the import of yeast Tim23 is not dependent on the TIM8·13 complex. Apparently, import of human Tim23 into yeast mitochondria and into mammalian mitochondria requires a higher membrane potential than import of yeast Tim23. This is probably due to a weaker import signal in the C-terminal portion of human Tim23 (Paschen et al., 2000). The biogenesis of human Tim23 may

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FIG. 5. Role of the DDP1–hTim13 complex in the import of human Tim23 and proposed pathomechanism of the Mohr-Tranebjaerg syndrome. Import into intact mitochondria (left panel): the Tim23 precursor (dashed line) is translocated across the TOM complex and trapped by the DDP1·hTim13 complex in the intermembrane space. The TIM22 complex interacts with the accumulated precursor and mediates efﬁcient insertion into the inner membrane at normal levels of and with reduced efﬁciency at low levels of . Loss of DDP1 function (right panel): in the absence of the DDP1–hTim13 complex (Mohr-Tranebjaerg syndrome), the hTim23 precursor cannot be trapped in the intermembrane space and accumulates bound to the receptors on the surface of the mitochondria (dashed line). Due to the reduced concentration of translocation intermediates in the intermembrane space, insertion of hTim23 into the inner membrane by the TIM22 complex is compromised.

therefore be more dependent on a functional DDP1·hTim13 complex than biogenesis of yeast Tim23. It can be speculated that mutations in DDP1 could signiﬁcantly affect the biogenesis of the TIM23 complex in humans. In the absence of a functional DDP1·hTim13 complex, the Tim23 precursor cannot be trapped in the intermembrane space of human mitochondria (Fig. 5). A direct interaction of the Tim23 precursor with the TIM22 complex might be rather inefﬁcient and the equilibrium shifted toward retrograde translocation. Thus, membrane insertion might require multiple rounds of interaction of the TIM22 complex with the TOM-bound precursor of Tim23. Accordingly, the

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Mohr-Tranebjaerg syndrome may be primarily a result of an impaired import of Tim23 into the inner membrane (Fig. 5). Thus, Mohr-Tranebjaerg syndrome is considered the ﬁrst example demonstrating that defects in the mitochondrial import machinery can lead to a mitochondrial disease, thereby suggesting a fundamentally new pathogenetic mechanism for progressive neurodegeneration. As the clinical features of MTS resemble typical defects in mitochondrial oxidative phosphorylation (OXPHOS), the underlying mechanism causing the disease phenotype may be similar, at least in part. The Tim23 is an essential component of the TIM23 complex, and it is required for the import of a variety of components necessary for the translocation, assembly, and integrity of the OXPHOS system of mitochondria. Therefore, it can easily be envisioned that a defect in targeting preproteins to the mitochondrial matrix may indirectly affect the mitochondrial OXPHOS activity and energy production by malfunctional shuttling of ATP or other metabolites required for functional integrity of mitochondria. This is supported by the fact that nerve cells, in particular those of the cochlea and the basal ganglia, are sensitive to insufﬁcient ATP supply and many mitochondrial diseases cause neurological movement disorders and inner ear deafness.

B. DEFECTS OF QUALITY CONTROL OF MITOCHONDRIAL INNER MEMBRANE PROTEINS—HEREDITARY SPASTIC PARAPLEGIA The biogenesis of mitochondria is not only dependent on the import and sorting of nuclear-encoded preproteins to their correct destination, but also on the removal of mistargeted, misfolded, or malfunctional preproteins. AAA-proteases are a conserved class of ATP-dependent proteases that mediate the degradation of integral membrane proteins in bacteria, mitochondria, and chloroplasts (Beyer, 1997; Langer et al., 2001). They combine proteolytic and chaperone-like activities, thereby forming a membrane-integrated quality-control system. Two proteolytic complexes are present in the mitochondrial inner membrane. These complexes are composed of homologous subunits but expose their catalytic sites to opposite membrane surfaces. The m-AAA-protease is active at the matrix side and is composed of Afg3 (also known as Yta10) and Rca1 (Yta12) (Arlt et al., 1996). The i-AAA-protease, which contains Yme1, probably in a homooligomeric complex, faces the intermembrane space (Leonhard et al., 1996). Inactivation of AAA-proteases causes severe defects in various organisms. Recently, the disease gene of an autosomal recessive form of hereditary spastic paraplegia (HSP) was shown to encode a mitochondrial protein named paraplegin, which is highly homologous to the yeast AAA-proteases Afg3,

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Rca1, and Yme1. Patients with mutations in paraplegin exhibit progressive spasticity of the lower limbs due to degeneration of corticospinal axons (Casari et al., 1998). In yeast, inactivation of Afg3 or Rca1 impairs both degradation of nonassembled inner membrane proteins as well as the assembly of respiratory chain complexes and of the ATP synthase (Paul and Tzagoloff, 1995; Arlt et al., 1996, 1998). Pleiotropic defects, including impaired respiration and abberant mitochondrial morphology, were also detected in yeast cells lacking the i-AAA-protease subunit Yme1 (Thorsness et al., 1993). In agreement with the observed requirement of AAA-proteases for respiratory chain assembly in yeast, muscle biopsies from patients harboring mutations in paraplegin revealed mitochondrial OXPHOS defects. Thus, an impaired quality control of mitochondrial inner membrane proteins due to compromised chaperone or protease function leads to impaired OXPHOS function and neurodegeneration in humans. Defects in paraplegin may cause an accumulation of nonassembled subunits of respiratory chain complexes or ATP–synthase, and may promote nucleation in neurodegeneration.

References

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SECTION II PRIMARY RESPIRATORY CHAIN DISORDERS

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MITOCHONDRIAL DISORDERS OF THE NERVOUS SYSTEM: CLINICAL, BIOCHEMICAL, AND MOLECULAR GENETIC FEATURES

Dominic Thyagarajan1 Department of Neurology Flinders Medical Centre Bedford Park, South Australia 5042, Australia

Edward Byrne Department of Neurology University of Melbourne Melbourne, Australia

I. II. III. IV. V.

VI.

VII.

VIII.

IX.

1

Introduction Historical Considerations Genetics and Pedigree Studies Clinical Features Important Clinical Questions A. Uneven Segregation of Mutant and Wild-Type mtDNA B. Interaction of mtDNA Mutation with Genetic Background C. Different Energy Requirements of Tissues Major Mitochondrial Syndromes A. Progressive Limb Myopathy without CPEO B. Recurrent Myoglobinuria C. Chronic Progressive External Ophthalmoplegia Syndromes D. KSS and Other CPEO Syndromes E. Neuropathy F. Encephalomyopathies Biochemical Features A. Complex I Deficiency B. Complex II Deficiency C. Complex IV Deficiency D. Cytochrome b Deficiency E. Complex V Deficiency F. Coenzyme Q Deficiency Diagnostic Approaches A. Initial Approach B. Mitochondrial Disease Workup C. Summary Treatment of Mitochondrial Disorders A. Physical and Supportive Therapies Author to whom correspondence should be addressed.

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Copyright 2002, Elsevier Science (USA). All rights reserved. 0074-7742/02 $35.00

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B. Metabolic Therapies C. Miscellaneous D. Gene Therapy References

I. Introduction

“Mitochondrial medicine,” a term coined by one of the founders of the ﬁeld, Rolf Luft, has shifted from the study of a few unusual metabolic disorders affecting muscle and brain to central biochemical and genetic dysfunction in important human disease processes (Luft, 1994; MorganHughes, 1994; Leonard and Schapira, 2000a,b). Knowledge of normal mitochondrial function applied to human disease permitted this evolution. Sophisticated histochemical and ultrastructural morphological techniques, reﬁnement of biochemical analysis of the respiratory chain, the discovery of mtDNA mutations in human disease, and techniques to transfer mtDNA (strictly, mitochondria) from one cell to another, opened new vistas. While technological advances have answered many questions, more have been raised. The problem of determining the signiﬁcance of minor histological changes especially in older patients, the identiﬁcation of artifactual abnormalities in biochemical studies, and the development of criteria to differentiate well-tolerated unusual mtDNA polymorphisms from disease causing mutations are but few of the problems overcome in recent years. The purpose of this chapter is to give a perspective on current knowledge of clinical features, biochemistry, and molecular biology of the mitochondrial diseases. We aim to provide a path for the interested physician and laboratory scientist to navigate the often complex road to accurate diagnosis. Treatments are discussed, but this is still an embryonic ﬁeld.

II. Historical Considerations

Understanding the pathophysiology of mitochondrial dysfunction has been closely linked to increased knowledge of normal mitochondrial function and has progressed through a series of major morphological, biochemical, and molecular biological stages, dependent on the techniques available at the time. Mitochondria were ﬁrst recognized in the latter part of the nineteenth century, as improved lenses became available. The name was proposed in 1898 by Bend from the Greek mitos (thread) and chondros

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(grain). A series of pioneering experiments largely by Wielend, Warburg, and Kielin in the 1920s led to the concept of cellular respiration, dependent upon a highly organized system of bound electron carriers in the inner mitochondrial membrane. Increased knowledge of mitochondrial structure paralleled advances in mitochondrial biochemistry. Supravital dye techniques, developed in the early years of the century, allowed mitochondria to be stained in fresh unﬁxed preparations. The modiﬁcation of one of these by Engel and Cunningham, the modiﬁed Gomori trichome stain (Engel and Cunningham, 1963) has proved extremely useful in the examination of frozen muscle sections for mitochondrial pathology. The widespread development of high-quality histochemistry laboratories in many university medical centers in North America and Europe provided a major stimulus to mitochondrial medicine as an important discipline. First the Gomori trichome stain and later the Seligman cytochrome oxidase reaction (Seligman et al., 1968) provided ready means of identifying syndromes in which mitochondrial dysfunction was likely. Despite the fact that oxygen metabolism had long been recognized as crucial in the energetics of brain, heart, and skeletal muscle, human mitochondrial dysfunction was not identiﬁed until 1962. Then, Luft and colleagues established that a hypermetabolic state of nonthyroidal origin was related to loose mitochondrial coupling between respiration and phosphorylation of ADP (Luft et al., 1962). Although only one further patient has been identiﬁed with this abnormality (DiMauro et al., 1976). Luft’s paper was seminal in that it described, for the ﬁrst time, both ultrastructure and Warburg manometry applied to the study of human material. A period of long, slow progress then followed. Many patients with mitochondrial morphological changes, biochemical evidence of electron transport chain (ETC) defects, and various clinical features were described. Shy and Gonatas (1964), investigated ultrastructural changes in muscle mitochondria and divided certain childhood myopathies into one group with proliferated mitochondria and normal appearance (pleoclonial myopathy), and another with enlarged, abnormal mitochondria and disoriented cristae (megaconial myopathy). Drachman (1968) and Kearns and Sayre (1958) described chronic progressive external ophthalmoplegia (CPEO) with other features. In 1972, Olson et al. (1972) reported seven patients with CPEO who had a distinctive subsarcolemmar clustering of skeletal muscle mitochondria on the Gomori modiﬁed trichrome stain that they called “raggedred” ﬁbers. Ultrastructurally, the mitochondria were enlarged, had abnormal cristae, and sometimes contained paracrystalline inclusions. It emerged that these “mitochondrial encephalomyopathies” (Shapira et al., 1977) or “mitochondrial cytopathies” (Egger et al., 1981) were clinically diverse, not necessarily associated with CPEO, and included disorders of vision (retinal

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degeneration, optic atrophy, cataract, and glaucoma), deafness, proximal myopathy or CPEO, neuropathy, encephalopathy, short stature, renal tubular disorders, endocrinopathies, and lactic acidosis. In some of these cases, speciﬁc ETC defects were identiﬁed biochemically, including in cytochrome b, ATPase, NADH–Coenzyme Q10 (CoQ) reductase, and cytochrome c oxidoreductase (COX). In the late 1970s and 1980s, many of the advances came from DiMauro’s group at Columbia University and MorganHughes’ group in London. DiMauro’s group identiﬁed cytochrome c oxidase deﬁciency in infants with lactic acidosis, and discriminated between fatal and benign forms (DiMauro et al., 1980, 1981). At the same time the Morgan-Hughes group (Morgan-Hughes, 1994) reﬁned the use of the oxygen-sensitive electrode and the cytochrome oxidation–reduction spectra analysis to probe respiratory chain function in isolated intact mitochondria in human muscle. Respiratory chain impairment in skeletal muscle was further deﬁned in a series of toxicity experiments in animal models. These models provided new insights into disease mechanisms (Byrne et al., 1985; Hayes et al., 1985). Site 1 respiratory chain inhibitors produced in the animal pathological fatigability related to the rapid depletion ﬁrst of phosphocreatinine and then of ATP which recovered slowly with rest. Failure of muscle contractility was followed by sarcolemmal inexcitability. If energy failure developed to an extreme level, muscle contracture developed (Byrne and Morgan-Hughes, 1989). Mitochondrial enzyme deﬁciencies other than ETC defects were characterized, including pyruvate dehydrogenase complex deﬁciency (Blass et al., 1970), carnitine palmitoyltransferase deﬁciency (DiMauro and MelisDiMauro, 1973), and carnitine deﬁciency (Engel and Angelini, 1973). A systematic biochemical classiﬁcation of mitochondrial disorders was devised (Morgan-Hughes, 1986; DiMauro et al., 1987), and included (1) substrate transport defects into the mitochondrial matrix, (2) substrate utilization defects in the mitochondrial matrix, (3) Kreb’s cycle defects, (4) ETC defects, and (5) defects of oxidation/phosphorylation coupling. Although classiﬁcations (1)–(3) are mitochondrial disorders in the strict sense, the term is often taken to mean defects of the ETC or oxidative phosphorylation coupling, the focus of this chapter. It is of interest that no further cases of Luft’s syndrome have been identiﬁed since the ﬁrst two, despite worldwide growth in the description of various mitochondrial disorders. Furthermore, none of the mitochondrial DNA mutations identiﬁed lead to loose coupling of the type seen in Luft’s syndrome. The clinical features of Luft’s syndrome with loose coupling and increase in mitochondrial volume resemble the toxic effects of agents such as dinitrophenol and it is possible that the syndrome Luft described had a toxic rather than genetic basis. This does not detract from the importance of these original papers.

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New techniques to probe the respiratory chain, provided additional insights into the mechanism of disease. Antibodies speciﬁc for respiratory chain complexes and their individual components (nuclear and mitochondrial DNA encoded) enabled determination of levels of protein. In many situations a general depression of all subunits was identiﬁed, whereas in other diseases, speciﬁc subunit deﬁciencies were found. It was apparent from histochemical studies that considerable variation existed between adjacent cells, particularly in skeletal muscle. This remained unexplained until mtDNA mutations and the peculiar phenomenon of “heteroplasmy” was discovered. A watershed came in the late 1980s with an understanding of the genetics of mitochondrial disorders. Egger and Wilson (1983) noted the excess of maternal inheritance in pedigrees with mitochondrial cytopathy, and maternal inheritance in Leber’s Hereditary Optic Atrophy (LHON). They postulated mitochondrial genetic inheritance, because mammalian mtDNA (discovered in 1963 by Nass and Nass) and sequenced in the human in 1981 (Anderson et al., 1981), was known to be maternally inherited (Hutchison et al., 1974). Abnormalities in the mitochondrial genome had been recognized in yeast species, especially large multigene deletions leading to petit variants (Kovac, 1974; Whittaker, 1979). In 1988, a speciﬁc point mutation of mtDNA in LHON (Wallace et al., 1988), and large-scale deletions in muscle mtDNA from patients with mitochondrial encephalomyopathies were found (Holt et al., 1988). The DiMauro group associated mtDNA deletions with the phenotype of CPEO (Moraes et al., 1989) and an explosion in genotype–phenotype correlation followed. There are now there are over 50 point mutations and hundreds of deletions of mtDNA known in various mitochondrial encephalomyopathies (Anonymous, 2000). In most but not all instances (LHON is a notable example), the mutant mtDNA coexists with the normal “wild type” (heteroplasmy). In general, mtDNA mutations impairing mitochondrial protein synthesis [transfer RNA (tRNA) mutations and deletions] are associated with the ragged-red ﬁbers on muscle biopsy, while a morphological clue is absent in mutations of the mitochondrial structural genes. Only 10% of mitochondrial protein are encoded by mtDNA, and it is possible that most mitochondrial disease originates in the nuclear DNA (nDNA). Zeviani et al. (1989) showed dominant inheritance of multiple mtDNA deletions in 1989, clearly implicating a nuclear factor. In 1995, Bourgeron et al. (1995) identiﬁed a mutation in the ﬂavoprotein subunit of complex II (nucleus encoded) in 2 siblings with recessively inherited Leigh syndrome (LS) and Suomalainen et al. (1995) showed linkage to chromosome 10q in autosomal dominant CPEO (adPEO) pedigrees with multiple mtDNA deletions. Since then, two other loci in adPEO have been determined by linkage analysis (Kaukonen et al., 1996, 1999). Mutations have been found in the heart/skeletal muscle of the adenine nucleotide

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transporter I (ANT1) gene in dominantly inherited PEO (Kaukonen et al., 2000) and in thymidine phosphorylase gene in a recessive form of PEO, myoneuro-gastrointestinal encephalomyopathy (MNGIE) (Nishino et al., 1999). Nuclear mutations have been found in complex I deﬁciency, affecting different nuclear subunits (Loeffen et al., 1998; van den Heuvel et al., 1998; Schuelke et al., 1999; Triepels et al., 1999). In two forms of COX deﬁciency, mutations have been found in different COX assembly genes (Tiranti et al., 1998; Zhu et al., 1998; Papadopoulou et al., 1999). As the nucleus-encoded defects affecting subunits of the respiratory chain and intergenomic signaling have been identiﬁed, there has been increasing interest in the role of mitochondrial abnormalities in the pathogenesis of common neurodegenerative diseases such as Parkinson’s disease (PD), Alzheimer’s disease (AD), Huntington’s disease (HD), and aging (Wallace et al., 1995; Leonard and Schapira, 2000b). In situ hybridization studies applied to skeletal muscle have provided additional insight into the distribution of mutant mtDNA at a single cell level. Large deletions were largely conﬁned to cytochrome c oxidase negative zones in muscle ﬁbers with wild-type mtDNA predominating in adjacent ﬁbers with normal COX activity (Collins et al., 1995).

III. Genetics and Pedigree Studies

Mitochondria are cellular organelles with a central role in energy metabolism. Their key role is to generate adenosine triphosphate (ATP) through ETC, embedded in the inner mitochondrial membrane. Pyruvate and fatty acids are transported into the mitochondrial matrix where oxidative pathways convert them to acetyl coenzyme A (acetyl-CoA). Acetyl-CoA is oxidized to the CO2 and H2O by the Kreb’s cycle, generating NADH and reduced ﬂavin mononucleotide (FMNH), which donate electrons to the ETC. The ETC comprises ﬁve multisubunit enzymes and two mobile electron carriers (coenzyme Q and cytochrome c) (Fig. 1). A series of redox reactions in the ETC results in the reduction of O2 to water and generates a pH gradient across the inner mitochondrial membrane. The H+ gradient generates proton ﬂow through the ﬁfth enzyme complex, which catalyzes the synthesis of ATP from ADP and inorganic phosphate (Pi). Unlike other organelles, mitochondria contain their own genetic material. In humans there are 2–10 copies of a double-stranded 16,569 kilobase (kb) circular DNA (Anderson et al., 1981). At fertilization, the sperm, containing 50–75 mitochondria, each with one copy of mtDNA, enters the oocyte, containing 105–108 mitochondria (and 105 copies of mtDNA in human oocytes), complete with mitochondria in the midpiece. In embryogenesis, the paternal contribution to the individual’s mtDNA is eliminated

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FIG. 1. The mitochondrial ETC.

by unknown mechanisms. The genetic mechanisms causing maternal inheritance are unknown, and may be exist in both somatic and germ cells (Manfredi et al., 1997). Mitochondrial DNA only encodes 13 subunits of the ETC, 22 tRNAs, and 2 ribosomal RNAs (rRNAs). The remaining 70 or so proteins of the ETC and the proteins required for replication, transcription, and translation of mtDNA are encoded by nuclear DNA (nDNA). Nucleus-encoded subunits of the ETC are synthesized in the cytoplasm, usually as larger precursor polypeptides with N-terminal presequences that direct them to mitochondria in an energy-dependent process. Nuclear and mitochondrial-encoded subunits assemble in the inner mitochondrial membrane after cleavage of the presequence by a Ca2+/Mg2+-dependent protease. The ETC is therefore a complex assembly of multisubunit enzymes encoded by two genetic systems, one with Mendelian and one with nonMendelian (maternal) transmission (Wallace, 1997). Mitochondrial diseases may thus be (a) autosomal dominant or autosomal recessive, when some nucleus-encoded subunit of the ETC or other protein important in biogenesis of the ETC is affected (Bourgeron et al., 1995; Loeffen et al., 1998; van den Heuvel et al., 1998; Schuelke et al., 1999), or (b) maternally inherited, when mtDNA is mutated. For reasons still inadequately explained, single large-scale rearrangements of mtDNA are usually, though not invariably, sporadic. Notable examples to this rule, include a family with diabetes and deafness in which there is maternal transmission of a large mtDNA deletion (Ballinger et al., 1992). Single large-scale rearrangements coexist with the wild-type, full-length mtDNA, a phenomenon called heteroplasmy.

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In general, they are not abundant in all tissues, tending to be present in highest amount in postmitotic tissues like muscle and brain (DiMauro and Bonilla, 1997; Marzuki et al., 1997). Like the mitochondrial genome itself, point mutations in mtDNA are generally maternally inherited. Heteroplasmy is a common feature of mtDNA point mutations. As in the case of mtDNA deletions, mutant load varies from tissue to tissue, and it can change with time in a particular tissue of an individual. Point mutations causing LHON are important exceptions. They are usually homoplasmic and found in all tissues. Another important exception is that of point mutations in the cytochrome b gene causing progressive exercise intolerance, proximal muscle weakness lactic acidosis, and in some cases, myoglobinuria. These are sporadic and are conﬁned to the muscle, and may be somatic mutations in the myogenic stem cells, arising after differentiation in the germ layer (Andreu et al., 1999). It is generally believed that the mutant load in a tissue and the metabolic demands of the tissue determine the detrimental effects of the mutation. Commonly, a certain mutant load must be reached before the tissue suffers— the concept of the “threshold effect” (DiMauro and Bonilla, 1997). Over 50 mtDNA point mutations causing disease are now described (Anonymous, 2000). Multiple mtDNA deletions follow Mendelian inheritance. Autosomal dominant forms are presumably caused by dysfunction of a protein important in the maintenance of stability or replication of mtDNA. The condition is genetically heterogeneous: at least three loci have been reported (Suomalainen et al., 1995; Kaukonen et al., 1996, 1999), and in one case, mutations in the ANT1 gene have been found in ﬁve families and one sporadic case (Kaukonen et al., 2000). Mutations in the thymidine phosphorylase gene cause an autosomal recessive form, MNGIE (Nishino et al., 1999). Another, probably autosomal, recessive defect of intergenomic signaling causes grossly reduced mtDNA copy number: mtDNA depletion (Moraes et al., 1991). However, mtDNA deletion may also be a phenomenon secondary to other pathologic processes (Poulton et al., 1995), including speciﬁc toxic states, e.g., treatment with Azidothymidine (AZT) (Arnaudo et al., 1991). In other examples, generally recessive, the mutation is in a gene encoding a component of the mitochondrial import machinery for carrier proteins, e.g., the deafness/dystonia peptide 1 (DDP1) gene in the X-linked Mohr-Trajenberg syndrome ( Jin et al., 1996), or a COX assembly protein, e.g., SCO2 in early onset COX deﬁciency, encephalopathy, and hypertrophic cardiomyopathy (Papadopoulou et al., 1999) or SURF1 in LS with generalized COX deﬁciency (Zhu et al., 1998; Tiranti et al., 1999). In an autosomal recessive form of hereditary spastic paraplegia linked to chromosome 16q, “ragged ﬁbers” are present in muscle, and there are mutations in a gene

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called “paraplegin.” The paraplegin product is highly homologous to the yeast mitochondrial ATPases, AFG3, RCA1, and YME1, which have both proteolytic and chaperone-like activities at the inner mitochondrial membrane (Casari et al., 1998). Freidreich’s ataxia, the most common cause of recessive ataxia, is associated with loss of function of frataxin, usually due to a homozygous intronic expansion. Frataxin is mitochondrial protein conserved through evolution. In yeast, knockout of the frataxin homologue causes mitochondrial iron accumulation. Thus, defective mitochondrial iron transport with free radical damage and oxidative stress with deﬁciency of aconitase and iron-sulfur proteins may be the pathogenic mechanism of disease (Puccio and Koenig, 2000). Mitochondrial genetic factors are implicated in neurodegenerations like AD and PD (Schapira et al., 1998; Leonard and Schapira, 2000b). Evidence for involvement of mitochondrial genetic factors is greatest in PD. Complex I activity is reduced in postmortem substantia nigra (but not other brain regions), platelets, and muscle. This complex I deﬁciency has been transferred from platelets of PD patients to zero cells (cells lacking mtDNA) by cybrid fusion, implying that the origin of the ETC defects in PD is mtDNA (Gu et al., 1998; Swerdlow et al., 1996). However, this does not necessarily establish a cause and effect relationship between mtDNA mutations and PD. Damage to mtDNA might be a bystander phenomenon secondary to some other factor such as direct oxidative damage. Several mtDNA mutations have been recognized in association with Parkinsonism (Chalmers et al., 1996; De Coo et al., 1999; Simon et al., 1999; Thyagarajan et al., 2000), but these are only in a handful of pedigrees. Whether sequencing of the mitochondrial complex I genes has clearly demonstrated the presence of pathogenic mtDNA mutations in larger groups of patients with idiopathic PD is a matter of controversy (Kosel et al., 1998; Simon et al., 2000). Clearly, PD is a heterogeneous disease, in which some of the nuclear gene abnormalities that have been found (Polymeropoulos et al., 1997) have no known role in function of the ETC. A good family history and thoughtful analysis of the pedigree can aid greatly in diagnosis. Father to offspring transmission excludes a mtDNA mutation, but a dominantly inherited mitochondrial disease is possible. One practical difﬁculty is that the number of affected individuals is often low, and complete ascertainment is not possible. The number of new mutations in LHON is low, and in Australia, where extensive records have been established, it is very often possible to make the genetic diagnosis in a new case simply by establishing between the patient and the well-characterized families bearing the three common point mutations. This understanding of the molecular genetics of mitochondrial disease has led to a classiﬁcation of mitochondrial ETC disorders (Table I) that has largely supplanted the biochemical classiﬁcations of the 1970s and 1980s.

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TABLE I MOLECULAR CLASSIFICATION OF MITOCHONDRIAL DISEASE Molecular defect mtDNA Single large-scale rearrangement Point mutation In structural genes In mitochondrial RNA genes nDNA coded subunits of the ETC Mutation in SDH ﬂavoprotein subunit Mutation in NDUFS4 subunit of complex I Mutation in NDUFV1 subunit of complex I Mutation in NDUFS8 subunit of complex I Mutation in NDUFS7 subunit of complex I Defects of intergenomic signaling Mitochondrial depletion Multiple deletions of mtDNA Dominant forms linked to 10q, 3p, 4p, and other loci

Mutations in ANT1 Mutations in thymidine phosphorylase Other recessive forms

Nuclear mutations affecting Mitochondrial biogenesis Mutations in SURF1 Mutations in SCO2 Mutations in the DDP1 gene Mutations in paraplegin Other Mutations in frataxin ? Mutations in mtDNA

Some phenotypic examplesa

Inheritance Nearly all sporadic

CPEO PS

Maternal Maternal

NARP, LS/FBSN, LHON, MELAS, MERRF, SNHL, cardiomyopathy, myopathy, multisystem disorders

Recessive

LS (Bourgeron et al., 1995)

Recessive

Fatal multisystem disorder (van den Heuvel et al., 1998) Leucodystrophy/myoclonic epilepsy (Schuelke et al., 1999) LS (Loeffen et al., 1998)

Recessive Recessive

LS (Triepels et al., 1999)

Recessive

Infantile encephalopathy, hepatopathy (Moraes et al., 1991)

Dominant

CPEO, psychiatric illness, cardiomyopathy, Parkinsonism (Suomalainen et al., 1995; Chalmers et al., 1996; Kaukonen et al., 1996, 1995)

Recessive

MNGIE (Nishino et al., 1999)

Recessive

Sensory neuropathy (Fadic et al., 1997), cardiomyopathy (Bohlega et al., 1996), Wolfram syndrome (Barrientos et al., 1996)

Recessive Recessive

LS (Tiranti et al., 1998; Zhu et al., 1998) Infantile cardioencephalomyopathy (Papadopoulou et al., 1999) Mohr-Trajenberg syndrome Hereditary spastic paraplegia (Casari et al., 1998)

X-linked Recessive

Recessive ?

Freidreich’s ataxia Parkinson’s disease and other neurodegenerative disorders (Leonard and Schapira, 2000b)

a PS: Pearson syndrome; NARP: ( ); LS/FBSN: Leigh syndrome/familial bilateral striatal necrosis, MELAS: mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes; MERRF: myoclonus epilepsy and ragged-red fibers; SNHL: ( ); SDH: ( ); NDUF: ( ).

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IV. Clinical Features

Mitochondrial diseases have many causes, including mutations in nuclear and in mitochondrial genes encoding ETC components, and in genes of intergenomic signaling. The disorders of substrate transport and utilization, pyruvate oxidation, and the citric acid cycle is not be discussed in this chapter. Most of the described disorders are caused by point mutations in the mitochondrial genome, but this may be an artifact of study as the mitochondrial genome is relatively small, and easily sequenced. These disorders affect mainly muscle and brain, although cardiac endocrine and other manifestations also occur. Syndromes can be divided into three broad groups with some overlap. The ﬁrst group involves mainly skeletal muscle involvement centered on CPEO and limb muscle fatigability. The second group involves multisystem manifestations with an emphasis of central nervous system involvement. The third group involves oligosymptomatic syndromes with an emphasis on a tissue other than skeletal muscle, a prime example being LHON. The clinical spectrum of respiratory chain cytopathies in terms of the individual clinical manifestations is enormous, as shown in Table II. This is constantly being expanded in surprising ways. The recognition, for example, that Madelung’s syndrome, a rare brown fat storage disorder, had a mitochondrial basis would not have been predicted from early knowledge of the clinical phenotypes. The range of clinical manifestations as set out TABLE II CLINICAL MANIFESTATIONS System/organ

Manifestations

CNS

Seizures, stroke-like episodes, dementia, sensorineural deafness, movement disorders including ataxia, myoclonus, dystonia, chorea, migraine, psychomotor regression/retardation, Parkinsonism

Skeletal muscle

Hypotonia, myopathy, ptosis, CPEO, recurrent myoglobinuria

Peripheral nerves

Neuropathy

Bone marrow

Pancytopaenia, sideroblastic anaemia

Kidney

De-Toni-Fanconi renal tubular acidosis

Endocrine

Type II diabetes mellitus, hypoparathyroidism, growth hormone deﬁciency

Heart

Cardiomyopathy, conduction defect

Gastrointestinal system

Pancreatic failure, pseudo-obstruction, hepatopathy

Eye

Retinal pigmentary degeneration, optic atrophy, cataract

Systemic

Systemic lactic acidosis

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in Table II overlaps considerably with non-mitochondrial disorders. Thus, it is necessary to make a careful search for subtle manifestations of an ETC disorder; for example, nerve deafness, atypical retinitis pigmentosa, or diabetes. Basic investigation may further assist delineation of a syndrome, e.g., basal ganglia calciﬁcation or cerebral atrophy on neuroimaging, or the detection of peripheral neuropathy on conduction tests, or cardiac conduction problems on ECG. Oligosymptomatic syndromes in the relatives of patients with severe phenotypes are well recognized, especially with MELAS and may evolve into a more complete phenotype with increasing age. The history of mitochondrial medicine is marked by the description of unique and surprising syndromes, each new one somewhat different from the last and posing new questions. An early “lumer” vs “splitter” debate,

TABLE III SOME MITOCHONDRIAL SYNDROMES Syndrome

Common clinical manifestations

MELAS

Short stature; migraine; dementia; senosorineural deafness; stroke-like episodes (often occipital and not conforming to metabolic territories), seizures, exercise intolerance, asthenic build and muscle weakness; diabetes mellitus and various other endocrinopathies; intracerebral calciﬁcation, cerebral atrophy.

MERRF

Myoclonus epilepsy; limb muscle weakness and wasting, particularly respiratory muscle weakness in older patients, ataxia, deafness, retinal pigmentary degeneration.

CPEO

Ptosis and progressive complex external ophthalmoplegia; limb muscle weakness and wasting; exercise intolerance; intracerebral calciﬁcation, white matter abnormalities on MRI.

KSS

CPEO with onset before age 20, retinal pigmentary degeneration, high CSF protein, heart block (almost invariable before age 50) white matter abnormalities on MRI.

LS/FBSN

Psychomotor retardation, poor suck/swallow in infancy and failure to thrive, signs of brainstem dysfunction (respiratory abnormalities, sudden death in infancy, eye movement disturbance, nystagmus); peripheral neuropathy; dystonia and other movement disorders, characteristic bilateral, symmetrical periventricular T2 signal hyperintensities on MRI in the deep gray matter; spongiform change, gliosis and microangiopathic necrosis in the deep gray matter.

LHON

Subacute visual failure, particularly in males (M:F ratio 9:1). Dystonia in some patients with the 14484 mutation.

MNGIE

Gastric hypomotility, CPEO, wasting and weakness, deafness.

PS

Infantile sideroblastic anemia

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centered on Kearns-Sayre syndrome (KSS), resolved in favor of the splitters when mitochondrial DNA abnormalities (ﬁrst identiﬁed by a prominent lumper) were found to be generally speciﬁc for unique syndromes (Berenberg et al., 1977). Some commonly recognized syndromes the reader is likely to encounter are listed in Table III. The list is by no means exhaustive. For the practicing clinician it is more important to have a good grasp of the possible clinical presentations, the usual syndromes, and a high index of suspicion than a long, list of acronyms committed to the memory. Overlap presentations, especially between MELAS and MERRF, have long been recognized, but the key syndromes are genetically distinct.

V. Important Clinical Questions

How such unique syndromes arise from a common process, ETC failure is far from clear and no explanation to date is entirely satisfactory. Proposed mechanisms include the following:

A. UNEVEN SEGREGATION OF MUTANT AND WILD-TYPE mtDNA The ratio of mutated and wild-type mtDNA species varies from tissue to tissue. A clear example is the tissue distribution of single and multiple mtDNA deletions among organs in CPEO syndromes (Shanske et al., 1990; Kawashima et al., 1994; Marzuki et al., 1997; Moslemi et al., 1999). At least in muscle, the mutant load also varies from cell to cell (Sciacco et al., 1994). In the case of some point mutations, there is a rough correlation between the mutant load in muscle and in other tissues, and the phenotype (Tatuch et al., 1992; Koga et al., 2000b); there is no clear evidence that regional involvement of the nervous system depends on distribution of the mutation alone. The stroke-like areas in MELAS and the symmetric deep gray matter lesions in LS are so distinct, it is likely that other factors are involved. Beal’s hypothesis that Ca2+-mediated excitoxic damage following glutamatergic pathways can explain only the striatal involvement in LS and the allied condition of FBSN, not the other neuroradiological patterns in seen in, say MELAS or KSS.

B. INTERACTION OF mtDNA MUTATION WITH GENETIC BACKGROUND There may be unique interactions between certain nuclear DNA genes and the mutant mtDNA species that shape the physiological response of

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tissue to ETC failure. Although this is often stated, and reasonably so, there has been very little evidence presented to support the hypothesis. There is, however, an example of mtDNA variant, a complex I mutation, modulating the disease expression in a large Queensland pedigree with LHON (Howell et al., 1991) by ameliorating a severe phenotype of infantile encephalopathy and LHON.

C. DIFFERENT ENERGY REQUIREMENTS OF TISSUES Certain tissues such as heart, brain, and skeletal muscle are very dependent on the ETC for energy, and it is not surprising that these tend to be involved in mtDNA related and other mitochondrial diseases. Other tissues with high aerobic metabolic requirements include the retina and pancreatic β cells. However, this cannot predict the diverse manifestations of mutations that would be predicted to have similar effects, e.g., mitochondrial tRNA mutations as a group. In fact, the different patterns of energy crisis are perhaps the most perplexing conundrum in the ﬁeld. The three best characterized CNS encephalopathies, KSS, MERRF, and MELAS, all involve defective mitochondrial protein translation; KSS by a large deletion including several tRNA genes; MERRF, with a point mutation in tRNALys; and MELAS, with a mutation in tRNALeu mutation. Yet not only is the topographic CNS involvement very different in each case, so is the type of dysfunction. A slow attrition of cerebellar Purkinje cells and cortical neurons results in ataxia and dementia in many patients with KSS; a violent energy crisis results in stroke-like episodes of MELAS; and a more subtle energy crisis with probable resultant membrane instability results in epilepsy in MERRF. Clearly there is much to be understood in this area.

VI. Major Mitochondrial Syndromes

A. PROGRESSIVE LIMB MYOPATHY WITHOUT CPEO Limb weakness coming on usually in teenage years, but sometimes later in life it can be the predominant feature of mitochondrial DNA disease. This is a relatively rare mitochondrial syndrome. Often there is little to be found on formal examination with only minimal ﬁxed weakness and no evidence of multisystem involvement. The patient’s prominent complaint is likely to be one of fatigue, sometimes with muscle pain. Baseline investigations,

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including creatinine kinase, show little or no abnormality. Electromyography (EMG) may reveal myopathic units, but again, ﬁndings are often subtle. Myasthenia may enter the physician’s mind because of the very prominent fatigability, and when tests for myasthenia are negative, a non-organic diagnosis may have been considered. Some patients may be prone to episodes of collapse with severe lactic acidosis on exercise, characterized by hyperventilation, and this may delay a diagnosis. A ﬁnding of unexplained lactic acidosis after low levels of exercise may be a clue. Muscle biopsy is the gold standard of diagnosis with the demonstration of typical abnormalities. Ragged-red ﬁbers are especially prominent in pure myopathic syndromes and COX stains may reveal excessive COX staining rather than COX negative ﬁbers (Collins et al., 1995; Petty et al., 1986). A particular category of skeletal myopathy without CPEO is the late-onset mitochondrial myopathy ( Johnston et al., 1995). This is characterized by insidious, slowly progressive weakness in the 60s and 70s often accompanied by muscle wasting. Muscle biopsy shows ragged-red ﬁbers, most of which is COX negative. Multiple mtDNA deletions can be shown by polymerase chain reaction (PCR) and by in situ hybridization shows transcription of mRNAs from deleted mtDNA species in a large number of muscle ﬁbers. The myopathy appears to result from the clonal expansion of mtDNA deletions with age, in individual muscle ﬁbers. Some deterioration in mitochondrial function in skeletal muscle is an inevitable part of the aging process and care must be taken not to overinterpret more subtle mitochondrial changes seen in both histological and biochemical studies in aged individuals (Byrne and Dennett, 1992; Trounce et al., 1989).

B. RECURRENT MYOGLOBINURIA Respiratory chain dysfunction is an uncommon cause of recurrent exercise-related myoglobinuria. By contrast, deﬁciency of the mitochondrial enzyme, carnitine palmitoyl transferase (CPT), typically presents with recurrent myoglobinuria. Deﬁciency of CPT and of the glycolytic and glycogenolytic enzymes is a much more common cause of this presentation than a mitochondrial problem. A small number of patients with respiratory chain cytopathies have presented with recurrent myoglobinuria in a setting of exercise-related muscle pain. An early report associated this syndrome with sporadic multiple mtDNA deletions (Ohno et al., 1991), but a more common cause is probably the recently described cytochrome b mutations (see above). Fixed weakness in such patients may be very mild and there is usually no extra ocular muscle involvement.

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C. CHRONIC PROGRESSIVE EXTERNAL OPHTHALMOPLEGIA SYNDROMES CPEO is by far the commonest mitochondrial syndrome diagnosed in neurology clinics. Most cases with CPEO, especially those with the multisystem KSS variant, have developed some evidence of ptosis by the teenage years. Although this may not be apparent until some years later, review of old photographs conﬁrms a relatively early onset. There are some cases that genuinely have an onset in middle life. Chronic progressive external ophthalmoplegia may begin very asymmetrically especially in late-onset cases, but typically affects both eyes simultaneously. When followed over many years, it takes a typical course with some involvement of all eye movement early in the course, and relentless progression to almost total ophthalmoplegia over 5–10 years. There is no particular early predilection for horizontal or vertical eye movements. Fatigability is not usually evident in either ptosis or eye movement, and there is usually little difﬁculty in differentiating this problem from the major diagnostic alternative, ocular myasthenia. Isolated progressive ptosis without external ophthalmoplegia is seen very rarely, and it is more typical of the extraocular involvement in the other main differential diagnosis of oculopharyngeal dystrophy. Ptosis progresses at the same rate as external ophthalmoplegia and most patients require ptosis surgery in their 20s. Some weakness of orbicularis oculi is found in almost all patients with CPEO, and severe weakness may be a contraindication to ptosis surgery. There is likely also to be mild weakness of neck ﬂexion and maybe some facial weakness. The patients are likely to have exercise intolerance, but proximal limb weakness may be very mild or absent until late in the piece (Petty et al., 1986).

D. KSS AND OTHER CPEO SYNDROMES Kearns-Sayre syndrome is the most easily recognizable mitochondrial syndrome and an archetype that has led to so many advances and understandings. As initially deﬁned, KSS is the development of ptosis and CPEO before 20 with retinitis pigmentosa and of raised CSF protein. This triad allowed reliable prediction of the later complete heart block, and suggested a need for either very careful cardiological monitoring or a prophylactic pacemaker. It is now recognized that cases with this typical phenotype can present a little later. The other phenotypic features include cerebellar ataxia, neuropathy, dementia, short stature, and nerve deafness. These are more variable, but affect most patients and one of the most striking things about the KSS syndrome is its great clinical uniformity.

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It is of interest that a great majority of patients with KSS have a large sporadic mtDNA deletion (Zeviani et al., 1988) whereas only about 50% of cases with non-KSS CPEO have a large deletion (Moraes et al., 1989). The particular predilection of the extraocular muscles for involvement in deletion-positive mitochondrial cytopathies is in contrast with the relative sparing of extraocular muscles in most tRNA related cytopathies, and remains unexplained. Other mitochondrial DNA abnormalities reported less commonly in CPEO include maternally inherited point mutations of mtDNA (Hattori et al., 1994; Seibel et al., 1994; Hammans et al., 1995). Duplications of mtDNA are found commonly in KSS and CPEO (Poulton et al., 1994), but when introduced into cell lines, duplications are not associated with ETC dysfunction and may be intermediates in the generation of deletions (Poulton et al., 1993). Chronic progressive external ophthalmoplegia may also show Mendelian inheritance, where the genetic hallmark is usually multiple mtDNA deletions (Hirano et al., 1994; Zeviani et al., 1989). These are clinically and genetically heterogeneous; and distinct clinical syndromes have been described (Cormier et al., 1991; Suomalainen et al., 1992; Hirano et al., 1994; Carrozzo et al., 1998). Recessive phenotypes include mitochondrial MNGIE (Hirano et al., 1994), autosomal recessive cardiomyopathy and ophthalmoplegia (ARCO) (Bohlega et al., 1996), optic atrophy, ptosis, myopathy and neuropathy (Mizusawa et al., 1988), or recurrent myoglobinuria (Ohno et al., 1991). MNGIE usually begins in childhood or adolescence, and it is characterized by severe gastrointestinal hypomotility with delayed gastric emptying and intestinal pseudo obstruction. Most patients also have deafness, neuropathy, and CPEO, often of mild degree. A striking feature is the wasted appearance of the patients. The autosomal-dominant form (adPEO) usually presents with adultonset progressive external ophthalmoplegia and myopathy (Suomalainen et al., 1992). Deafness and cataract are also common features of this syndrome. Depression was a prominent feature in one pedigree. Tremor and levodopa-responsive Parkinsonism has been reported in some pedigrees (Chalmers et al., 1996). In some patients a severe sensory ataxic axonal neuropathy is part of the syndrome (Fadic et al., 1997). The multiple mtDNA deletions are generally found in muscle, which also shows COX negative and ragged-red ﬁbers, but have also been described in other autopsied postmitotic tissues. In a unique case, the multiple deletions were found in lymphoblasts (Cormier et al., 1991). Autosomal-dominant PEO is genetically heterogenous. At least three loci exist (Suomalainen et al., 1995; Kaukonen et al., 1996, 1999). Quite recently,

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mutations in the heart/skeletal muscle form of the ANT1 gene have been found in one form (Kaukonen et al., 2000). Mutations in the thymidine phosphorylase gene have been discovered in MNGIE (Nishino et al., 1999). Thymidine phosphorylase activity was reduced in the patients, suggesting a loss of function and that the loss of maintenance of mtDNA might relate to disturbed thymidine metabolism, but the pathophysiology of the dominant and recessive forms is still unclear.

E. NEUROPATHY Isolated peripheral neuropathy is uncommon in mitochondrial disease, but more subtle degrees of neuropathy are often seen in multisystem mitochondrial disorders. This is rarely the dominant clinical feature. Subclinical neuropathy is evident in about a quarter of patients (Mizusawa et al., 1991; Chu et al., 1997). Patients with more severe neuropathies are much less common. Neuropathies are typically sensorimotor. Both distal axonopathy and segmental demyelination is recognized in different cases, with axonal breakdown being much more common. Striking ultrastructural abnormalities have been identiﬁed in Schwann cells in electromicrograph (EM) studies (Yiannikas et al., 1986). Mutant mtDNA is recognized in peripheral nerves as in other tissues. Sural nerve biopsies are seldom terribly helpful diagnostically.

F. ENCEPHALOMYOPATHIES 1. Myoclonus Epilepsy with Ragged-Red Fibers This is an important cause of myoclonus epilepsy. The myoclonus may be generalized or multifocal, and it may be associated with tonic-clonic seizures. A periodic course with exacerbations lasting weeks or months alternating with periods of relatively good control is typical. Most patients have some other features of a multisystem disorder especially a pancerebellar syndrome (often mild) and nerve deafness. In almost all cases, muscle biopsy reveals typical ragged-red and COX negative ﬁbers. Weakness is seldom prominent clinically. About 80% of patients have a point mutation in the tRNALys gene at position 8344 (Shoffner et al., 1990) (Most of the remaining patients have a point mutation at either position 8356 or 8363, again in the tRNALys (Ozawa et al., 1997; Silvestri et al., 1992). The 8344 mutation is associated with reduced synthesis of large molecular weight mitochondrial subunits (Noer et al., 1991).

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2. Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-Like Episodes Perhaps the most striking of all mtDNA-related syndromes is the MELAS syndrome. A young child with a history of severe migraine and usually nerve deafness presents somewhere between the age of three or four and 15 years of age with a devastating neurological illness evidenced by increasing drowsiness often with hemiparesis. It may be possible to demonstrate a hemianopia or the child may be blind. Magnetic resonance imaging (MRI) scan reveals marked unilateral or bilateral posterior cerebral edema extending beyond the posterior circulation. Neurosurgical decompression may be lifesaving. Typically the episode will settle with minimal clinical deﬁcit, although the child may be left with a hemianopia. Diagnosis is usually achieved during the acute episode either by mtDNA analysis on blood or by muscle biopsy. In spite of treatment, further episodes usually follow with increasing neurological deﬁcit often with cortical blindness and eventually with a fatal outcome. Less severe cases are now well recognized with patients remaining relatively well—well into adult life. Onset may occur in adulthood. Quite commonly, other features will be present in the patient or maternal family members such as migraine, diabetes, short stature, deafness, or seizures. The diabetes–deafness syndrome is a special case (van den Ouweland et al., 1994). The majority of patients with MELAS syndrome have a point mutation in the tRNALeu gene usually at the 3243 position (Goto et al., 1992). Other tRNA point mutations have also been documented (Nishino et al., 1996). In contrast to MERRF, mutations in MELAS have been identiﬁed in several other tRNA genes and in complex I genes (Lertrit et al., 1992; Pulkes et al., 1999). It is clear that in the MELAS syndrome there is a much wider array of clinical manifestations than in the MERRF syndrome. 3. Madelung Syndrome Multiple symmetrical lipomatosis is characterized by large lipomas around the neck and shoulder girdle. Some patients also have nerve deafness and a degree of neuropathy (Berkovic et al., 1991). This syndrome may be the only manifestation of the MERRF mutation A8344G (Holme et al., 1993), and has been also reported with single (Campos et al., 1996) and multiple deletions (Klopstock et al., 1994) of mtDNA. The MERRF mutation has been demonstrated in the association of photomyoclonus, ataxia, and symmetric lipomatosis (Ekbom’s syndrome) (Traff et al., 1995). 4. Severe Infantile Myopathies These are probably the most severe manifestations of mitochondrial disease with presentation in early infancy profound weakness hypotonia and progressive respiratory failure. The baby is found to have lactic acidosis.

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Cardiac involvement may be prominent. Most patients have a fatal outcome, but the DiMauro group recognized a benign form, and it is crucial to distinguish that from the fatal form for prognostic reasons. In the benign form, due to reversible COX deﬁciency, infants improve spontaneously within the ﬁrst year of life (DiMauro et al., 1981). Fatal infantile mitochondrial myopathy is associated with a range of mitochondrial deﬁciencies (Nagai et al., 1993; Tanaka et al., 1986; Hoppel et al., 1987), including deﬁciencies in all respiratory complexes. Differential diagnosis can be made by muscle immunohistochemistry: the fatal infantile myopathy is characterized by absence of the nuclear DNA-encoded subunit VIIa,b of COX, while in the benign myopathy both VIIa,b and the mtDNA-encoded subunit II are absent (Tritschler et al., 1991). Some cases of fatal infantile myopathy are associated with mitochondrial DNA depletion (Moraes et al., 1991). 5. Leigh-Type Encephalopathy Leigh syndrome/familial bilateral striatal necrosis is a progressive neurodegeneration particularly of infants, which appears to result from defective energy metabolism from a number of causes, including ETC chain disorders arising from nuclear and mtDNA mutations (DiMauro and DeVivo, 1996). A variety of mutations in the mtDNA including in tRNA genes (Howell et al., 1996; Koga et al., 2000a; Chalmers et al., 1997; Santorelli et al., 1997; Shtilbans et al., 2001), structural mtDNA genes, particularly the ATPase 6 gene (Tatuch et al., 1992; Thyagarajan et al., 1995; Makino et al., 2000; Carrozzo et al., 2001; Kirby et al., 2001; Wilson et al., 2001), and mtDNA deletions (Santorelli et al., 1996) cause LS/FBSN. Recessively inherited LS may be due to complex I deﬁciency (Rahman et al., 1996; Loeffen et al., 1998; Triepels et al., 1999), COX deﬁciency due to COX assembly gene mutations (Zhu et al., 1998; Tiranti et al., 1999; Sue et al., 2000), or complex II deﬁciency (Bourgeron et al., 1995; Makino et al., 2000). Recessively inherited LS may also be due to mutations in other genes important in mitochondrial energy metabolism, e.g., the E1 α subunit of pyruvate dehydrogenase (Matthews et al., 1993). Very rare cases of LS may present in adolescence and early adult life. VII. Biochemical Features

Exploration of biochemical phenotype was a key step in convincing the medical world of the reality of respiratory chain failure as a cause of human disease. The oxygen-sensitive electrode provided a ready means of testing the integrity of the respiratory chain with substrates that feed in at various sites. It led to a biochemical classiﬁcation of respiratory chain

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cytopathies, often supported by cytochrome oxidation/reduction spectra and respiratory complex assay in isolated mitochondria or whole tissue homogenate. In the 1970s and middle 1980s, the standard classiﬁcation of respiratory chain disorders rested on biochemical phenotype. The usefulness of the biochemical classiﬁcation for mtDNA mutation related respiratory inefﬁciencies is now less clear. Most mtDNA mutations affect subunit synthesis either through a mutation in a key tRNA gene or through deletion of a series of tRNA genes. tRNA mutations may, among other mechanisms, lead to inefﬁciencies in amino acid incorporation most marked in larger subunits (Noer et al., 1991). Deletion mutations do not allow synthesis of any subunits of any deleted tRNAs from the deleted molecule, although synthesis becomes possible by complementation by the wild-type molecule. The ratio of wild-type to mutant deleted molecules appears to determine the extent of ETC compromise.

A. COMPLEX I DEFICIENCY This is one of the most commonly identiﬁed respiratory chain phenotypes, related perhaps to the large number of subunits encoded by both the mitochondrial and the nuclear genomes, the size of complex, and perhaps in previous times due to technical difﬁculties in the assay of complex I function and interpretation of results. Interpretation difﬁculties are compounded by the presence of tissue-speciﬁc deﬁciencies (Kirby et al., 1999). Although respiratory chain assays performed in muscle and cultured ﬁbroblasts are generally comparable, assay in both tissues is recommended to help detect the possibility of a tissue-speciﬁc deﬁciency (Loeffen et al., 2000). Complex I deﬁciency in muscle is especially characteristic of the MELAS syndrome. Interestingly, in the relatively mild clinical phenotype of LHON, in which homoplasmic mutations are generally in mitochondrial-encoded complex I genes, the complex I deﬁciency and its effect on ATP synthesis is quite variable (Brown, 1999). In isolated complex I deﬁciency, without the presence of known mtDNA mutations, the phenotype is generally a Leigh-type encephalopathy, fatal infantile lactic acidosis, neonatal cardiomyopathy with lactic acidosis, macrocephaly with progressive leucodystrophy, and unspeciﬁed progressive or stable encephalomyopathies (Loeffen et al., 2000). In nuclear gene mutations affecting complex I function, the reported phenotypes are a Leigh-type encephalopathy, a fatal, progressive encephalopathy with lactic acidosis (van den Heuvel et al., 1998), or a leucodystrophy with myoclonus epilepsy (Schuelke et al., 1999). Complex I deﬁciency is often part of a combined deﬁciency. This can occur in a number of situations including mtDNA point mutations and

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deletions. In the absence of known mtDNA abnormalities, it may be associated with fatal infantile encephalomyopathy (Nagai et al., 1993). A particular area of difﬁculty has been interpreting the modest complex I deﬁciency consistently found in some patients with PD, in muscle, brain, and platelets (Schapira, 1999), and its relevance to the pathophysiology of the disease.

B. COMPLEX II DEFICIENCY As complex II is encoded entirely by the nucleus, preservation of complex II activity or even an increase in activity is a valuable clue for a mtDNA defect. A mutated ﬂavoprotein subunit of complex II, causing autosomal recessive LS, was the ﬁrst deﬁned nuclear gene abnormality in the ETC (Bourgeron et al., 1995).

C. COMPLEX IV DEFICIENCY Patchy complex IV deﬁciency detected histochemically usually accompanies of mtDNA mutations affecting protein synthesis. It is a robust spectrophotometric assay, and a high proportion of COX inactivity in muscle is accompanied by a fall in total muscle COX activity. Major falls in COX activity are characteristic of severe infantile mitochondrial diseases where a nuclear gene mutation in a key structural or regulatory gene leads to a very severe COX deﬁciency in all muscle ﬁbers. Examples are the benign and malignant forms of severe infantile myopathy (DiMauro et al., 1980, 1981) (see above). Generalized COX deﬁciency due to mutations in COX assembly genes present with LS in the case of SURF1 mutations and encephalopathy with hypertrophic cardiomyopathy in the case of mutations in SCO 2 (Sue et al., 2000).

D. CYTOCHROME b DEFICIENCY Isolated cytochrome b deﬁciency was ﬁrst reported in 1984 in a patient with lifelong ptosis and fatigable weakness (Hayes et al., 1984). Further cases were not commonly reported until somatic mutations, conﬁned to the muscle in the mitochondrial cytochrome b gene, were associated with cytochrome b deﬁciency and a clinical syndrome of exertional myalgia and lactic acidosis with or without myoglobinuria (Andreu et al., 1999).

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E. COMPLEX V DEFICIENCY There is an interesting relationship between the known mutations in the mitochondrial ATPase genes and the effect on ATP synthesis. Mutations have only been described in the ATPase 6 gene. Mutations at nucleotide (nt) 8993 and 9176 are common causes of LS/FBSN (Santorelli et al., 1993; Campos et al., 1997; Makino et al., 2000). At each of these positions, there exist two mutations, viz. T8993G, T8993C and T9176C and T9176G. The T8993G mutation causes a more severe defect of ATP synthesis than the T8993C mutation (Vazquez-Memije et al., 1998). Similarly, the T9176C mutation produces no detectable reduction in ATP synthesis in ﬁbroblasts carrying high load of the mutation (Thyagarajan et al., 1995), while the T9176G severely affects the ATP synthesis in skin ﬁbroblasts. Differences in the clinical phenotype, however, appear small. The T899G mutation appears to alter the stability and altered assembly of the enzyme complex, most likely due to changes in the properties of subunit a of the membrane sector part of the ATP synthase (Houstek et al., 1995).

F. COENZYME Q DEFICIENCY Ogasahara and colleagues ﬁrst identiﬁed muscle coenzyme Q deﬁciency in a familial mitochondrial encephalomyopathy (Ogasahara et al., 1989). Since then the syndrome of coenzyme Q deﬁciency appears to consist of various combinations of ataxia, seizures, mental retardation and proximal muscle weakness, pyramidal signs, and exertional fatigue with lactic acidosis. The syndrome is important to recognize as seizures may respond to administration of coenzyme Q10 (Musumeci et al., 2001).

VIII. Diagnostic Approaches

A. INITIAL APPROACH What does the laboratory need from the referring clinician? What does the clinician need from the laboratory? Mitochondrial diagnosis is a complicated area with many clinical nuances, and a different diagnostic approach may be appropriate in different clinical situations. It is necessary for a close rapport between expert clinician and expert laboratory to optimize diagnostic yield. The clinician must know what test to order, what tissues to test, and what the results mean.

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For the clinician:

r Identify the syndrome you think the patient has or may have. Do you know the tissues that are affected and the best route to conﬁrm a diagnosis? r Deﬁne the clinical syndrome as clearly as possible, e.g., does this look like a well-deﬁned encephalomyopathy, for example CPEO, KSS, MELAS, MERRF, NARP, or LS/FBSN? r Is there any family history to support a particular inheritance pattern? Maternal inheritance suggests a mtDNA problem, while Mendelian inheritance a nDNA problem. Often the family history is not helpful. The penetrance of mtDNA mutations may be low or low levels of heteroplasmy may be asymptomatic. The signiﬁcance of oligosymptomatic relatives may not be clear in interpretation of the family history. r Do you suspect a mtDNA mutation? If so, is this likely to be manifest in blood or require a tissue biopsy (usually skeletal muscle) to conﬁrm the diagnosis. This varies from syndrome to syndrome. In many of the point mutation related disorders (LHON, MERRF, MELAS), the mutation is detectable at low levels in most tissues including those obtainable noninvasively such as hair follicles or blood (Kotsimbos et al., 1994). Only a minority of CPEO syndromes is associated with mtDNA point mutations. In most sporadic CPEO and almost all KSS cases, large heteroplasmic deletion can be identiﬁed. Such mutations are seldom identiﬁed in blood and generally require analysis of muscle tissue obtained by needle or open biopsy. r How likely is the diagnosis of mitochondrial disease clinically? One of the diagnostic problems the clinician faces in mitochondrial medicine is that the clinical symptoms are protean. The likelihood of a mitochondrial diagnosis being conﬁrmed is high where there is a typical multisystem syndrome such as MELAS or MERFF. With oligosymptomatic presentations, nonneurological presentations, or atypical multisystem CNS disorders, a mitochondrial cytopathy may be part of a long differential diagnosis. How far should diagnosis be pursued in the mitochondrial area depends very much on the degree of suspicion the clinician has. Most laboratories screen for only the common few of the pathogenic point mutations. To take matters forward, it is necessary for the physician to reappraise the likelihood of a mitochondrial cytopathy after investigations for other diagnoses are available, and if the diagnosis is still unclear, a major mitochondrial workup may well be appropriate. r What does a major mitochondrial workup involve? Diagnosis of a mitochondrial cytopathy, especially a new, one is usually established by

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pulling together information from various diagnostic areas. This is discussed in the next section. For the laboratory scientist: The diagnosis of mitochondrial cytopathies is very much a two-way effort requiring good communication between the laboratory scientist and the clinician. Laboratory scientists may be in a difﬁcult position in this area if this communication is not maintained. A blood specimen or blood-derived mtDNA specimen may be sent to the laboratory and the request form may read “query mitochondrial cytopathy for mtDNA testing.” What diagnosis does the physician suspect? Is the specimen sent and are the tests requested an appropriate approach to conﬁrm or refute that diagnosis? This is difﬁcult to ascertain without a full clinical picture. Several options are available to the diagnostic laboratory. It may be reasonable to screen for major recognized point mutations (and deletions if muscle tissue is provided), and pass the information on to the referring physician with the comment that a cytopathy has not been excluded. A more useful (although not always possible) approach is to contact the referring physician and further evaluate the physician’s diagnostic concerns. Is MELAS, MERRF, or another syndrome the concern? Does the patient have an obscure multisystem disorder, and is the possibility of a mitochondrial cytopathy part of a wide diagnostic search? It may be appropriate to discuss with the physician the possibility of obtaining a muscle biopsy for more extensive histochemical, biochemical, and DNA tests. It is probably inappropriate to offer a full genome screen unless there is some direct supportive evidence that the patient has a mitochondrial problem. As well as providing a better diagnostic service, laboratories that establish a close rapport with referring physicians are more likely to contribute to the evolving knowledge in this ﬁeld by identifying new mitochondrial cytopathies. B. MITOCHONDRIAL DISEASE WORKUP 1. Overview of Clinical Information Is the clinical syndrome consistent with a mitochondrial cytopathy? This may be difﬁcult as mitochondrial medicine now has such a broad scope. Is there any suggestion of a family history (especially useful if maternal transmission looks likely)? 2. Lactate Measurement Where skeletal muscle pathology is prominent, there may be an excessive serum lactate response with light exercise. A resting serum lactate level is

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not terribly useful, and aerobic exercise testing requires a laboratory with special expertise and good control values. This type of testing was very useful in the early days of mitochondrial medicine, but is now seldom necessary. Cerebrospinal ﬂuid (CSF) lactate and pyruvate analysis with calculation of a ratio is a useful test in obscure encephalopathies where a mitochondrial basis is suspected. It requires prior arrangement with the laboratory for assay and rapid transport of CSF specimen to the laboratory on ice. 3. Ancillary Investigations Demonstration of multisystem features typical of mitochondrial disease may support the diagnosis, i.e., retinitis pigmentosa, deafness, diabetes, cardiac conduction problems, among others. 4. Other Neurological Investigations CSF protein may be elevating in KSS. Neuroradiological features of MELAS are well deﬁned with nonvascular territory, posterior emphasis stroke-like areas, early and later cerebellar atrophy, and basal ganglia calciﬁcation. Diffuse white matter problems may be evident in KSS or MERRF. Symmetric T2 hyperintense lesions in the deep gray matter on MRI characterize Leigh syndrome. 5. Muscle Biopsy Which muscle should be biopsied? Even when clinical involvement is largely conﬁned to the extraocular muscles, skeletal muscle involvement can usually be detected in any proximal limb muscle. A needle biopsy is conveniently taken from vastus lateralis and is to be preferred in laboratories comfortable with processing needle biopsy cores. With a large Bergstrom biopsy needle, 200 mg of muscle can be obtained in three passages, and morbidity and discomfort are negligible. Open biopsies can be taken from a wider range of muscles including deltoid, biceps and triceps in the upper limb, and quadriceps in the leg. Open biopsy may be indicated where the examiner ﬁnds focal weakness involving a particular muscle group. What muscle specimens to prepare? A muscle biopsy for full mitochondrial workup is typically the central aspect of the patient’s investigation, and it is important that the procedural physician or surgeon ensures that arrangements have been made beforehand for all relevant tests. This usually involves the carrying out of the procedure in a tertiary center. Standard tests that should all be requested include the following: 1. Histochemistry. A fresh core of about 80 mg in size is placed in a sealed jar and transferred immediately to the laboratory on ice (not dry ice). At the laboratory, the core is frozen in liquid nitrogen

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precooled isopentane and transferred to a −70◦ freezer prior to section in a cryostat. 2. Electron Microscopy. Half a needle biopsy core (approximately 35 mg of tissue) is placed in gluteraldehyde for EM. With open biopsy, the EM specimen should be ﬁxed in clamps prior to section and transfer to gluteraldehyde. 3. Biochemistry. A fresh specimen is taken for biochemistry (one core/70 mg). Again, this is placed in a sealed jar and transferred on ice to the laboratory. 4. Molecular Genetics. A specimen for mitochondrial DNA studies is also taken (half a needle biopsy core/35 mg). In most centers, these investigations are carried out at different laboratories, and the tragedy of lost specimens will be avoided if one laboratory, usually the histochemistry laboratory, is responsible for further distribution of specimens. This also ensures that the available material is used optimally. Occasionally, for unavoidable technical reasons, the amount of material available may be reduced, and it may be necessary for the laboratory to confer with the clinician in order to attach priority to the tests. 6. Histochemistry This has been the mainstay of diagnosis in mitochondrial medicine since the wide popularization of the modiﬁed Gomori trichome stain. Reliable enzyme histochemistry, especially the Seligman cytochrome oxidase method, further reﬁned diagnosis. All muscle biopsies will be subject to a range of histochemical analysis including Sudan black or other lipid stain, trichrome reaction, NADH tetrazolium reductase, succinate dehydrogenase, cytochrome oxidase, and ATPase reactions. There may be a mild excessive lipid in some cases as the only abnormality—PDH deﬁciency, for example. Typically ragged-red ﬁbers are often identiﬁed in mtDNA mutations affecting protein synthesis but are likely to absent in point mutations of structural genes. The percentage of ragged-red ﬁber ranges from only 4 or 5 ﬁbers in a biopsy to 30–40% of all ﬁbers. A small number of ragged-red ﬁbers may lack speciﬁcity as they may accumulate with aging. In a younger patient, even a very small number of ragged-red ﬁbers raise the possibility of a mitochondrial cytopathy, but this ﬁnding assumes greater signiﬁcance if supported by other abnormalities suggesting mitochondrial dysfunction in the histochemical examination. Mitochondrial aggregates are readily seen with NADH tetrazolium reductase and succinate dehydrogenase reactions, and many more abnormal ﬁbers may be evident with these techniques than with the Gomori trichrome reaction. The single most useful histochemical reaction is undoubtedly the cytochrome oxidase stain. The COX-negative

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ﬁbers accumulate from the age of about 40, and very small numbers of COX-negative ﬁbers require cautious interpretation. The key feature histochemically in most mtDNA-related cytopathies is the presence of a subpopulation of COX-negative ﬁbers more prevalent than expected in a patient of that age. The number of COX-negative ﬁbers usually greatly exceeds the number of ragged-red ﬁbers. Less commonly, as in some limb myopathies with cytochrome b deﬁciency or rarely in MELAS cases, ﬁbers with increased COX activity may be prominent. Infantile myopathies with selective COX deﬁciency are characterized by a total lack of activity histochemically in all ﬁbers in the biopsy. In such cases a nuclear rather than a mitochondrial genetic disorder is usually responsible. The COX stain is so much more useful than other histochemical reactions because it is probably a much cleaner respiratory chain probe. NADH tetrazolium reductase, for example, has some activity in the outer mitochondrial membrane. Double staining with succinate dehydrogenase and COX offers an elegant way of conﬁrming that mitochondria exist in COXnegative ﬁbers. Unequivocal histochemical abnormalities mean the patient does indeed have a mitochondrial disease. Furthermore, the demonstration of energy mosaicism on histochemistry with patchy ragged-red and COXnegative ﬁbers suggest that the patient has a mtDNA mutation affecting mitochondrial protein synthesis. A global histochemical complex deﬁciency may point to a nuclear DNA mutation. Cases are well recognized where histochemistry is not diagnostic but where other studies such as respiratory complex assay or mtDNA studies have conﬁrmed the diagnosis of mitochondrial disease. Leber’s Hereditary Optic Atrophy and other point mutations affecting structural genes are classic examples, and it is important to realize that negative histochemical investigations do not rule out a mitochondrial disorder. 7. Electron Microscopy Electron microscopic studies played a pivotal role in establishing the concept of mitochondrial disease. Demonstration of a large excess of mitochondria or of large mitochondria with bizarrely orientated cristae, often with striking paracrystalline inclusions were salient features in early publications. Interpretation problems may arise with EM in routine diagnostic practice. Low levels of ultrastructurally abnormal mitochondria are found in many muscle biopsies, and they lack speciﬁcity. EM abnormalities almost always abound only in cases where the technically easier and much more rapidly available histochemical techniques have already conﬁrmed a mitochondrial process. In practice, EM is not employed uniformly by all laboratories. Where available, it provides an elegant means of supporting the histochemical diagnosis. Great care must be taken in the interpretation

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of more subtle EM abnormalities especially where histochemical studies have been negative because overinterpretation can lead to diagnostic error. 8. Biochemical Studies Major advances in the understanding of mitochondrial disease followed the application of biochemical techniques, familiar in the undergraduate laboratory, to human muscle biopsies. Skilled application of the difﬁcult technique of Warburg manometry to isolated intact muscle mitochondria led to the identiﬁcation of the ﬁrst mitochondrial disease and the successor of Warburg manometry, the oxygen-sensitive electrode, was used to delineate a range of focal respiratory chain blocks. Although still very useful in pinpointing respiratory chain problems, polarographic techniques are now mainly of research rather than everyday clinical use. The very large amount of material needed in earlier clinical studies (3–4 g of skeletal muscle) has now been greatly reduced by miniaturization of both the electrode and mitochondrial extraction laboratory equipment. Cybrid experiments require great expertise and are conﬁned to major research centres. Spectrophotometric respiratory complex assay, on the other hand, requires very small amounts of test material and has replaced polarographic study as the routine biochemical test. Fresh (or frozen) biopsy material is essential for these studies, and skeletal muscle is the tissue of choice. The demonstration of a signiﬁcant deﬁciency in a key respiratory complex usually indicates a primary mitochondrial cytopathy, but care must be taken in the following areas. Secondary deﬁciencies may occur with other disease processes, and the respiratory chain ﬁndings must be interpreted in light of the patient’s overall picture. For example, a muscle that is necrotic or that has extensive ﬁbrotic or fatty replacement may have low respiratory chain activity. Not all patients with unequivocal mitochondrial problems have respiratory chain deﬁciencies in routine biochemical studies. In KSS, for example, where both histochemical and mtDNA studies give evidence of a clear cytopathy, routine complex assays may be normal. This relates to a compensatory enlargement of the mitochondrial mass and also to the fact that respiratory chain failure is not evenly distributed through the muscle. Thus, in some cases histochemistry is a more precise probe for mitochondrial dysfunction than whole tissue biochemical studies. Mitochondrial respiratory assays have suffered from problems of artifact problems or inadequate control data in the past. Complex assays should be carried out in reference laboratories with control ranges established locally. Other investigations including cytochrome oxidase reduction spectra and spin resonance for delineation of iron–sulfur protein content are specialized techniques that in general require larger amounts of biopsy

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material. These are research rather than everyday techniques in routine clinical diagnostic practice. 9. Mitochondrial DNA Analysis This is a central diagnostic area. In a characteristic syndrome, ﬁnding a disease causating mutation is a fast track to conﬁrmation of diagnosis, and no testing, other than on blood, be needed. Where the phenotype is less characteristic, and other tests (i.e., those above) support a mitochondrial problem, a detailed mitochondrial DNA investigation is appropriate. This includes a screen for recognized pathogenic mutations on available tissues (ideally a ﬁxed postmitotic tissue such as skeletal muscle). Large deletion mutations are usually only evident in skeletal muscle, and the appropriate investigation is a Southern blot. Where no mutation is found, a case conference should be held between laboratory and clinical team. If a unique cytopathy is suspected, it is appropriate to carry out a whole genome screen either by sequencing or by a validated screening protocol. This is expensive and time-consuming, and should only be done where other investigations strongly support the presence of a mitochondrial cytopathy. 10. Signiﬁcance of Novel Mutations What makes a mutation likely to be pathogenic and what is the role of cybrid experiments? Most non-pathogenic population polymorphisms have now been identiﬁed and are available in standard databanks. Proposed criteria for pathogenicity are summarized in Table IV. Another way of deﬁnitively establishing a mutation as pathogenic is to carry out a cybrid transfer (King and Attardi, 1989) and to conﬁrm that the putative mutation has a signiﬁcant respiratory chain phenotype.

TABLE IV CRITERIA FOR PATHOGENICITY OF A NOVEL MUTATION Detection of the mutant mtDNA in affected individuals Non detection in unaffected individuals, including normal individuals from diverse ethnic backgrounds. Identiﬁcation of the mutation in at least two unrelated families with a similar or identical clinical phenotype. Heteroplasmy in the proband or relatives Point mutations should either be located in a peptide coding reading frame and alter an amino acid (missense) or alter a tRNA or rRNA residue in a conserved region. Any deletion or duplication. OR Conﬁrmation in by cybrid transfer of a major biochemical respiratory chain phenotype.

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C. SUMMARY In typical cases, mitochondrial disease diagnosis is very straightforward. It may simply involve mitochondrial DNA testing on a blood sample. In complex cases or unusual cases, diagnosis is probably one of the most demanding areas in modern medical practice and requires a close collaboration between the clinical and laboratory team, interpreting results from different diagnostic areas.

IX. Treatment of Mitochondrial Disorders

Despite the rapid recent advances in the understanding of mitochondrial disorders, advances in therapy have been slow. Therapies for mitochondrial disease are pharmacologic and non-pharmacologic (summarized in Table V). Because mitochondrial diseases are comparatively uncommon, are genetically and clinically heterogeneous, vary so greatly in severity and course and because we lack validated clinical outcome measures, there are no good randomized, double-blind controlled treatment trials. Thus, pharmacologic therapy is very difﬁcult to evaluate. Most reports of success are based on single or few patients. Where randomized trials have been done, the treated group has been mixed. 31P magnetic resonance spectroscopy of brain or muscle (Barbiroli et al., 1997a,b, 1999) or noninvasive tissue oximetry during exercise (Abe et al., 1999) may be useful in vivo biochemical end points, but do not necessarily inform us on clinical effects.

A. PHYSICAL AND SUPPORTIVE THERAPIES General principles of neurological care apply to patients with mitochondrial disease. 1. Exercise Short-term aerobic training, consisting of eight weeks of treadmill exercise at 70–85% of estimated maximum heart rate reserve-showed improvements in estimated aerobic capacity, heart rate, and blood lactate, and 31P nuclear magnetic resonance (NMR) spectroscopy showed increased oxidative capacity of muscle in patients with mitochondrial myopathies compared with normals and nonmetabolic myopathy disease controls (Taivassalo et al., 1998, 1999). Concentric exercise training may also result in “gene shifting” from satellite cells to mature myoﬁbers (see below) (Taivassalo et al., 1999b).

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TABLE V TREATMENT OF MITOCHONDRIAL DISEASE Intervention

Type

Examples

Nonpharmacologic Aerobic exercise Pacemaker insertion Pharmacologic Quinones Vitamins

Corticosteroids Miscellaneous

Gene therapy (presently conceptual)

Ubiquinone (CoQ10) Idebenone Menadione (vitamin K3) Phylloquinone/phytonadione (vitamin K1) Thiamine Riboﬂavin Dichloroacetate Carnitine Succinate Creatine Chloramphenicol

Genetic complementation

Protein-DNA chimera

Protein complementation Sequence-speciﬁc inhibition Other

Recoded mitochondrial genes Peptide nucleic acids Induced muscle regeneration Preimplantation selection Forced paternal inheritance

2. Offending Agents—Drugs and Anaesthetic Agents Anticonvulsant therapy in patients with seizures should be modiﬁed because the deleterious effects of valproic acid (VPA) on mitochondrial energy metabolism. Valproic acid decreases plasma carnitine levels, which may inhibit oxidation of fatty acids (Ohtani et al., 1982), and it impairs pyruvate uptake by brain mitochondria (Benavides et al., 1982), pyruvate oxidation in hepatocytes (Turnbull et al., 1983), and ETC (Haas et al., 1981). The rare coma that may result from VPA intoxication may be treated by direct hemoperfusion (Matsumoto et al., 1997). Infections should be vigorously treated, but certain antibiotics (e.g., aminoglycosides) may impair mitochondrial protein synthesis, particularly those acting on the mitochondrial rRNA, which is similar to the prokaryotic rRNA (Prezant et al., 1993; Thyagarajan et al., 2000). Gentamicin is contraindicated in deafness due to the A1555G mutation.

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Most antiviral agents inhibit mitochondrial DNA polymerase. Azidothymidine may cause a mitochondrial myopathy associated with carnitine deﬁciency and mtDNA depletion, and ﬁaluridine can cause a fatal hepatocerebral syndrome (Lewis and Dalakas, 1995). Acylovir has no reported mitochondrial toxicity. Patients with KSS may suffer anesthesiological complications because of sensitivity to the muscle relaxants, etomidate and thiopentone. Sudden third-degree AV conduction block may occur in the absence of an artiﬁcial pacemaker and lead to death, particularly with halothane anaesthesia; isoﬂurane is preferable (Lauwers et al., 1994). Depressed ventilatory drive and impaired responses to hypercapnia and hypoxemia may complicate the course in a ventilated patient (Barohn et al., 1990). In KSS, pacemaker insertion should be considered early to prevent fatality from cardiac conduction block that is ultimately almost invariable. Successful cardiac transplantation has been reported in KSS (Tranchant et al., 1993). 3. Counseling Prenatal diagnosis and genetic counseling is problematic in mitochondrial genetic disorders. This follows the genetic principles outlined above. In the NARP 8993 mutation, there is some relationship between the mutant load in the mother and the risk of an affected offspring (White et al., 1999a). A similar relationship exists in the MERRF 8344 mutation. Although there is only limited information on the predictive capability of chorionic villous sampling (CVS), it may be used in asymptomatic women with relatively low mutant load of these mutations since the available data indicate that embryonic and extraembryonic tissues bear similar mutant loads (White et al., 1999b). These women should be prepared to consider termination of pregnancy. Women with high mutant loads may wish to consider oocyte donation. Preimplantation diagnosis not routine although it is used routinely in other nuclear genetic disorders (Harper and Wells, 1999). B. METABOLIC THERAPIES 1. CoQ10 and Other Quinones Coenzyme Q10 has been the most fully evaluated. In rats, exogenous CoQ10 accumulates in the inner mitochondrial membrane and promotes mitochondrial enzyme activity (Nakamura et al., 1980). However, in two humans with mitochondrial myopathies, it failed to accumulate in muscle though serum levels increased (Zierz et al., 1990). It diffuses in the mitochondrial membrane bilayer independently of other redox components,

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and if administered orally, it is readily absorbed with a plasma half-life of 33.9 h. Normal serum levels are 637 ± 84 ng/mL, but may be inﬂuenced by gender, alcohol, serum triglycerides, and exercise ingestion of statins. There are no important adverse effects even when used in doses of 100 mg/day for up to six years (Ogasahara et al., 1986; Nishikawa et al., 1989; Bresolin et al., 1990; Kaikkonen et al., 1999; Overvad et al., 1999). Quinones may act by 1. providing a path for electrons to bypass defective ETC complexes and sustain the H+ gradient, 2. functioning as antioxidants (Beyer and Ernester, 1990), and 3. stabilizing components of the respiratory chain. In several case reports, CoQ10 has been extremely beneﬁcial for muscle and brain symptoms in familial, probably autosomal, recessive deﬁciency of CoQ10. In collected reports of patients with mitochondrial encephalomyopathies, a beneﬁcial effect of CoQ10 has been reported (Ogasahara et al., 1985, 1986, 1989; Goda et al., 1987; Ihara et al., 1989; Nishikawa et al., 1989; Shoffner et al., 1989a; Zierz et al., 1989; Bendahan et al., 1992; Arpa et al., 1994; Hirano et al., 1996; Servidei et al., 1996; Barbiroli et al., 1997, 1999; Liou et al., 2000). In KSS, doses of 3 mg/kg/day and 60–150 mg/day lowered serum lactate (Ogasahara et al., 1985, 1986; Bresolin et al., 1988) and improved eye movements (Ogasahara et al., 1985, 1986), and cardiac parameters (Ogasahara et al., 1986). In doses of 30–90 and 300 mg/day, CoQ10 monotherapy was reported to improve some parameters in MELAS, including pancreatic cell dysfunction (Liou et al., 2000), tissue oximetry (Abe et al., 1999) and serum lactate (Yamamoto et al., 1987; Abe et al., 1999), others (Ihara et al., 1989). Single patients with COX deﬁciency improved in strength (Yamamoto et al., 1987; Arpa et al., 1994) and 31P-NMR spectroscopic ﬁndings (Nishikawa et al., 1989). However, beneﬁts were not conﬁrmed in other reports of KSS (Zierz et al., 1990; Tranchant et al., 1993) and in 44 patients with various mitochondrial encephalomyopathies (Bresolin et al., 1990), in a double-blind multicentre trial. In another open trial, using CoQ10 and a vitamin cocktail, there were no objective, reproducible clinical beneﬁts or changes in oxidative metabolism in 16 patients with various mitochondrial encephalomyopathies despite a substantial increase in serum CoQ10 (Matthews et al., 1993a). In an open trial in 8 patients with various mitochondrial encephalomyopathies, 31P NMR showed improved mean postexercise ratio of phosphocreatine (PCr) to inorganic phosphate, but this was the effect of a single responder (Gold et al., 1996). Idebenone is a benzoquinone derivative that has been studied only in occasional cases of MELAS (Ihara et al., 1989; Ikejiri et al., 1996).

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2. Menadione: (Vitamin K 3), Phylloquinone (Vitamin K1), and Ascorbate In a patient with complex III deﬁciency (see Fig. 1), Eleff et al. reasoned they could bypass the ETC block by using menadione (40–80 mg/day) and ascorbate (4 g/day) as electron acceptors and reducers of cytochrome c oxidase (Eleff et al., 1984). 31P NMR showed an increase in the PCr/Pi ratio at rest and an increase in its rate of recovery after exercise (Eleff et al., 1984; Argov et al., 1986). Withdrawal of vitamin K resulted in increased fatigue and weakness, which improved with in 24 h of recommencing therapy (Argov et al., 1986). Shoffner et al. (1989a) used phylloquinone (25 mg/day) and ascorbate (4 g/day) to produce a 62% improvement in retinal cone function in a single patient with CPEO. Addition of CoQ10 produced an additional 46% improvement. Phylloquinone may be preferred to menadione: it is lipophilic, while menadione must ﬁrst be alkylated to be lipophilic and biologically active; it is concentrated more in mitochondria; and there are no reported side effects, while menadione has been reported to produce hemolytic anemia and hyperbilirubinemia, and kernicterus in newborns. A larger trial of 16 patients in which ascorbate and menadione were used in combination with CoQ10 and other vitamins in patients with assorted mitochondrial disorders showed no beneﬁt (Matthews et al., 1993). 3. Thiamine (Vitamin B1) and Riboﬂavin (Vitamin B2) Thiamine pyrophosphate is a coenzyme for pyruvate decarboxylase. The rationale for its use is that it may lower pyruvate and lactate levels, and stimulate NADH production, making more reducing equivalents available for the ETC via complex I. In large doses of 300 mg/day, thiamine reduced lactate and pyruvate levels in 3 patients with KSS, but failed to produce important clinical beneﬁt (Lou, 1981). One patient with myopathy, lactic acidosis, cardiomyopathy, and cardiac failure responded to thiamine and prednisone (Mastaglia et al., 1980). Thiamine was part of a cocktail containing ﬂavin mononucleotide and intravenous cytochrome c, which produced clinical improvement in muscle fatigability, and severity of stroke-like episodes in eight of nine patients with mitochondrial encephalomyopathies, four of whom had MELAS (Tanaka et al., 1997). However, a larger study, in which thiamine (100 mg/day for two months) was part of a vitamin and CoQ10 cocktail, showed no beneﬁt in 16 patients with different mitochondrial encephalomyopathies (Matthews et al., 1993). High doses are well tolerated except in occasional cases of hypersensitivity. Riboﬂavin is a precursor of the electron transport cofactors ﬂavin monophosphate (complex I) and ﬂavin adenine dinucleotide (complex II). At a dose of 100 mg/day, it improved exercise capacity in a patient with complex I deﬁciency (Arts et al., 1983). Penn et al. (1992) noted that in

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a patient with complex I deﬁciency and a known tRNALeu mutation, encephalopathy ceased with nicotinamide and riboﬂavin treatment. Phosphocreatine (PCr)/ATP recovery rates fell in parallel with sural nerve sensory amplitudes. A sustained clinical response was noted in an infant with complex I deﬁciency and a myopathy (Ogle et al., 1997). In a larger trial of riboﬂavin in 6 patients with complex I deﬁciency with encephalomyopathy and pure myopathy, the 2 patients with pure myopathy improved clinically, but only 1 of the patients with an encephalomyopathy improved, and there was no good correlation between clinical response and normalization of complex I activity (Bernsen et al., 1993). 4. Steroids Low doses of glucocorticoids have improved muscle strength, lowered lactate levels and improved other clinical features in case reports of mitochondrial encephalomyopathies (Shapira et al., 1975; Mastaglia et al., 1980; Montagna et al., 1988; Gubbay et al., 1989). However, steroids should be used with caution. Methylprednisolone inhibits the oxidation of NAD-linked substrates between the primary NADH dehydrogenase ﬂavoprotein and coenzyme Q, and inhibits succinate oxidation in vitro, suggesting that any therapeutic effects mitochondrial disease result from indirect rather than direct effects on the mitochondrial membrane. Furthermore, there is one report of fatal ketoacidosis and hyperglycaemia in 2 patients with KSS who received a brief course of corticosteroids (Curless et al., 1986).

C. MISCELLANEOUS 1. Dichloroacetate (DC A) In mitochondrial encephalomyopathies, high intracerebral lactate levels, evident on magnetic resonance spectroscopy (MRS), may contribute to neuronal death. Dichloroacetate, which stimulates conversion of lactate to CO2 and acetyl-CoA, has been used to lower lactic acidemia in adult and congenital lactic acidosis (Stacpoole et al., 1988), although a controlled trial has shown the effect is clinically insigniﬁcant and does not lengthen survival (Stacpoole et al., 1992). In a patient with MELAS and a stroke-like episode, who clinically improved with DCA treatment, an elevated lactate–creatine ratio in the “stroke” region decreased on MRS studies with improvement. During a second episode, the lactate–creatine ratio rose from baseline in a region of the brain that was normal on magnetic resonance imaging (MRI) scans (Pavlakis et al., 1998). In other anecdotal reports of MELAS treated with DCA alone (Saijo et al., 1991; Saitoh et al., 1998), or in combination

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with thiamine (Kuroda et al., 1997), there have been clinical improvements in addition to reduction of lactic acidemia. Similar observations have been made in Pearson’s marrow–pancreas syndrome (Seneca et al., 1997), muscle COX deﬁciency (Burlina et al., 1993), and other disorders of the ETC (North et al., 1996; Tulinius et al., 1989). The only randomized, doubleblind study of DCA in mitochondrial encephalomyopathies was a small, short-term, placebo-controlled, crossover trial in 11 patients with various mitochondrial disorders in whom blood lactate and several indices of brain oxidative metabolism on proton MRS improved after one week, but 31P-NMR spectroscopy, clinical symptoms, the neurologic examination, and quantitative muscle strength testing did not change (De Stefano et al., 1995). 2. Carnitine Carnitine deﬁciency may be found in the muscle of a third and in the plasma of half of patients with mitochondrial myopathies (Campos et al., 1993a,b). Evidence suggests that an increased NADH/NAD+ ratio generated by reduced ﬂux through the respiratory chain inhibits oxidation, producing secondary carnitine deﬁciency (Infante and Huszagh, 2000). In an open trial of L-carnitine (50–200 mg/kg/day in four daily doses) in patients with “mitochondrial myopathy” and plasma carnitine deﬁciency, muscle weakness improved in 19 of 20 patients, failure to thrive in 4 of 8, encephalopathy in 1 of 9, and cardiomyopathy in 8 of 8 patients (Campos et al., 1993b). There are similar anecdotal reports in the literature, but there are no placebo-controlled, randomized trials. 3. Succinate Succinate is a (TCA) cycle intermediate that donates electrons directly to the ETC (Fig. 1). Treatment of a single patient with complex I deﬁciency with 6 g/day resulted in disappearance of stroke-like episodes (Kobayashi et al., 1987), and respiratory failure resolved in a patient with combined deﬁciency of complexes I, IV, and V on a regimen of 300 mg/day of CoQ10 and 6 g/day of succinate (Shoffner et al., 1989b). 4. Creatine Another treatment strategy is increasing ﬂux through non-mitochondrial energy pathways. Increasing ﬂux through glycogenolysis/glycolysis may be expected to increase lactic acidemia, but ATP may be regenerated from PCr using creatine without increasing lactic acid production. This has been exploited in a short-term, randomized, double-blind, crossover trial in 7 patients, 6 with MELAS (Tamopolsky et al., 1997) of 5 g, reducing to 2 g, b.d. creatine monophosphate. A variety of strength measurements were

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used as end points, and the trial indicated an improvement in strength in high-intensity anaerobic and aerobic activities, but no effect in low intensity aerobic activities. The treatment may be useful in weaning the fatigued patient from a ventilator. 5. Chloramphenicol In Luft’s disease, the hypermetabolism has been reduced by Lugol’s iodine and methylthiouracil (Luft et al., 1962) and by inhibition of mitochondrial protein synthesis by chloramphenicol (DiMauro et al., 1976).

D. GENE THERAPY Gene therapy for mitochondrial disorders is in its infancy. A major obstacle to all the somatic gene therapy approaches is the delivery of a therapeutic gene into the mitochondrial matrix in cells throughout the body, including the brain. Some potential strategies have been reviewed (Taylor et al., 1997b). Prospects for somatic gene therapy include the following: 1. Genetic Complementation by Delivered Genes Expressed in the Mitochondrion Heteroplasmic mtDNA mutations are functionally recessive. There is a “threshold effect” in which a proportion of mutant mtDNA is required before the mutation has biochemical and clinical consequences. By intramitochondrial genetic complementation, even a small reduction in mutant DNA may correct the ETC defect. Conceptually, transport of other small nucleic acid species across both mitochondrial membranes known in some species, e.g., tRNAs in yeast (Entelis et al., 1998) and 5S rRNA in mammalian cells (Magalhaes et al., 1996) may be exploited for gene delivery to the mitochondrial matrix, but this has yet to shown in mammalian cells. 2. Protein Complementation by Recoded Mitochondrial Genes Expressed in the Cytoplasm Another approach, pioneered by Nagley et al. in yeast (1988), has been to insert a recoded, corrected copy of the defective mitochondrial gene coupled to a leader sequence in the nucleus, and express it in the cytosol. The recoded gene product is targeted to mitochondria by the attached protein import sequence. The cytosolically synthesized protein was correctly imported into mitochondria and functionally assembled into the ATPase complex; phenotypic rescue occurred. A similar approach designed to correct the homologous human ATPase 6 gene mutation has been tried using mouse mtDNA and the N-terminal leader sequence for the Fp subunit of succinate dehydrogenase (Sutherland et al., 1994, 1995). However, when

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the recoded gene-targeting sequence was expressed in NIH3T3 ﬁbroblasts, the construct was found to be toxic to the host cells. 3. Sequence-Speciﬁc Inhibition of Mutant mtDNA Replication Yet another approach has been taken by Taylor et al. (Taylor et al., 1997a; Chinnery et al., 1999) to selectively inhibit replication of the mutant mtDNA using sequence-speciﬁc peptide nucleic acid (PNAs) complementary to the mutant sequence of a mtDNA base change or deletion breakpoint. The antigenomic PNAs speciﬁcally inhibited replication of mutant but not wildtype mtDNA templates in an in vitro replication runoff assay, and the PNAs were taken up into cultured human myoblasts. 4. Other Genetically Based Therapies (a) Induced Muscle Regeneration. In injured muscle, satellite cells, the myogenic precursor cells are activated and proliferate to form new muscle ﬁbers. Because there is varied mutant load in different tissues due to segregation of mutant mtDNA molecules during embryogenesis and the mitotic activity of the cells, the normally quiescent satellite cells may contain much lower mutant mtDNA loads that myoﬁbers. Clark et al. (1997) used bupivacaine to cause necrosis of muscle ﬁbers, leaving satellite cells intact in a patient with a tRNALeu(CUN) mutation that was absent in satellite cells, and showed reversal of the genetic defect in the injected muscle. A similar effect has been reported in concentric exercise training, presumably because the signals for muscle growth and repair stimulate satellite cell fusion with mature myoﬁbers (Taivassalo et al., 1999b). However, an attempt to correct ptosis by the same approach has been unsuccessful in 5 patients (Andrews et al., 1999). (b) In Vitro Fertilization with Preimplantation Selection. One group has shown skewed segregation of the NARP mutation in oocytes from women, with mutant load of the mutation ranging from none to >95% (Blok et al., 1997). This is not conﬁrmed, but if the case, it is technically possible to harvest eggs from an affected woman, fertilize them in vitro, determine if the embryos contain the mutation by single-cell PCR, and implant only those free of mutation. Sampling error makes this approach fraught with danger. (c) Forced Paternal Inheritance. Maternal inheritance of the mtDNA is almost universal in the animal kingdom, but the mechanisms are unknown. It appears that at conception, mitochondria from paternally derived sperm may be recognized and actively destroyed. Manipulation of this mechanism may allow interference with the vertical transmission of mtDNA mutations from mother to child. In somatic cells, the elimination of paternally derived mitochondria occurs in 48 h (Manfredi et al., 1997). If this also occurs in

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the egg and it is possible to promote admixture of mtDNA between the sperm and oocyte soon after conception, the zygote may be genetically “rescued.” However, the mechanisms of maternal transmission of mtDNA are still obscure. In summary, this and other forms of genetic therapy are mostly conceptual and in the earliest stages of development. However, they may be the future of treatment in this group of devastating disorders.

References

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SECTION III SECONDARY RESPIRATORY CHAIN DISORDERS

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FRIEDREICH’S ATAXIA

J. M. Cooper1 and J. L. Bradley Department of Clinical Neuroscience Royal Free & University College Medical School London, NW3 2PF, United Kingdom

I. Features of Friedreich’s Ataxia A. Clinical Features B. Pathological Features C. Genetic Features II. Effect of FRDA Gene Mutations A. Transcription B. Frataxin Protein III. Models of FRDA A. Y east B. Bacterial C. Transgenic Mice IV. FRDA Molecular Mechanisms A. Iron Metabolism B. Mitochondrial Function C. Oxidative Stress V. Therapeutic Intervention A. Iron Chelation Therapy B. Antioxidant Therapy C. Mitochondrial Targeted D. Gene Therapy VI. Conclusion References

I. Features of Friedreich’s Ataxia

A. CLINICAL FEATURES Friedreich’s ataxia (FRDA) usually presents in adolescence (onset between 2 and 51 years; Durr et al., 1996), and clinical features include a progressive limb and gait ataxia, absence of deep tendon reﬂexes, extensor plantar responses, and loss of position and vibration sense in the lower limbs 1

Author to whom correspondence should be addressed.

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and dysarthria. Hypertrophic cardiomyopathy and skeletal abnormalities (including scoliosis and pes cavus) are relatively common, while diabetes and decreased visual acuity also have an increased incidence (Harding, 1981; Durr et al., 1996). The time period between disease onset and loss of ambulation is quite variable (3–44 years), and the disease usually results in premature death (21–69 years) (Harding, 1981). Sensory nerve action potentials and spinal somatosensory evoked potentials decrease with disease duration and are often absent, while motor nerve conduction velocities are usually less affected. There are several clinical ratings scales for patients with ataxia in general, including the International Cooperative Ataxia Ratings Scale (ICARS) (Trouillas et al., 1997). However, these have not been validated for FRDA. Several studies have compared the relationship between the genetics and clinical presentation (Durr et al., 1996; Filla et al., 1996), but there has been very little detailed analysis of the natural history of FRDA and of which factors may inﬂuence disease progression.

B. PATHOLOGICAL FEATURES Pathologically the most obvious ﬁndings are loss of large sensory neurones in the dorsal root ganglia and deterioration of the sensory posterior and Clarke’s columns, spinocerebellar tracts, and corticospinal motor tracts of the spinal cord (Harding, 1981). The peripheral neuropathy, which appears to be of a “dying back” nature, affects the longest and largest myelinated ﬁbers (Hughes et al., 1968). Demyelination, particularly of the large ﬁbers from the dorsal root ganglia (DRG), occurs in the posterior columns. Only mild neuronal loss is seen in the cerebellum. Cardiac hypertrophy is relatively common, and iron deposition in the heart has also been reported, although not in all patients (Lamarche et al., 1993; Bradley et al., 2000).

C. GENETIC FEATURES Friedreich’s ataxia is inherited in an autosomal recessive pattern with over 95% of patients having a homozygous expansion of a GAA triplet repeat in intron 1 of the FRDA gene on chromosome 9 (Campuzano et al., 1996). Normal alleles usually have between 6 and 34 GAA repeats (Montermini et al., 1997), but this can be expanded to between 67 and 1700 in patients or carriers (Durr et al., 1996). Most of the remaining patients are compound heterozygotes with the GAA expansion in one allele and point mutations in the other. Twenty-three different mutations have been identiﬁed including

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5 frameshift, 10 missense (exons 3, 4, 5a), 4 splice mutations (introns 3 and 4), 3 mutations affecting the initiation codon, and 1 nonsense mutation. Missense mutations have only been found in the C-terminal half of the protein (L106S, D122Y, G130V, I154F, L156P, R165C, W173G, L182F, L182H, H183R) (Pook et al., 2001), suggesting this may contain a functional domain(s). Most of the missense mutations cause nonconservative changes in highly conserved regions and tend to give a typical to severe phenotype, similar to the frameshift mutations (Cossee et al., 1999; De Michele et al., 2000; Forrest et al., 1998). Two mutations in less well-conserved amino acids give milder phenotypes (L106S, G130V) and two mutations give atypical clinical features (D122Y, R165C) (Cossee et al., 1999; Bidichandani et al., 1997; Bartolo et al., 1998; Forrest et al., 1998). Some patients that exhibit the clinical symptoms of FRDA do not show linkage to chromosome 9, suggesting there may be a second locus (Kostrzewa et al., 1997). In addition patients with vitamin E deﬁciency (AVED), caused by a mutation of the α-tocopherol transfer protein gene on chromosome 8, have a very similar phenotype to FRDA, and these patients may respond to vitamin E therapy (Cavalier et al., 1998). In FRDA patients variations in the size of the GAA repeat in intron 1 have been identiﬁed between generations. Contractions in size occur with paternal transmission, while both contraction and expansion occur with maternal transmission. Parental age also has an inﬂuence, the contraction being more marked with increasing paternal age, and a greater expansion occurring with increasing maternal age (De Michele et al., 1998). The number of GAA repeats is less in sperm DNA than blood DNA in a given individual, suggesting a postzygotic mechanism, which is also supported by the observation that the GAA contraction was smaller between generations than between blood and sperm DNA. Somatic mosaicism in the size of the GAA expansion has been reported with heterogeneity in repeat sizes in different CNS tissues from the same patient (Montermini et al., 1997). This suggests mitotic instability, with the repeat size appearing to follow the developmental origin of the tissue. This may contribute to the contraction and expansion in GAA repeat number observed in serially passaged cells (Bidichandani et al., 1999). While this could inﬂuence disease expression, the level of frataxin in a particular tissue is likely to be the most important factor in determining which tissues are affected. FRDA is the commonest inherited ataxia and prior to the availability of genetic diagnosis was thought to have a prevalence of approximately 1 in 50,000. It is now possible to include patients that would previously have been excluded by clinical criteria. Now patients with late onset (Bidichandani et al., 2000), retained reﬂexes (Klockgether et al., 1996), spastic paraplegia (Gates et al., 1998), pure sensory ataxia (Berciano et al., 1997), and chorea

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(Hanna et al., 1998) have been shown to have the genetic abnormality. The carrier frequency has been revised and estimated to be between 1: 60 to 1: 90 and a prevalence of approximately 1 in 30,000 (Cossee et al., 1997).

II. Effect of FRDA Gene Mutations

A. TRANSCRIPTION The FRDA gene contains seven exons (1–5a, 5b, 6) spanning 80 kilobases (kb) of genomic DNA. The 1.3-kb transcript containing exons 1–5a is the most abundant, and it generates a protein containing 210 amino acids. There is an alternative transcript that contains exon 5b, in place of 5a, and encodes a protein of 171 amino acids, but its signiﬁcance is unknown (Campuzano et al., 1996). Exon 6 appears to be noncoding. The highest mRNA levels are found in tissues with a relatively high mitochondrial content including heart, pancreas, liver, and skeletal muscle. In neuronal tissues, spinal cord has the highest levels with comparatively lower levels in the cerebellum and very little in the cerebral cortex (Campuzano et al., 1996). The relatively high frataxin mRNA levels in the spinal cord and heart may partly explain their clinical involvement in the disease, and the lack of involvement of other tissues with relatively high frataxin levels, such as liver, may partly reﬂect their regenerative capacity following cell loss. Frataxin expression appears to be required for development, as knockout of the FRDA gene was embryonically lethal (Cossee et al., 2000). This is further supported by the relatively high levels of frataxin mRNA found in a range of tissues during development including spinal cord, DRG, heart, liver, skeletal muscle, and skin ( Jiralerspong et al., 1997; Koutnikova et al., 1997). Homozygous GAA repeat expansions result in decreased frataxin mRNA levels in patients lymphoblasts and ﬁbroblasts (Bidichandani et al., 1998; Wong et al., 1999). This could relate to the GAA expansion affecting mRNA splicing, disruption of an enhancer element in the intron or blockade of transcription. There was no evidence of abnormally spliced mRNAs or of enhancer activity in the part of intron 1 containing the normal GAA repeat sequence (Bidichandani et al., 1998). The initiation and splicing of exons 1 and 2 of the frataxin mRNA were unaffected by GAA repeats between 9 and 270, but there was a decrease in the amount of mature transcript suggesting the larger GAA repeats interfered with transcript elongation (Ohshima et al., 1998). Indeed, increasing the GAA expansion alone was sufﬁcient to decrease transcription in vitro (Grabczyk and Usdin, 2000). This may be caused by the GAA/TTC repeats, which have been shown to

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form unusual DNA structures, including DNA triplexes, leading to blockade of transcription (see review by Patel and Isaya, 2001). The size of the GAA repeat appears to inﬂuence the clinical phenotype with a signiﬁcant inverse relationship between the size of the smaller GAA repeat and the age of onset (Durr et al., 1996; Filla et al., 1996). However, as with other triplet repeat diseases the large scatter of data associated with this correlation precludes the prediction of the disease course for any individual.

B. FRATAXIN PROTEIN In normal tissues the frataxin protein levels broadly reﬂected the frataxin mRNA content with the highest levels in the heart, skeletal muscle, spinal cord, and cerebellum (Campuzano et al., 1997). In agreement with the lower levels of frataxin mRNA, there were lower frataxin protein levels in skeletal muscle, cerebellum, and cerebral cortex from patients with FRDA. In addition, frataxin protein levels were decreased in proportion to the GAA repeat size, with frataxin level correlating with the size of the smallest GAA expansion in FRDA lymphoblasts (Campuzano et al., 1997). This supports the inverse relationship between GAA size and age of onset. Consequently, knowledge of the normal function of frataxin needs to be established for a better understanding of the disease mechanism. Analysis of the protein sequence failed to identify any similarities with domains in other proteins with known function; however two interesting features were identiﬁed. First, frataxin has a predicted N-terminal mitochondrial-targeting sequence (Campuzano et al., 1997) and second, there is a stretch of evolutionarily highly conserved amino acids in exons 4 and 5a (Dhe-Paganon et al., 2000). The X-ray crystal structure of human frataxin has been determined, and it suggests there is a conserved contiguous anionic patch of 12 amino acids on the surface of the protein [between amino acids (aa) 92–124], a feature also seen in ferritin (Dhe-Paganon et al., 2000). Fifteen conserved residues between aa 124–165 provide a nearly neutral ﬂat surface and are consistent with interaction with another protein. Iron is not present in the bacterially expressed protein, but after incubation with iron, X-ray diffraction analysis suggested frataxin could bind one molecule of iron. However, the residue associated with this association (His155) is not conserved, which led to the suggestion that an oligomer of frataxin may bind iron in a similar way to ferritin (Dhe-Paganon et al., 2000). Human pathological mutations occur in the protein core (L106S, I154F, L156P, W173G, L182F, L182H, H183R), within the anionic patch (D122Y), or in the ﬂat external surface (G130V, R165C). One of the mutations in the external ﬂat surface (G130V) was consistently associated with a mild phenotype, while the other mutation

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affecting this surface (R165C) and a mutation affecting an amino acid in the anionic patch (D122Y) gave relatively severe but atypical symptoms (see Section I.C.). This suggests missense mutations of the protein core may be more inﬂuential upon the general function of the protein while the other missense mutations may only modify speciﬁc functions.

III. Models of FRDA

A. Y EAST The identiﬁcation of a yeast frataxin homologue (yfh1p) has enabled several groups to manipulate the YFH1 gene in yeast to study its function and identify possible functions for frataxin. The YFH1 gene was disrupted or knocked out in several yeast models (Babcock et al., 1997; Koutnikova et al., 1997; Foury and Cazzalini 1997). This resulted in mitochondrial iron accumulation, impaired mitochondrial respiratory chain (MRC) function, decreased mitochondrial DNA levels, and increased susceptibility to oxidative stress induced by hydrogen peroxide. Consequently, yfh1p may be involved either in mitochondrial iron homeostasis or in antioxidant defense mechanisms. It is not known whether the decreased MRC activities are solely attributable to the loss of mtDNA or whether other mechanisms are involved.

B. BACTERIAL The most conserved portion of the frataxin protein has a signiﬁcant homology with the cyaY protein of γ -purple bacteria, consistent with their common ancestry with mitochondria. Knockout of the cyaY gene in Escherichia coli failed to affect viability, iron content, or susceptibility to hydrogen peroxide (Li et al., 1999a). This suggests that the bacterial cyaY may have a different function to its mitochondrial counterpart.

C. TRANSGENIC MICE Knockout of the FRDA gene in a transgenic mouse model was found to be embryonically lethal and therefore not a useful model to study disease mechanisms (Cossee et al., 2000). However, two conditional gene-targeting models were more successful (Puccio et al., 2001). In these models, homozygous knockout of the frataxin gene was only generated in selected tissues

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and not always in all cells. The ﬁrst step involved generating two transgenic mice lines that were heterozygous for a deletion of FRDA exon 4. These two lines both carried a cre recombinase transgene but expressed in different tissues. It was under the control of muscle creatine kinase (MCK) in one, and neuron speciﬁc enolase (NSE) in the other. These mice were crossed with mice homozygous for the conditional allele of FRDA containing loxP sequences ﬂanking exon 4. Exon 4 is deleted following recombination between the loxP elements on the FRDA conditional allele, which occurs wherever the cre transgene is expressed. Consequently, only homozygous deleted FRDA alleles are present in selected cells in the offspring carrying the cre/FRDA-deletion/FRDA-conditional allele. This gave rise to mice lacking a full-length FRDA transcript in heart and skeletal muscle (MCK cre mice), or decreased levels in the brain, liver, and kidney and absent level in the heart (NSE cre mice). There was no embryonic lethality, although the mice died prematurely (NSE mice approximately 24 days, MCK mice approximately 76 days). Clinically, the NSE mice expressed a rapidly progressive movement disorder from approximately 12 days while the MCK mice exhibited weight loss at 7 weeks followed by progressive signs of muscle fatigue. Both models exhibited signs of cardiac hypertrophy (Puccio et al., 2001). In the hearts, morphologically abnormal mitochondria and decreased succinate dehydrogenase staining were apparent by 2–3 weeks (NSE mice) and 7–10 weeks (MCK mice) of age. Mitochondrial complexes I–III, and aconitase activities were decreased at 7–10 weeks in MCK mice and at death in the NSE mice. However, these features were not apparent in the skeletal muscle of either model. Iron deposits were seen in the MCK mice heart at 10 weeks, but not in the NSE mice. This data suggests that iron deposition follows the respiratory chain, aconitase, and clinical defects, and it is therefore likely to be a secondary feature. While this is a useful model, it differs from typical FRDA in that it is a mixture of extremes: some cells possessed relatively normal levels of frataxin while others were devoid of frataxin. Low residual levels of frataxin may play an important role in the cell and ameliorate pathogenesis. Frataxin knock in mice, heterozygous for a 230-GAA repeat in the FRDA gene, were either crossed to give double heterozygous knockin mice or crossed with FRDA heterozygous knockout mice to give knockout/knockin mice. The knockin mice had 75% residual frataxin while the knockin/ knockout mice expressed between 25 and 36% of wild-type frataxin levels. However, up to one year of age iron levels were normal and there were no apparent clinically abnormalities, except in one mouse, which died at one year of age and showed a threefold increase in heart iron and ﬁbrosis (Miranda et al., 2002). Iron loading for two months did not facilitate either iron accumulation or clinical disability in these mice. This suggests that

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frataxin levels need to be decreased to below 25% of normal to give any detectable pathology. The insertion of longer GAA repeats are presumably required to give more severe pathology.

IV. FRDA Molecular Mechanisms

A. IRON METABOLISM 1. Mitochondrial Iron in FRDA There is increasing evidence that mitochondrial iron metabolism is disrupted in models of FRDA and in selected tissue samples from FRDA patients. However, it is not yet clear where in the disease mechanism it should be placed. The yeast frataxin homologue (yfh1p) was identiﬁed as a suppresser, which rescued a mutant yeast strain unable to grow on iron-limited medium (Babcock et al., 1997). This may be attributable to activation of the highafﬁnity iron transport system in these mutants, even in iron-replete conditions when this system is not usually detected (Babcock et al., 1997). The YFH1-deletion mutants exhibited a twofold increase in cellular iron and a tenfold increase in mitochondrial iron (Foury and Cazzalini 1997; Babcock et al., 1997), suggesting yfh1p may be involved with mitochondrial iron uptake or efﬂux. In agreement with the yeast models, there is evidence of iron accumulation in FRDA tissues. Iron deposits were detected in heart tissue from some but not all FRDA patients (Lamarche et al., 1993; Bradley et al., 2000). Iron imaging using magnetic resonance imaging (MRI) suggested iron levels were increased in the dentate nucleus in FRDA (Waldvogel et al., 1999). In addition ﬁbroblasts cultured from FRDA patients had a mildly increased mitochondrial iron content (Delatychi et al., 1999). Currently it is not clear what role frataxin has within mitochondria or why iron accumulates when frataxin levels are decreased. In the presence of ferrous iron, the yeast homologue yfh1p appears to form multimers consisting of approximately 60 subunits and more than 3000 atoms of iron (Adamec et al., 2000). Similar ﬁndings have been shown for human frataxin expressed in yeast cells and provisionally for frataxin in mouse heart (Cavadini et al., 2002). This data concurs with that from X-ray crystallography of human frataxin, which suggested that frataxin may bind both proteins and iron (Dhe-Paganon et al., 2000). It is possible frataxin is required for mitochondrial iron storage or to keep it in a bioavailable form. While there is a

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mitochondrial ferritin that may also store iron it appears to have limited tissue distribution (Levi et al., 2001). It is not clear why loss of frataxin leads to mitochondrial iron accumulation if frataxin is merely involved with iron storage. One possibility is that the loss of available iron may facilitate increased iron uptake in an attempt to alleviate this situation. In yeast lacking yfh1p (YFH1 deletion), chelating the iron in the medium using bathophenanthroline disulfate (BPS) prevented mitochondrial iron accumulation (Foury, 1999). Under these conditions the MRC activities were improved, although aconitase activity remained low. This suggested that the mitochondrial iron accumulation was responsible for the decrease in MRC function, but either free radical generation or abnormal Fe–S synthesis may still be affecting aconitase activity. This is in agreement with a similar study where the number of petite mutants in yeast cells lacking yfh1p increased as extracellular iron levels increased (Radisky et al., 1999). Increased sensitivity to iron was reported in cultured ﬁbroblasts from FRDA patients exhibiting a 66% decrease in frataxin mRNA (Wong et al., 1999), implying an increased vulnerability to extracellular iron concentrations. However, this sensitivity has not been observed in similar experiments in our own laboratory (Bradley, unpublished observations). 2. Cellular Iron Regulation Iron has an important role in many cellular processes, but because it can have severe detrimental effects its levels are tightly regulated. Iron is transported in serum bound to transferrin, and it is taken up into cells following binding to the transferrin receptors on the cell surface and internalization into endosomes. Iron exits the endosomes using the Nramp2 transporter and is then used for biosynthetic processes or stored in ferritin. In general, the regulation of cellular iron levels, and also many proteins involved with iron metabolism, is mediated by iron-responsive proteins (IRP), which binds iron-responsive elements (IRE) in the 3 or 5 untranslated regions (UTR) of the mRNA of speciﬁc proteins. Iron-response protein 1 is the most widespread of these proteins, which is located in the cytosol and has aconitase activity (Kennedy et al., 1992). It possesses a 4Fe–4S cluster, which under conditions of low cellular iron, loses an iron molecule leaving a 3Fe–4S cluster. Aconitase activity is lost but its IRP function is activated and it can bind to IREs. A variety of mRNAs contain IREs including transferrin receptor, ferritin, aminolevulinate synthase in erythroid cells, and mitochondrial aconitase. The IRE resides in either the 5 or 3 UTR of the mRNA, and binding of the IRP either prevents translation or stabilizes the mRNA for enhanced translation, respectively. Consequently, low cellular iron levels give rise to lower ferritin levels and increased transferrin receptor levels. This leads to increased iron uptake and decreased storage. The opposite process

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occurs in conditions of high cellular iron content. Increased oxidative stress has also been reported to inactivate aconitase activity and activate IRP1 activity (Hentze and Kuhn, 1996), suggesting that oxidative stress may increase cellular iron uptake. This may have important implications for iron accumulation in FRDA where aconitase activity is decreased (see Section IV.C). However, a contradictory report suggests free radicals inactivate IRP1 activity and therefore do not inﬂuence iron metabolism (Brazzolotto et al., 1999). How iron is transported into the mitochondria and what regulates mitochondrial iron uptake is generally not well understood. Mitochondria play a pivotal role both in Fe–S cluster and in heme biosynthesis, and therefore iron uptake needs to be tightly regulated with these processes. There is evidence that in erythroid cells at least iron may be transferred directly from the endosomes into miochondria (Richardson and Ponka, 1997), and the regulation of mitochondrial iron uptake for heme biosynthesis may differ to that for Fe–S synthesis (see Sections IV.A.3 and IV.A.4) 3. Heme Biosynthesis Mitochondria are the site of the rate-limiting step of heme biosynthesis, which is catalyzed by aminolevulinate synthase (ALAS). Aminolevulinate leaves the mitochondria and is converted via several steps to coproporphyrinogen III in the cytosol. Coproporphyrinogen III is then further metabolized in the mitochondria, ending up with the incorporation of ferrous iron catalyzed by ferrochelatase. Heme provides the prothetic group in a variety of proteins involved with oxygen transport (hemoglobin, myoglobin), mitochondrial respiration (cytochromes a, b, c), drug metabolism (cytochrome P450), steroid biosynthesis (prostaglandin endoperoxide synthase), cellular antioxidant defenses (catalase, glutathione peroxidase), and signal transduction (NOS, guanylate cyclase). Ferrochelatase is bound to the mitochondrial inner membrane, where it may be linked to the uptake of iron. In fact, there is a suggestion that heme export from the mitochondrion may be in exchange for iron uptake (Romslo, 1983). This may explain why in the absence of heme biosynthesis there is no accumulation of mitochondrial iron (Tangeras, 1986). Ferrochelatase requires reduced iron (Fe2+), and there is evidence that it is directly linked to complex I, which provides the reducing potential (Taketani et al., 1986). In yeast, ferrochelatase only uses iron as it is transported into the mitochondria and iron stored in mitochondria is not available to it (Lange et al., 1999). This would suggest that iron uptake for heme biosynthesis is under different control to that required for Fe–S cluster synthesis. However, because ferrochelatase possesses a 2Fe–2S cluster, heme biosynthesis is dependent upon Fe–S cluster synthesis to some degree.

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Erythroid cells are involved with hemoglobin synthesis, and the process differs in certain aspects to the “housekeeping” heme biosynthesis seen in other cells. In erythroid cells ALAS is regulated by IRE to regulate protoporphyrin synthesis in the absence of iron. In addition there appears to be no link between mitochondrial iron uptake and heme synthesis in erythroid cells where mitochondrial iron accumulates if heme biosynthesis is inhibited (Cox et al., 1994). In FRDA there is no evidence that heme biosynthesis is affected. First, heme-containing proteins (cytochrome oxidase) are normal in FRDA (Bradley et al., 2000), and second, the mitochondrial iron accumulation in FRDA is not typical of abnormalities associated with heme biosynthesis in nonerythroid cells. As the mechanisms of mitochondrial iron uptake for heme synthesis may be speciﬁc for this pathway, it suggests the iron accumulation in FRDA mitochondria is associated with a separate pathway of iron uptake. 4. Iron–Sulfur Cluster Synthesis Phylogenic proﬁles analyse the distributions of genes in genomes. Genes that occur together on the genome may have related functions (Marcotte et al., 1999; Huynen et al., 2000) and therefore phylogenic proﬁles are useful in predicting function. It is possible the proteins may interact or be part of the same biological pathway. The analysis of 56 genomes has shown that frataxin or its homologues (e.g., yfh1, CyaY ) has the same phylogenetic distribution as genes encoding proteins involved with Fe–S cluster synthesis (e.g., hscA and hscB in proteobacteria and JAC1 and SSQ1 in Saccharomyces cerevisiae). Historically, they have disappeared together and also transferred laterally together from the mitochondrial genome, suggesting frataxin may also be involved with iron–sulfur cluster synthesis (Huynen et al., 2001). In eukaryotes, mitochondria have been shown to have an essential role in Fe–S cluster synthesis (Lill et al., 1999). Many proteins involved with Fe–S cluster synthesis have been characterized in yeast (Fig. 1) (Lill and Kispal, 2000), and many are homologous to bacterial proteins involved with nitrogen ﬁxation (nif) or iron–sulfur cluster synthesis (isc) (Zheng et al., 1998). The nfs1p is an essential protein in yeast and is similar to the nifS and iscS proteins in bacteria, which are involved with the generation of sulphur from cysteine (Zheng et al., 1994). The isu1p and isu2p proteins are similar to the bacterial iscU and the N-terminal portion of nifU, which contains cysteine residues involved in binding the iron substrate used to generate the Fe–S cluster (Agar et al., 2000). This suggests that, in yeast, isu1p and isu2p may bind iron and act as a “workbench” for Fe–S cluster assembly. In bacteria nifS is involved with the further synthesis of the iron– sulfur cluster on nifU (Yuvaniyama et al., 2000); however, this role has not

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FIG. 1. The proposed pathway for Fe–S cluster synthesis in the mitochondrion. The yeast proteins known to be involved with Fe–S cluster synthesis are shown in italics. The functions are based upon homology with bacterial proteins and yeast knockout studies. Yfh1p (frataxin) has been proposed to be involved with mitochondrial iron storage and possibly maintaining its bioavailability. It is possible this function is important for Fe–S cluster synthesis.

yet been assigned in yeast. The function of nfu1p in yeast is not known, but it has homology to the C-terminus of the bacterial nifU, and although it is not essential itself, if lost in conjunction with isu1p, Fe–S cluster synthesis is inhibited (Schilke et al., 1999). The ferredoxin yah1p (yeast adrenodoxin homologue 1) is required for Fe–S cluster synthesis, and it may be required for the input of reducing equivalents at several steps including generation of elemental sulfur or release of the Fe–S cluster from its substrate. Yah1p itself may be reduced by arh1p (adrenodoxin reductase homologue 1) (Manzella et al., 1998). The jac1p and ssq1p are chaperones involved with Fe–S cluster synthesis, and they may be important to stabilize the apoproteins prior to the insertion of the Fe–S cluster (Strain et al., 1998). In yeast, isa1p and isa2p contain highly conserved cysteine residues required for Fe–S cluster synthesis (Kaut et al., 2000). Their function is not known, but unlike isu, yah1p, nfs1p, and arh1p, they are not essential; therefore, they could be involved with efﬁcient transport of iron to isu proteins or the transfer of the Fe–S cluster to the apoprotein. There are still many functions yet to be assigned to this pathway

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including the proteins involved in the release of the 2Fe–2S cluster from isu proteins and the proteins involved with the conversion to 4Fe–4S clusters. It is not only Fe–S clusters in mitochondrial proteins that are synthesized in the mitochondrion but also extramitochondrial Fe–S proteins. Yeast cells deﬁcient in atm1p (an ATP binding cassette (ABC) ATPase) accumulated mitochondrial iron and did not incorporate the Fe–S into cytosolic proteins, although mitochondrial Fe–S proteins were assembled correctly (Kispal et al., 1999). It would appear that atm1p exports components of the Fe–S cluster required for the extramitochondrial Fe–S protein assembly. The nfs1p, yah1p, isa1p, and isa2p are all required for extramitochondrial Fe–S cluster synthesis, suggesting that export of the Fe–S cluster occurs after these steps (Lange et al., 2000; Kaut et al., 2000; Pelzer et al., 2000). It is interesting to note that in humans perturbations of cytosolic Fe–S cluster synthesis caused by mutations in hABC7, a homologue of atm1p, resulted in sideroblastic anemia and ataxia, mitochondrial iron accumulation, and decreased cytosolic Fe–S protein maturation (Bekri et al., 2000; Allikmets et al., 1999). Human, homologues of these yeast proteins have not yet been identiﬁed. However, it is possible that frataxin plays a role in mitochondrial Fe–S cluster synthesis—for example, in the supply or transfer of available iron. Frataxin deﬁciency would then result in decreased Fe–S center synthesis, leading to decreased MRC and aconitase function, and ultimately mitochondrial iron accumulation. Signiﬁcant mitochondrial iron accumulation occurs when the following genes have been inactivated in yeast; ATM1, NFS1, SSQ1, and YAH1 (Kispal et al., 1997; Knight et al., 1998; Li et al., 1999b; Lange et al., 2000). The loss of NFU1/ISU1 or ISA1/ISA2 have more mild effects (Schilke et al., 1999; Jensen and Culotta, 2000). The iron accumulation in yfh1p mutants contrasts with that from the mice models (see Section III.C) and patient studies where the data supports the suggestion that signiﬁcant iron accumulation may be a late event in FRDA. The suggestion that frataxin is involved in Fe–S cluster synthesis would be consistent with the secondary role of mitochondrial iron accumulation as this is a feature of yeast mutants where iron–sulfur synthesis is affected (Section IV.A.4). B. MITOCHONDRIAL FUNCTION Many mitochondrial proteins have an N-terminal presequence that is cleaved during importation (Lithgow, 2000). In the case of frataxin, the ﬁrst 55 amino acids are cleaved at two sites, between aa 41–42 and 55–56 by the mitochondrial processing peptidase in two steps (Branda et al., 1999; Cavadini et al., 2000). This predicted mitochondrial location was conﬁrmed when expressed frataxin was tagged and shown to colocalize with mitochondrial

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markers in human cervical carcinoma cells (HeLa) and African green monkey kidney cells (COS) cells (Babcock et al., 1997; Koutnikova et al., 1997; Priller et al., 1997). In transfected HeLa cells, frataxin was found to associate with the mitochondrial inner membrane (Campuzano et al., 1997), although in yeast the frataxin homologue (yf h1p) was localized to the matrix space (Branda et al., 1999). Several FRDA point mutations (I154F, G130V) appear to inﬂuence frataxin processing (Koutnikova et al., 1998), possibly by altering its secondary structure and therefore mitochondrial uptake or recognition for cleavage. Many biological pathways are found in the mitochondrion including heme biosynthesis, iron–sulfur cluster biosynthesis, urea cycle, β oxidation of fatty acids, carbohydrate oxidation, oxidative phosphorylation of ADP to ATP, calcium homeostasis, and apoptosis. The role frataxin plays in the mitochondrion is not known, but the initial observations that decreased frataxin levels resulted in dysfunction of the mitochondrial respiratory chain came with the yeast YFH1 mutants, which grew poorly on nonfermentable substrates. The defect of respiration involved abnormal cytochrome oxidase, NADH cytochrome c reductase, and ATPase activities (Foury and Cazzalini, 1997; Koutnikova et al., 1997). However, the concurrent loss of mtDNA in these cells may underlie these MRC defects, which may be secondary to increased oxidative damage. Dysfunction of the MRC in FRDA received further support when a severe defect of complexes I–III of the MRC was observed in postmortem heart, and to a lesser degree in skeletal muscle from FRDA patients (Bradley et al., 2000). An earlier report on two heart samples found similar data (Rotig et al., 1997). While mtDNA levels were also decreased in the heart and to a lesser degree in skeletal muscle samples, the decrease was not believed to be sufﬁcient to markedly affect MRC function and was likely to be secondary to oxidative damage (Bradley et al., 2000). The pattern of respiratory chain dysfunction is reminiscent of that caused by oxidative stress. Oxidative damage in the manganese superoxide dismutase (Mn SOD) knockout transgenic mouse model, caused a severe defect of complexes I–III of the MRC and aconitase activity (Melov et al., 1999). Likewise, similar biochemical features were apparent in Huntington’s disease, where oxidative stress may be secondary to excitotoxicity (Tabrizi et al., 2000). However, all the activities decreased in FRDA contain Fe–S clusters, and therefore it has been proposed that an abnormality of Fe–S cluster synthesis may also be responsible for these defects (see Section IV.B). In the conditional knockout transgenic mice, the observed decreases in complexes I–III and aconitase activities mimic that seen in the FRDA heart samples. This adds support to the importance of these ﬁndings, and as they precede iron accumulation, they are not merely secondary to oxidative damage but may be due to decreased Fe–S synthesis.

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There is no evidence of MRC dysfunction in cells grown from FRDA patients; this includes ﬁbroblasts, lymphoblasts, and myoblasts (Rotig et al., 1999; Bradley, unpublished observations). This suggests MRC dysfunction may be apparent only following prolonged exposure to frataxin deﬁciency or that cells in culture either have residual frataxin levels above the pathological levels or have less dependence upon frataxin for their normal function. 31 Phosphorous magnetic resonance spectroscopy (31P-MRS) is an in vivo technique that can measure, among other parameters, the high-energy phosphorous compounds (phosphocreatine, PCr, and ATP) in heart and skeletal muscle. In skeletal muscle in particular, the analysis of the rate at which PCr recovers following exercise (Vmax ) is a measure of the efﬁciency of oxidative phosphorylation (Kemp et al., 1993), and in heart the PCr/ATP ratio is a good measure of energy availability (Ingwall et al., 1985). 31P-MRS, analysis of FRDA patients has revealed markedly decreased oxidative phosphorylation in the heart (Lodi et al., 2001b) and skeletal muscle, with the latter correlating with the size of the smallest GAA repeat (Lodi et al., 1999). These data underline the role of mitochondrial dysfunction in FRDA and suggest it is playing a primary role in disease pathogenesis.

C. OXIDATIVE STRESS The ﬁrst suggestion that an increased susceptibility to oxidative stress could be involved in FRDA came with the observation that vitamin E deﬁciency, caused by mutations of the α-tocopherol transfer protein gene, can result in a similar phenotype (Cavalier et al., 1998). Consequently, increased lipid peroxidation due to a deﬁciency of this primary lipid-soluble, cellular chain braking antioxidant caused similar pathology to FRDA. It is interesting to note that vitamin E levels are particularly high in mitochondria (Buttriss and Diplock, 1998), which are a major cellular source of free radicals, and inhibition of the MRC is a target in long-term vitamin E deﬁciency (Thomas et al., 1993). This makes an interesting parallel with FRDA where deﬁciency of frataxin, a mitochondrial protein, leads to MRC dysfunction and oxidative damage in FRDA (Bradley et al., 2000). Data from the yeast YFH1 knockout models suggested that loss of the frataxin homologue increased the sensitivity to hydrogen peroxide (Babcock et al., 1997; Foury and Cazzalini, 1997). However, rather than reﬂecting the antioxidant capacity of the cells, it is possible that this increased sensitivity merely reﬂected the increased mitochondrial iron content, which then promoted Fenton chemistry and the decomposition to the hydroxyl radical and ensuing cell damage. There are several reports suggesting the presence of increased oxidative damage in FRDA patients. Aconitase activity is particularly sensitive to

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free radical damage (Hausladen and Fridovich, 1994). Consequently, the decreased aconitase activities reported in heart and skeletal muscle from FRDA patients (Rotig et al., 1997; Bradley et al., 2000) may reﬂect elevated free radical damage, in addition to a possible defect of Fe–S cluster synthesis. Plasma malondiadehyde (MDA) levels were raised in 11 FRDA patients indicative of increased lipid peroxidation (Emond et al., 2000). Free glutathione levels were decreased in blood from FRDA patients, although total glutathione levels were normal, suggesting extensive gluathionylation of proteins in response to oxidative stress (Piemonte et al., 2001). Urine levels of 8-hydroxy-2 -deoxyguanosine (8OH2 dG) were raised in 29 FRDA patients, suggesting elevated oxidative damage to DNA. However, plasma dihydroxybenzoic acid was not increased in FRDA patients, and it was suggested this could be related to the insensitivity of the assay (Schultz et al., 2000). Cultured ﬁbroblasts from FRDA patients showed an increased susceptibility to hydrogen peroxide, but only under conditions that cause little cell death in control cells (Wong et al., 1999). Total cellular iron levels were increased in these cells, but the difference was not statistically signiﬁcant. It is possible the elevated iron levels seen in some cells from FRDA patients could be giving rise to the increase in sensitivity of the cells to oxidative stress. Likewise, if Fe–S cluster synthesis is impaired, the resulting decrease in MRC function could lead to elevated free radical generation and oxidative stress (Hasegawa et al., 1990). However, reports suggest frataxin deﬁciency may result in a delayed antioxidant defense, and they propose the increased oxidative damage then leads to the other biochemical features reported, although what role frataxin has in this mechanism is not known ( Jiralerspong et al., 2001).

V. Therapeutic Intervention

Given the evidence of mitochondrial iron accumulation, oxidative damage, and mitochondrial respiratory chain abnormality in patients with FRDA, the emphasis for therapeutic intervention has focused on iron chelation, antioxidant protection, and mitochondrial energy enhancement. With the relative paucity of data relating to the validation of rating scales for the assessment of FRDA patients and the natural history of FRDA, there is a lack of coordination of the assessment of therapies for FRDA patients. Pseudo markers of FRDA have been used to assess either the degree of cardiac hypertrophy (echocardiography) (Lodi et al., 2001a; Hausse et al., 2002) heart or skeletal muscle energetics (31P-MRS) (Lodi et al., 2001a),

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or markers of oxidative damage (Schultz et al., 2000). The assessment of clinical symptoms has been assessed using ICARS in one study (Lodi et al., 2001a) and also in the assessment of ataxia in patients with AVED taking vitamin E (Gabsi et al., 2001).

A. IRON CHELATION THERAPY Iron accumulation has been reported in yeast and mice models as well as patient tissues; consequently, removing or sequestering this iron may have therapeutic beneﬁts. Even though the iron accumulation may be a late secondary event, reducing the level of this iron may help prevent secondary oxidative damage, provided that iron availability for other functions is maintained. Iron chelation has been shown to restore mitochondrial iron levels and prevent MRC dysfunction in the deleted YFH1 yeast model (Foury, 1999). The conventional iron chelator, desferrioxamine (DFO), is relatively hydrophilic with poor permeability across the plasma membrane. It has poor intestinal absorption and cannot effectively mobilize iron from iron-loaded mitochondria ( Jin et al., 1989; Richardson et al., 2001). However, using succinylacetone to induce mitochondrial iron accumulation in reticulocytes, several iron chelators, including 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH), have been shown to effectively mobilize mitochondrial iron, and they may be useful in FRDA therapy (Richardson et al., 2001). There have been no published accounts of iron chelation therapy in FRDA; however, FRDA patients have normal serum iron and ferritin levels, and therefore general iron chelation may have potential problems (Wilson et al., 1998). An iron chelator targeted to the mitochondrion may be a more feasible approach.

B. ANTIOXIDANT THERAPY FRDA patients have been treated with a variety of antioxidants including Idebenone (Schultz et al., 2000; Schols et al., 2001; Hausse et al., 2002) coenzyme Q10 and vitamin E (Lodi et al., 2001a). Anecdotal reports of the use of N-acetyl cysteine also suggest beneﬁts (http://internaf.org/ataxia/ nacupd.html). Idebenone is a short-chain analogue of coenzyme Q10, is well tolerated by humans, crosses the blood–brain barrier (Nagai et al., 1989), has been reported to be a relatively good antioxidant (Mordente et al., 1998), and has been used in a variety of diseases with some beneﬁts (Ranen et al., 1996; Gutzmann and Hadler, 1998). The effect of idebenone upon cardiac

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hypertrophy in FRDA patients was assessed using echocardiography, but other clinical effects were not reported. After six months treatment, cardiac hypertrophy was decreased in up to half the patients tested, although this was not always associated with improved fraction shortening (Hausse et al., 2002). It is difﬁcult to interpret this data because of the inherent variability of echocardiographic analyses. This will be resolved if these cardiac improvements are maintained over prolonged periods of treatment. Another assessment of idebenone failed to identify improvements in skeletal muscle 31 P-MRS or echocardiographic parameters (Schols et al., 2001), although this may reﬂect the short time scale used. Vitamin E is a naturally occurring lipid-soluble antioxidant distributed throughout cellular membranes but predominantly in mitochondrial membranes. It is obtained in the diet, and vegetable oils and nuts provide a particularly rich source. Vitamin E treatment has been shown to increase vitamin E levels in a variety of tissues including brain, muscle, and heart (Zhang et al., 1995). It has been used to treat cardiovascular disease, Parkinson’s disease, cancers, and AVED with varying degrees of success (Bostick et al., 1993; Stephens et al., 1996; Shoulson, 1998; Gabsi et al., 2001); however, its efﬁcacy has been assessed only in conjunction with coenzyme Q10 with FRDA patients. Coenzyme Q10 (CoQ10) is naturally found in cells, and up to 5 mg/day may be consumed in an average diet (very rich in soybean oil, meat, and ﬁsh). Coenzyme Q10 is readily taken up into the blood (Folkers et al., 1994), the brain (Matthews et al., 1998), and liver (Zhang et al., 1995), although other reports suggest dietary CoQ10 levels do not inﬂuence tissue CoQ10 levels in the rat (Reahal and Wrigglesworth, 1992). Coenzyme Q10 may reduce vitamin E, and therefore when combined in a therapy may act synergistically (Ernster et al., 1995). This was found to be the case in protecting rats against atherosclerosis (Thomas et al., 2001), and as part of a long-term therapy for FRDA patients where heart and skeletal muscle energetics were signiﬁcantly improved after three months (Lodi et al., 2001a). Provisional three-year follow-up data from this study showed the enhanced energy levels were maintained, clinical parameters were stabilized or improved in 8 out of 10 patients, and fraction shortening had improved (Hart et al., manuscript in preparation). The evidence of MRC dysfunction in FRDA suggests that drugs that enhance mitochondrial ATP synthesis may also be beneﬁcial. Coenzyme Q10 acts as an electron carrier in the respiratory chain, and consequently part of its action could be to enhance cellular energy synthesis. This would appear to be the case in the pilot trial using CoQ10 (Lodi et al., 2001a).

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C. MITOCHONDRIAL TARGETED The primary abnormality in FRDA is almost certainly localized to the mitochondrion and therefore a therapy speciﬁcally targeting the mitochondrion would be expected to be most beneﬁcial. By coupling antioxidants to the triphenylphosphonium cation, they can be speciﬁcally targeted to the mitochondrion. This approach has been used for vitamin E and found to result in an 80-fold increase in mitochondrial vitamin E content (Smith et al., 1999). Such compounds have not yet been used in patients, but are likely to be of particular beneﬁt in FRDA.

D. GENE THERAPY The discovery that the expanded GAA repeat results in transcription blockade raises the possibility of ﬁnding drugs that may interfere with the “sticky DNA” structures allowing for improved transcription and translation of frataxin. Likewise, gene therapy replacing the dysfunctional frataxin gene would be beneﬁcial, but because of the systemic nature of the disease, will require extensive delivery to a wide range of cells. Before this can become a possibility for FRDA, a number of general issues relating to gene therapy need to be overcome.

VI. Conclusion

Clues to the molecular mechanisms underlying FRDA are currently dominated by the evidence that iron accumulation may be a relatively late event in the disease process, and frataxin may bind iron and either act as a mitochondrial iron store or keep it in a bioavailable form. As such, frataxin may play an important role in the process leading to Fe–S cluster synthesis (Fig. 2). Consequently, it has been proposed that decreased frataxin levels lead to a decrease in the level of bioavailable iron and a subsequent decrease in Fe–S cluster synthesis. The resultant decreases in MRC complexes I–III and aconitase activities lead to a decrease in ATP synthesis and an increase in free radical production from the inhibited MRC. This suggests the accumulation of mitochondrial iron is due to the attenuation of Fe–S cluster synthesis, as seen for mutations of the Fe–S pathway in yeast cells, which contributes to the increased susceptibility to free radicals (Fig. 2). This proposed mechanism still awaits the demonstration

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FIG. 2. Possible interrelationship between frataxin deﬁciency and the other parameters identiﬁed in FRDA. Frataxin may act as an iron store in mitochondria, and when deﬁcient frataxin fails to maintain a bioavailable form of iron that causes a decrease in Fe–S cluster synthesis. Depleted Fe–S proteins will inhibit the MRC and aconitase activities leading to decreased ATP synthesis and increased free radical generation from the MRC. Increased free radical generation will further inhibit the MRC and aconitase activities and lead to oxidative stress. Abnormal Fe–S cluster synthesis will lead to iron accumulation which will exacerbate the oxidative stress through Fenton chemistry. There is a suggestion that antioxidant defenses may also be impaired.

that Fe–S cluster synthesis is abnormal in FRDA patients. It has also been proposed that frataxin may inﬂuence the antioxidant protective mechanisms of the mitochondria; however, this mechanism is as yet ill deﬁned.

Acknowledgments

J.L.B. would like to thank Ataxia and the National Lottery for continued ﬁnancial support.

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Pelzer, W., Muhlenhoff, U., Diekert, K., Siegmund, K., Kispal, G., and Lill, R. (2000). Mitochondrial Isa2p plays a crucial role in the maturation of cellular iron-sulfur proteins. FEBS Lett. 476, 134 –139. Piemonte, F., Pastore, A., Tozzi, G., Tagliacozzi, D., Santorelli, F. M., Carrozzo, R., et al. (2001). Glutathione in blood of patients with Friedreich’s ataxia. Eur. J. Clin. Invest. 31, 1007– 1011. Pook, M. A., Al-Mahdawi, S. A. H., Thomas, N. H., Appleton, R., Norman, A., Mountford, R., et al. (2000). Identiﬁcation of three novel frameshift mutations in patients with Friedreich’s ataxia. J. Med. Genet. 37, E38. Priller, J., Scherzer, C. R., Faber, P. W., MacDonald, M. E., and Young, A. B. (1997). Frataxin gene of Friedreich’s ataxia is targeted to mitochondria. Ann. Neurol. 42, 265–269. Puccio, H., Simon, D., Coss´ee, M., Criqui-Filipe, P., Tiziano, F., Melki, J., et al. (2001). Mouse models for Friedreich’s ataxia exhibit cardiomyopathy, sensory nerve defect and Fe-S enzyme deﬁciency followed by intramitochondrial iron deposits. Nat. Genet. 27, 181–186. Radisky, D., Babcock, M. C., and Kaplan, J. (1999). The yeast frataxin homologue mediates mitochondrial iron efﬂux. Evidence for a mitochondrial iron cycle. J. Biol. Chem. 274, 4497–4499. Ranen, N. G., Peyser, C. E., Coyle, J. T., Bylsma, F. W., Sherr, M., Day, L., et al. (1996). A controlled trial of idebenone in Huntington’s disease. Mov. Disord. 11, 549–554. Reahal, S., and Wrigglesworth, J. (1992). Tissue concentrations of coenzyme Q10 in the rat following its oral and intraperitoneal administration. Drug. Metab. Dispos. 20, 423–427. Richardson, D. R., Mouralian, C., Ponka, P., and Becker, E. (2001). Development of potential iron chelators for the treatment of Friedreich’s ataxia: Ligands that mobilise mitochondrial iron. Biochim. Biophys. Acta 1536, 133–140. Richardson, D. R., and Ponka, P. (1997). The molecular mechanisms of the metabolism and transport of iron in normal and neoplastic cells. Biochim. Biophys. Acta 1331, 1–40. Romslo, I. (1983). Iron uptake by mitochondria. In “Structure and function of iron storage and transport proteins” (Urushizaki, I., Aisen, P., Listowski, I., and Drysdale, J. W., eds.), p. 493. Elsevier, Amsterdam. Rotig, A., de Lonlay, P., Chretien, D., Foury, F., Koenig, M., Sidi, D., et al. (1997). Aconitase and mitochondrial iron-sulphur protein deﬁciency in Friedreich’s ataxia. Nat. Genet. 17, 215–217. Schilke, B., Voisine, C., Beinert, H., and Craig, E. (1999). Evidence for a conserved system for iron metabolism in the mitochondria of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 96, 10206–10211. Schols, L., Vorgerd, M., Schillings, M., Skipka, G., and Zange, J. (2001). Idebenone in patients with Friedreich ataxia. Neurosci. Lett. 306, 169–172. Schulz, J. B., Dehmer, T., Sch¨ols, L., Mende, H., Hardt, C., Vorgerd, M., et al. (2000). Oxidative stress in patients with Friedreich’s ataxia. Neurology. 55, 1719–1721. Shoulson, I. (1998). DATATOP: A decade of neuroprotective inquiry. Parkinson Study Group. Deprenyl And Tocopherol Antioxidative Therapy of Parkinsonism. Ann. Neurol. 44, S160–S166. Smith, R. A., Porteous, C. M., Coulter, C. V., and Murphy, M. P. (1999). Selective targeting of an antioxidant to mitochondria. Eur. J. Biochem. 263, 709–716. Stephens, N. G., Parsons, A., Schoﬁeld, P. M., Kelly, F., Cheeseman, K., and Mitchinson, M. J. (1996). Randomised controlled trial of vitamin E in patients with coronary disease: Cambridge Heart Antioxidant Study (CHAOS). Lancet 347, 781–786. Strain, J., Lorenz, C. R., Bode, J., Garland, S., Smolen, G. A., Ta, D. T., et al. (1998). Suppressors of superoxide dismutase (SOD1) deﬁciency in Saccharomyces cerevisiae. J. Biol. Chem. 273, 31138–31144.

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Tabrizi, S. J., Workman, J., Hart, P. E., Mangiarini, L., Mahal, A., Bates, G., et al. (2000). Mitochondrial dysfunction and free radical damage in the Huntington R6/2 transgenic mouse. Ann. Neurol. 47, 80–86. Taketani, S., Tanaka-Yoshioka, A., Masaki, R., Tashiro, Y., and Tokunaga, R. (1986). Association of ferrochelatase with Complex I in bovine heart mitochondria. Biochim. Biophys. Acta 883, 277–283. Tangeras, A. (1986). Effect of decreased ferrochelatase activity on iron and porphyrin content in mitochondria of mice with porphyria induced by griseofulvin. Biochim. Biophys. Acta 882, 77–84. Thomas, P. K., Cooper, J. M., King, R. H., Workman, J. M., Schapira, A. H., Goss-Sampson, M. A., et al. (1993). Myopathy in vitamin E deﬁcient rats: Muscle ﬁbre necrosis associated with disturbances of mitochondrial function. J. Anat. 183, 451–461. Thomas, S. R., Leichtweis, S. B., Pettersson, K., Croft, K. D., Mori, T. A., Brown, A. J., et al. (2001). Dietary cosupplementation with vitamin E and coenzyme Q(10) inhibits atherosclerosis in apolipoprotein E gene knockout mice. Arterioscler. Thromb. Vasc. Biol. 21, 585–593. Trouillas, P., Takayanagi, T., Hallett, M., Currier, R. D., Subramony, S. H., Wessel, K., et al. (1997). International cooperative ataxia rating scale for pharmacological assessment of the cerebellar syndrome. J. Neurol. Sci. 145, 205–211. Waldvogel, D., van Gelderen, P., and Hallett, M. (1999). Increased iron in the dentate nucleus of patients with Friedrich’s ataxia. Ann. Neurol. 46, 123–125. Wilson, R. B., Lynch, D. R., and Fischbeck, K. H. (1998). Normal serum iron and ferritin concentrations in patients with Friedreich’s ataxia. Ann. Neurol. 44, 132–134. Wong, A., Yang, J., Cavadini, P., Gellera, C., Lonnerdal, B., Taroni, F., et al. (1999). The Friedreich’s ataxia mutation confers cellular sensitivity to oxidant stress which is rescued by chelators of iron and calcium and inhibitors of apoptosis. Hum. Mol. Genet. 8, 425–430. Yuvaniyama, P., Agar, J. N., Cash, V. L., Johnson, M. K., and Dean, D. R. (2000). NifS-directed assembly of a transient [2Fe-2S] cluster within the NifU protein. Proc. Natl. Acad. Sci. USA 97, 599–604. Zhang, Y., Aberg, F., Appelkvist, E. L., Dallner, G., and Ernster, L. (1995). Uptake of dietary coenzyme Q supplement is limited in rats. J. Nutr. 125, 446–453. Zheng, L., White, R. H., Cash, V. L., and Dean, D. R. (1994). Mechanism for the desulfurization of L-cysteine catalyzed by the nifS gene product. Biochemistry. 33, 4714–4720. Zheng, L., Cash, V. L., Flint, D. H., and Dean, D. R. (1998). Assembly of iron-sulfur clusters. Identiﬁcation of an iscSUA-hscBA-fdx gene cluster from Azotobacter vinelandii. J. Biol. Chem. 273, 13264–13272.

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WILSON DISEASE

C. A. Davie1 and A. H. V. Schapira University Department of Clinical Neurosciences Royal Free and University College Medical School Royal Free Campus, Rowland Hill Street London NW3 2PF, United Kingdom

I. The Role and Transport of Copper in Health A. Function B. Transport II. Aceruloplasminemia and Menkes’ Disease A. Aceruloplasminemia B. Menkes’ Disease III. Experimental Models of Wilson Disease IV. Mitochondrial Dysfunction in Animal Models V. Mitochondrial Dysfunction in Human Disease VI. Molecular Basis for the Variation in Phenotype VII. Diagnosis VIII. Cranial Magnetic Resonance Imaging and Spectroscopy in Wilson Disease IX. Treatment A. Chelating Therapy B. Inhibition of Copper Absorption from the Intestine C. Liver Transplantation X. Conclusion References

I. The Role and Transport of Copper in Health

A. FUNCTION Copper is an essential element for several cellular processes in humans. Speciﬁc cuproproteins use the redox nature of the metal to permit electron transfer reactions in a number of important metabolic pathways. Copper is an essential converted component for several enzymes. These include the electron transport protein, cytochrome c oxidase. Copper is an important component of cytochrome oxidase (complex IV) of the mitochondrial respiratory chain. Copper also may substitute for iron in redox reactions 1

Author to whom correspondence should be addressed.

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generating free radicals (Halliwell and Gutteridge, 1989). Other important enzyme pathways dependent on copper include the free radical scavenger, superoxide dismutase, and the neurotransmitter, dopa-β-mono-oxygenase. The latter is an enzyme involved in the catecholamine synthesis pathway. The reactivity of copper, in part, accounts for the potential toxicity of this metal when cellular homeostasis is disturbed. To prevent such toxicity, pathways are in place that are essential for delivering copper to speciﬁc sites within the cell. The trafﬁcking of copper along these pathways is mediated by a class of proteins called copper chaperones.

B. TRANSPORT Copper is absorbed from the upper intestine binding to albumin. In this state it is transported to the liver, the principal organ of copper homeostasis. It is within hepatocytes that copper accumulation occurs and where regulation of copper excretion occurs depending on the intracellular concentration of the metal. This regulation is accomplished by the copper transporting ATPase encoded at the Wilson disease (WD). A locus, which is localized to the trans-Golgi network. As the concentration of copper increases within the hepatocyte, this ATPase moves from the trans-Golgi network to a cytoplasmic vesicular compartment near the cannalicular membrane. As copper then accumulates within this vesicular compartment, the decrease in cytoplasmic copper triggers a redistribution of the ATPase back to the trans-Golgi network and copper excretion into bile. In health, this mechanism provides a responsive mechanism to maintain intracellular copper levels and ensures that excess copper is safely disposed of. It is also within hepatocytes that copper is incorporated into ceruloplasmin, a glycoprotein containing six copper atoms. The Wilson ATPase is required for the delivery of copper to the secretory pathway of hepatocytes. Impairment of this mechanism results in a marked reduction in ceruloplasmin synthesis. Most of the ingested copper is excreted in bile, thus preventing copper toxicity. Trace amounts of copper are excreted via the kidneys except in cases of copper overload. In the situation where there is excess copper in either the intestine or the liver, then the metal produces a complex with, in the ﬁrst instance, glutathione and then with metallothioneins. Metallothioneins are proteins that are induced by heavy metals and provide some storage capacity for copper. Copper does not exist in a free state in cells, and it is invariably bound to a peptide or a protein. The presence of excess bound copper in tissue leads to the production of free radicals. This leads to depletion of glutathione and oxidisation of lipids, and cytoskeletal proteins with subsequent DNA disruption.

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Two major disturbances of copper metabolism in WD are reduced incorporation of copper into ceruloplasmin and a decrease in the biliary excretion of copper. Most of the copper in plasma is in the form of ceruloplasmin. In the majority of patients with untreated WD, serum ceruloplasmin is greatly reduced and nonceruloplasmin-bound copper is increased.

II. Aceruloplasminemia and Menkes’ Disease

A. ACERULOPLASMINEMIA A complete absence of ceruloplasmin is found in the disorder of aceruplasminaemia (Harris et al., 1995). Aceruloplasminemia is an autosomal recessive disorder of iron metabolism resulting in diabetes, retinal degeneration, and neurological symptoms. Analysis of affected individuals reveals absent serum ceruloplasmin and evidence of excess iron accumulation in association with inherited mutations of the ceruloplasmin gene. Serum ferritin concentration is markedly elevated in aceruloplasminaemia and liver biopsy reveals increased hepatic iron content with abundant iron in hepatocytes and reticuloendothelial cells. Clinical symptoms include progressive dementia, extrapyramidal disorders, cerebellar ataxia, and diabetes mellitus, all of which may appear in adulthood. The dentate nucleus, thalamus, putamen, caudate nucleus, and liver may show low signal intensities on T1- and T2-weighted magnetic resonance imaging (Morita et al., 1995). Pathological examination of the central nervous system has revealed severe destruction of the basal ganglia and dentate nucleus, with considerable iron deposition in neuronal and glial cells, whereas the cerebral cortex tends to show mild iron deposition in glial cells without neuronal involvement. An electron microscopic study with energy-dispersive X-ray analysis has shown iron deposition in both the neural and glial cells of the brain.

B. MENKES’ DISEASE Menkes’ disease is caused by a defect in the copper transporter ATP7A present on the X chromosome at Xq13. It occurs with a frequency of approximately 1 in 200,000. The Menkes’ disease ATPase transports copper across the placenta, the gastrointestinal tract and the blood–brain barrier. The clinical features of this disease arise from copper deﬁciency. Patients

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have hypopigmented hair with an unusual structure (often called steely or kinky hair) caused by reduced keratin crosslinking, a process dependent on an uncharacterized copper oxidase (Kaler, 1994). Connective tissue abnormalities arise, including aneurysms, hyperelastosis, and brittle bones (Bankier, 1995). Intellectual development is frequently affected. Epilepsy and infantile spasms are common (Sfaello et al., 2000). Clinical manifestations begin in the ﬁrst few months of life or even in the neonatal period. Hypothermia, hypotonia, poor weight gain, seizures, and neurodevelopmental delay or regression are seen. Outcome is poor, with death occurring usually by three years of age (Tumer and Horn, 1997). A characteristic facial appearance with steely hair suggest the diagnosis. Neuroimaging usually shows cortical atrophy, extra-axial ﬂuid collections and progressive and extensive degeneration of grey matter with secondary demyelination. A number of milder clinical variants occur (including the occipital horn syndrome), probably due to partial expression of the copper transporter protein. A low serum copper level and a high 64Cu uptake in ﬁbroblasts conﬁrm the diagnosis. Menkes’ disease has on occasion been successfully treated with parenteral administration of copper in the form of copper histidine, but this needs to be started within one month of birth (Sarkar et al., 1993).

III. Experimental Models of Wilson Disease

There are a number of commonly used animal models for WD, though none of these express a neurological phenotype. The toxic milk mouse is a spontaneously arising mutation characterized by severe copper deﬁciency in newborn mice fed on milk from the mutant mother. Adult toxic milk mice develop hepatic copper overload together with decreased serum ceruloplasmin. A transgenic mouse model has been developed with a deletion of the murine WD gene (Buiakova et al., 1999). These mice produce signiﬁcant hepatic copper overload by 6–8 weeks. The Long Evans Cinnamon (LEC) rat is a spontaneously arising mutation that contains an intragenic deletion of the rat orthologue of the WD gene. These rats develop marked hepatic copper overload together with decreased serum ceruloplasmin and impaired biliary copper excretion. This phenotype can be reversed by expression of the human WD gene in the liver mediated via a recombinant adenovirus (Terada et al., 1998). These animals develop acute hepatitis and fulminant liver failure that is responsive to copper chelating therapy.

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The Bedlington terrier also produces a hepatic phenotype. This model does not have a defect in the ATP7B gene and the gene defect remains to be identiﬁed.

IV. Mitochondrial Dysfunction in Animal Models

A number of studies in animal models have found supportive evidence for mitochondrial dysfunction in WD. Studies in the LEC rat suggest that copper toxicity is mediated via lysosomal uptake and incomplete degradation of copper-loaded metallothionein, which then promotes lipid peroxidation and hepatocellular damage (Klein et al., 1998). Subcellular fraction analysis showed copper accumulation predominantly in lysosomes (Terada et al., 1995; Schilsky, 1996), but also increased levels of copper accumulation in mitochondria (Klein et al., 1998). Copper overload in healthy rats also results in oxidative damage to liver (Sokol et al., 1990) but only an isolated deﬁciency of mitochondrial complex IV (Sokol et al., 1993). This suggests that oxidative stress plays an important role in the liver injury in experimental hepatic copper overload (Sokol et al., 1993). Ultrastructural studies have shown various morphological mitochondrial changes in the mitochondria from the liver of the LEC rat (Sternlieb et al., 1995). A number of key mitochondria enzymes require copper as a cofactor. The Cu2+-mediated regulation of cytochrome P450-dependent steroidogenic activity has been observed in the adrenal mitochondria of the rat (Veltman and Maines, 1986). It has also been shown that copper at low concentrations stimulates the activity of ferrochetalase, the key enzyme in heme synthesis, but that high concentrations of copper were inhibitory for biosynthesis of haem (Wagner and Tephly, 1975). LEC rats fed a diet supplemented with vitamin E developed abnormal liver function later than controls, whereas those on vitamin-E-deﬁcient diets had defective liver function before controls—supporting a role for free-radical-mediated damage in this model (Yamazaki et al., 1993). The mitochondrial respiratory chain is an important source of reactive oxygen species and high intramitochondrial copper concentrations might be expected to increase free-radical generation. The link between production of mitochondrial-reactive oxygen species and abnormalities of the respiratory chain and oxidative phosphorylation (OXPHOS) system has been shown in mice. Knockout of the gene encoding the mitochondrial freeradical-scavenging enzyme manganese superoxide dismutase 2 (SOD2) results in deﬁciency of respiratory-chain complexes I–III and aconitase and neuronal death (Melov et al., 1999).

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V. Mitochondrial Dysfunction in Human Disease

A number of studies in humans suggest that mitochondrial dysfunction plays an important role in the pathophysiology of WD. It has been demonstrated that the WD protein is present in cells in two forms, the 160-kDa and the 140-kDa products. The 160-kDa product is targeted to the trans-Golgi network (Hung et al., 1997). One study has suggested that the 140-kDa product is located in mitochondria (Lutsenko and Cooper, 1998), although this observation awaits conﬁrmation. Thus, the WD protein is a P-type ATPase with an unusual subcellular localization. Mitochondrial targeting of the WD protein would suggest an important role for copper-dependent processes taking place in this organelle. The molecular mechanism underlying the copper-induced changes in the subcellular distribution of the WD protein is unknown. It has been suggested that the N-terminal domains of the protein, which speciﬁcally bind copper, may play an important role in copper-dependent targeting of the WD protein to various cell membranes. Three distinct patterns of structural abnormalities of mitochondria have been identiﬁed in the hepatocytes of asymptomatic and symptomatic WD patients before treatment. Comparison of the types of abnormal hepatocellular mitochondria displayed by ﬁve pairs and one trio of asymptomatic siblings revealed remarkably similar types of abnormalities in each family, indicating that these structural changes are genetically determined (Sternlieb, 1992). Interestingly, these characteristic morphological abnormalities are also seen in the mitochondria of LEC rats (Sternlieb et al., 1995). Gu et al. (2000) studied liver tissue from patients with WD, and patients with and without liver disease. The liver tissue was taken at the time of liver transplantation or liver resection. The authors studied mitochondrial function and aconitase activity in WD liver tissue, and compared the results with those in a series of healthy controls and patients without WD. There was evidence of severe mitochondrial dysfunction in the livers of patients with WD. Enzyme activities were decreased as follows: complex I by 62%, complex II + III by 52%, complex IV by 33%, and aconitase by 71%. These defects did not appear to be secondary to penicillamine use, cholestasis, or poor hepatocellular synthetic function. There thus seems to be quite a widespread defect of energy metabolism in WD. The pattern of enzyme defects suggests that free-radical formation and oxidative damage, probably mediated via mitochondrial copper accumulation, are important in WD pathogenesis. The pattern of enzyme defect observed in the liver of patients with WD from the study by Gu et al. (2000) is similar to that reported in the mouse

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model with an inactive superoxide dimutase (SOD2) gene (Melov et al., 1999)—an established model of oxidative stress and damage. The OXPHOS and aconitase defects in WD may reﬂect free radical-induced damage to mitochondrial function. This is supported by a study showing increased lipid peroxidation in the liver of three patients with WD (Sokol et al., 1994). Furthermore in a separate study, 50% of WD patients showed single or multiple mitochondrial DNA deletions from hepatic tissue compared to only 2 of 67 older controls (Mansouri et al., 1997). Such deletions might be seen in response to oxidative stress or in senescent tissue, and it has been suggested that the mitochondrial DNA deletions may have arisen through copperinduced oxidative damage. One study has shown low plasma ascorbate and urate concentrations in untreated patients with WD, again suggesting increased free-radical generation (Ogikara et al., 1995). Low levels of plasma vitamin E have been demonstrated in WD patients (von Herbay et al., 1994). It has also been demonstrated that a higher concentration of copper accumulates in hepatocellular mitochondria in humans with WD compared to patients with other chronic liver diseases (Sokol et al., 1994). Studies on the subcellular distribution of copper in WD have shown it to be present in the particulate fraction (Goldﬁscher and Sternlieb, 1968) and signiﬁcantly increased in a hepatic mitochondrial fraction in WD (Sokol et al., 1994). Increased concentrations of mitochondrial copper in WD may be the direct result of defective intracellular copper transport, which in turn is caused by dysfunction of the P-type ATPase. For instance, the WD protein might function as a copper transporter at both the trans-Golgi network and at the mitochondrial membrane, and mutations in ATP7B may cause mitochondrial copper overload through defective export. Copper accumulation might be associated with increased oxidative stress and damage within target tissues where copper concentrations are high. This is not dissimilar to Friedreich’s ataxia, where frataxin deﬁciency is associated with intramitochondrial iron accumulation, free-radical damage, and OXPHOS and aconitase defects (see preceding Friedreich’s ataxia chapter). Furthermore, mutations in the Menkes’ protein, with which the WD protein shares substantial homology, are known to cause failure of plasma-membrane localization of this protein (Goodyer et al., 1999) and therefore impaired copper trafﬁcking. However, it is possible that some of these defects in mitochondrial function occur as a secondary effect related to ongoing biochemical disturbances such as cholestasis, impaired hepatic protein synthesis or even as a consequence of treatment with chelating therapy. It seems likely, however, that mutations in the ATP7B gene result in abnormal copper transport and high concentrations of intramitochondrial copper, which in turn induce free-radical-mediated damage.

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VI. Molecular Basis for the Variation in Phenotype

Wilson disease is an autosomal recessive disorder with an incidence of between 1 in 35,000 and one in 100,000. The associated carrier frequency is approximately 1 in 90. The age of disease onset in WD is highly variable from the age of two to over ﬁfty. The clinical presentation can also be highly variable (Robert and Cox, 1998): acute or chronic liver disease, acute hemolytic crisis, psychiatric manifestations with acute psychosis, and progressive neurological disease without clinically evident hepatic disease. In those patients presenting with neurological manifestations (Walshe and Yealland, 1992), it may present with a complex tremor, which is present at rest increasing posturally. Other well-recognized neurological presentations include dysarthia and dysphagia, dystonic posturing, an akinetic rigid syndrome, choreoathetosis, cerebellar ataxia, action myoclonus, or a gradual impairment in cognition. Unlike Parkinson’s disease, the disorder of motor function tends to start in the bulbar muscles and progress caudally. Patients can on occasion present with seizures. It is a truism that Kayser-Fleischer (KF) rings will be present with the onset of neurological symptoms. However, these rings, which are due to copper deposition in Descemet’s membrane in the cornea, may only be picked up with slit-lamp examination, particularly in brown-eyed individuals. Although a useful diagnostic ﬁnding, KF rings are not speciﬁc to patients with WD, occurring also in adults with predominantly cholestatic chronic liver disease. Kayser-Fleischer rings will normally disappear with effective chelating therapy. Another ophthalmological ﬁnding is the presence of sunﬂower cataracts. The reason for such diversity of clinical presentation is not entirely clear but is in part related to the large number of genetic mutations that have been associated with the disease. Over 200 mutations have been identiﬁed and are listed in a database (Kenney and Cox, 2001). The most common mutation, His1069Gln, accounts for between 10 and 70% of all mutations identiﬁed in patients of European and North American origin (Maier-Dobersberger et al., 1997). In other continents such as Asia, however, this mutation is rare. The mutations described include single base insertions and deletions, nonsense, and splice site mutations (Riordan and Williams, 2001). The gene for WD found on chromosome 13 and termed ATP7B was ﬁrst discovered in 1993 shortly after the identiﬁcation for the gene for Menkes’ disease with which it shares a high degree of homology (Tanzi et al., 1993). The correlation between genotype and phenotype, however, is complicated by the fact that many WD patients are compound heterozygotes, i.e., they carry different ATP7B mutations on each chromosome 13. Patients who are

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homozygous for severe mutations have an earlier onset of disease. One study in Icelandic families suggests that milder mutations may present with later onset neurological disease (Thomas et al., 1995).

VII. Diagnosis

In those patients presenting with liver disease, neurological impairment, and KF rings, the diagnosis is straightforward. Unfortunately, this is seldom the case and a high index of suspicion is necessary. Routine liver function tests may be impaired. Serum ceruloplasmin levels are usually very low but can be normal, particularly in those patients with a hepatic presentation (Steindl et al., 1998). The use of low ceruloplasmin for diagnostic purposes is therefore unreliable. Ceruloplasmin is an acute phase protein, and its serum level can be elevated in hepatic inﬂammation, in pregnancy, and in patients taking oestrogen. Furthermore, a low ceruloplasmin level is not speciﬁc for WD. Ceruloplasmin synthesis is reduced in acute liver failure and from decompensated cirrhosis due to any cause. Serum ceruloplasmin levels are also reduced in nephrotic syndrome and protein losing enteropathies. Furthermore, low ceruloplasmin levels can be observed in heterozygote carriers of the disease. In most patients, serum copper concentration is low. The nonceruloplasmin bound copper is elevated. Measurements of urinary copper excretion may be useful. Elevated urinary excretion of copper in a 24-h collection is highly suggestive of WD (assuming lack of contamination from such sources as tap water). Several separate 24-h collections of urine should be made. It can be particularly useful to perform a 24-h urine collection with concomitant administration of oral penicillamine 500 mg 12 hourly. This produces a considerable increase in patients with WD compared to controls. Hepatic copper content greater than 250 μg/g dry weight is regarded as diagnostic of WD. Some patients and heterozygote carriers may have a less marked elevation.

VIII. Cranial Magnetic Resonance Imaging and Spectroscopy in Wilson Disease

In patients with a neurological or psychiatric presentation, Magnetic Resonance Imaging (MRI) can show a number of abnormalities (King et al., 1996; Saatci et al., 1997). On T2-weighted images, WD is suggested by atrophy; putaminal lesions with a pattern of symmetric, bilateral, concentric-laminar T2 hyperintensity; and the involvement of the pars compacta of the substantia

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nigra, the periaqueductal gray matter, the pontine tegmentum, and the thalamus. The hepatic component of WD may cause increased T1 signal intensity in basal ganglia. In the adult age group, the basal ganglia lesions may be different from those in the pediatric group; the putaminal lesions may not be present; the globus pallidus and substantia nigra may show increased hypointensity on T2-weighted images. Cortical and subcortical lesions also may be present with a predilection for the frontal lobe. The paramagnetic effects of copper are detected only in untreated patients. The majority of patients who are neurologically symptomatic will have abnormal brain imaging. Patients with a longer duration of (treated) disease have less severe changes in signal intensity. The MR lesions can be reversed with successful chelating therapy. Magnetic resonance imaging seems to be of limited value in follow-up. Magnetic resonance spectroscopy (MRS) is an in vivo and in vitro technique that allows the quantitation of a number of brain metabolites. The ability of MRS to measure neurochemicals in brain volumes less than 1 mL provides a unique “window” into metabolic and neurodegenerative processes. MRS is especially useful because it allows quantiﬁcation of different chemicals in a single study, which can be repeated many times. N-acetyl aspartate (NAA) is one such amino acid that can be measured in vivo using MRS. N-acetyl aspartate is predominantly localized to neurones and has been used as a measure of neuronal viability. A number of studies have shown reversible changes in NAA in human disease. N-acetyl aspartate is synthesized within mitochondria. The concentration of NAA is reduced when mitochondrial function is inhibited in vitro. Similarly, a reduction of NAA has been observed in clinical mitochondrial disorders. Furthermore, reversibility of NAA has been demonstrated following clinical recovery in patients with mitochondrial encephalopathy with lactic acidosis and strokelike episodes (MELAS) (Kamada et al., 2001). It may be feasible, therefore, to use changes in NAA as a surrogate marker of neuronal viability and as a means of assessing the efﬁcacy of therapeutic agents to prevent cell death. A number of studies have shown a reduction of NAA from the striatum in patients with WD (Alanen et al., 1999; Kraft et al., 1999). A study by Page et al. (2001) has shown a far greater absolute reduction of NAA from the striatum in WD patients presenting with a neurological phenotype compared to WD patients with no neurological features. The lower levels of NAA in the patients with a neurological phenotype suggests persistent neuronal dysfunction as a result of copper deposition in the striatum. Given that NAA is synthesized by mitochondria, the reduction of NAA detected from the striatum may be indicative of mitochondrial dysfunction rather than neuronal death. It is not yet established whether this in vivo technique could provide a surrogate measure of neuronal recovery in patients receiving treatment therapies for WD.

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IX. Treatment

Current treatment regimes for WD are based on the restoration of copper homeostasis by the systemic chelation of copper or the prevention of copper absorption from the gut. The earlier that this can be achieved, the greater the chance of avoiding permanent neurological impairment. The prognosis is extremely good for patients treated early, and even after the development of symptoms, full recovery back to normal may still be achieved.

A. CHELATING THERAPY 1. Penicillamine The most frequently used chelating therapy is penicillamine introduced by J. M. Walshe in 1965 (Walshe and Patston, 1965). Penicillamine increases urinary excretion of copper, thus preventing further deposition of copper. Therapy with penicillamine is initiated with a test oral dose, and if tolerated, is given four times daily. Most patients will be asymptomatic within four months. However, neurological symptoms and signs have been reported to worsen in some patients following the initiation of therapy. This occurs as a result of deposition of mobilized hepatic copper within the basal ganglia. Most, though not all, such patients will recover with continued use (Walshe and Yealland, 1993). Once neurological improvement occurs and total body copper has been reduced, the dose is halved and continued as lifelong maintenance treatment. Compliance is extremely important since some patients develop rapid worsening of liver or neurological function with sudden cessation of the drug. This is likely to occur secondary to copper release from storage tissue (Schilsky, 1996). Side effects are fairly frequent and can be severe. These include thrombocytopenia, leucopenia, and rarely, aplastic anemia. A myasthenia gravis like syndrome may occur, as may a condition resembling systemic lupus erythematosus. Other serious adverse effects include nephrotic syndrome and Goodpasture’s syndrome. The development of such events requires immediate withdrawal of the drug. Less serious side effects include rashes, diarrhea, arthralgia, and loss of taste. Approximately 20–30% of WD patients develop side effects on penicillamine requiring a change of treatment (Walshe, 1989). Severe neurological disease may not resolve entirely on treatment. 2. Trientene The second line chelating therapy is trientene. This differs from penicillamine by lacking sulphydryl groups. Although it is less potent than penicillamine, this does not appear to affect its clinical efﬁcacy. It tends

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to be well tolerated with little signiﬁcant toxicity. It may produce gastric disturbance, iron deﬁciency due to iron chelation, or a sideroblastic anemia. The adverse effects of penicillamine resolve and do not recur with trientene. Penicillamine and trientene have both been used successfully in pregnancy, and should remain the mainstay of treatment in such circumstances (Sternlieb, 2000). Pyridoxine supplementation should be given during pregnancy with concurrent penicillamine or trientene treatment. In addition to chelation therapy, dietary copper should be restricted.

B. INHIBITION OF COPPER ABSORPTION FROM THE INTESTINE 1. Zinc Zinc is a more recent therapeutic option for WD and acts by interfering with absorption of copper from the gastrointestinal tract, inducing metallothien in enterocytes. Metallothien preferentially binds copper present in the gut because it has greater afﬁnity for copper than zinc. As a bound product, copper is excreted as enterocytes are shed. Gastric upset is a common but relatively minor side effect. This side effect may be exacerbated by the need to take zinc separately from meal times since food interferes with its effectiveness. Although one study showed a similar efﬁcacy and fewer side effects than penicillamine (Czlonkowska et al., 1996), there have been reported cases of neurological deterioration occurring while on zinc therapy (Walshe and Munro, 1995). 2. Terathiomolybdate Ammonium tetrathiomolybdate works by forming a complex with protein and copper. It interferes with copper absorption from the gut. It is able to be absorbed itself and it can bind to plasma copper with high afﬁnity. It may have a role in a few speciﬁc patients since, unlike penicillamine, it is not associated with early neurological deterioration at initiation.

C. LIVER TRANSPLANTATION Liver transplantation tends to be reserved for patients presenting with severe decompensated liver disease refractory to medical therapy or patients presenting in fulminant hepatic failure. This corrects the metabolic disorder to the extent that chelating therapy is no longer necessary. Neurological improvement following liver transplantation has been reported (Polson et al., 1987; Chen et al., 1997) but experience remains limited.

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There may clearly be a role for agents that protect against mitochondrial dysfunction in WD. It is certainly possible that factors producing oxidative stress may inﬂuence the course of the disease. Potential cytoprotective mechanisms include inducing cellular stores of glutathione, and taking vitamin E and/or coenzyme Q10. The signiﬁcant defects in oxidative function and mitochondrial dysfunction discussed earlier provide a strong case for assessing the beneﬁt of the co-administration of free radical scavengers and penicillamine in the treatment of WD.

X. Conclusion

Copper is an essential trace metal that plays a fundamental role in the biochemistry of the human nervous system. Wilson disease is an inherited disorder of copper metabolism, and the dramatic neuropsychiatric phenotypes of the disease underscore the toxicity of this metal when neuronal copper homeostasis is perturbed. A growing body of evidence has demonstrated mitochondrial dysfunction in WD. Our understanding of copper homeostasis in WD may provide further insights into other disorders of the CNS. Gain-of-function mutations in the cytosolic copper enzyme superoxide dismutase result in the motor neuron degeneration of amyotrophic lateral sclerosis, and current evidence suggests a direct pathogenic role for copper in this process (Lyons et al., 2000). Recent studies have also implicated copper in the pathogenesis of neuronal injury in Alzheimer’s disease (White et al., 1999) and the prionmediated encephalopathies (Pauly and Harris, 1998). Further elucidation of the mechanisms of copper trafﬁcking and metabolism within the nervous system will be of direct relevance to WD, but also may be of great value to our understanding of the pathophysiology and the treatment of a wide range of neurodegenerative diseases. References

Alanen, A., Komu, M., Penttinen, M., and Leino, R. (1999). Magnetic resonance imaging and proton MR spectroscopy in Wilson’s disease. Br. J. Radiol. 72, 749–756. Bankier, A. (1995). Menkes disease. J. Med. Genet. 32, 213–215. Buiakova, O. I., Xu, J., Lutsenko, S., Zeitlin, S., Das, K., Das, S., et al. (1999). Null mutation of the murine ATP7B (Wilson’s disease) gene results in intracellular copper accumulation and late-onset hepatic nodular transformation. Hum. Mol. Genet. 8, 1665–1671.

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Chen, C. L., Chen, Y. S., Lui, C. C., and Hsu, S. P. (1997). Neurological improvement of Wilson’s disease after liver transplantation. Transplant. Proc. 29, 497–498. Czlonkowska, A., Gajda, J., and Rodo, M. (1996). Effects of long-term treatment in Wilson’s disease with D-penicillamine and zinc sulphate. J. Neurol. 243, 269–273. Gu, M., Cooper, M., Butler, P., Walker, A. P., Mistry, P., Dooley, J. S., and Schapira, A. H. V. (2000). Oxidative-phosphorylation defects in liver of patients with Wilson’s disease. Lancet 356, 469–474. Goldﬁscher, S., and Sternlieb, I. (1968). Changes in the distribution of hepatic copper in relation to the progression of Wilson’s disease (hepatolenticular degeneration). Am. J. Pathol. 53, 883–901. Goodyer, A. I., Jones, E. E., Monaco, A. P., and Francis, M. J. (1999). Characterization of the Menkes protein copper-binding domains and their role in copper-induced protein relocalization. Hum. Mol. Genet. 8, 1473–1478. Halliwell, B., and Gutteridge, J. M. C. (1989). “Free Radicals in Biology and Medicine,” New York, Oxford University Press. Harris, Z. L., Takahashi, Y., Miyajima, H., Serizawa, M., MacGillivray, R. T., and Gitlin, J. D. (1995). Aceruloplasminemia: Molecular characterization of this disorder of iron metabolism. Proc. Natl. Acad. Sci. USA 92, 2539–2543. Hung, I. H. M., Suzuki, M., Yamaguchi, Y., Yuan, D. S., Klausner, R. D., and Gitlin, J. D. (1997). Biochemical characterization of the Wilson disease protein and functional expression of HaH1, a novel human gene involved in copper homeostasis. J. Biol. Chem. 272, 21461– 21466. Kaler, S. G. (1994). Menkes disease. Adv. Paediatr. 41, 262–303. Kamada, K., Takeuchi, F., Houkin, K., Kitagawa, M., Kuriki, S., Ogata, A., et al. (2001). Reversible brain dysfunction in MELAS:MEG and 1H MRS analysis. J. Neurol. Neurosurg. Psychiat. 70, 675–678. Kenney, S., and Cox, D. W. Wilson disease mutation database. http://www.medgen.med. ualberta.ca/database.html. King, A. D., Walshe, J. M., Kendall, B. E., Chinn, R. J., Paley, M. N., Wilkinson, I. D., et al. (1996). Cranial MR imaging in Wilson’s disease. Am. J. Roentgenol. 167, 1579–1584. Klein, D., Lichtmannegger, J., Heinzmann, U., Muller-Hocker, J., Michaelsen, S., and Summer, K. H. (1998). Association of copper to metallothionein in hepatic lysosomes of Long-Evans cinnamon (LEC) rats during the development of hepatitis [see comments]. Eur. J. Clin. Invest. 4, 302–310. Kraft, E., Trenkwalder, C., Then Bergh, F., and Auer, D. P. (1999). Magnetic resonance spectroscopy of the brain in Wilson’s disease. J. Neurol. 246, 693–699. Lutsenko, S., and Cooper, M. J. (1998). Localization of the Wilson’s disease protein product to mitochondria. Proc. Natl. Acad. Sci. USA 95, 6004–6009. Lyons, T. J., Nersissian, A., Huang, H., Yeom, H., Nishia, C. R., Graden, J. A., et al. (2000). The metal binding properties of the zinc site of yeast copper-zinc superoxide dismutase: Implications for amyotrophic lateral sclerosis. J. Biol. Inorg. Chem. 5, 189–203. Maier-Dobersberger, T., Ferenci, P., Polli, C., Balac, P., Dienes, H. P., Kaserer, K., et al. (1997). Detection of the His1069Gln mutation in Wilson disease by rapid polymerase chain reaction. Ann. Int. Med. 127, 21–26. Mansouri, A., Gaou, I., Fromenty, B., Berson, A., Letteron, P., Degott, C., et al. (1997). Premature oxidative aging of hepatic mitochondrial DNA in Wilson’s disease. Gastroenterology 113, 599–605. Melov, S., Coskun, P., Patel, M., Tuinstra, R., Cottrell, B., Jun, A. S., et al. (1999). Mitochondrial disease in superoxide dismutase 2 mutant mice. Proc. Natl. Acad. Sci. USA 96, 746– 751.

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Morita, H., Ikeda, S., Yamamoto, K., Morita, S., Yoshida, K., Nomoto, S., et al. (1995). Hereditary ceruloplasmin deﬁciency with hemosiderosis: A clinicopathological study of a Japanese family. Ann. Neurol. 37, 646–656. Ogihara, H., Ogihara, T., Miki, M., Yasuda, H., and Mino, M. (1995). Plasma and antioxidant status in Wilson’s disease. Pediatr. Res. 37, 219–226. Page, R. A., Davie, C. A., MacManus, D., Dooley, J., Walshe, J., Miller, D. H., et al. (2001). Magnetic resonance spectroscopy of patients with Wilson’s disease. J. Neurol. Neurosurg. Psychiat. 70, 274–275. Pauly, P. C., and Harris, D. (1998). Copper stimulates endocytosis of the prion protein. J. Biol. Chem. 273, 33107–33110. Polson, R. J., Rolles, K., Calne, R. Y., Williams, R., and Marsden, D. (1987). Reversal of severe neurological manifestations of Wilson’s disease following orthoptic liver transplantation. Q. J. Med. 64, 685–691. Riordan, S. M., and Williams, R. (2001). The Wilson’s disease gene and phenotypic diversity. J. Hepatol. 34, 165–171. Robert, E. A., and Cox, D. W. (1998).). Wilson disease. Balliere’s Clin. Gastroenterol. 12, 237–256. Saatci, I., Topcu, M., Baltaoglu, F. F., Kose, G., Yalaz, K., Renda, Y., and Besim, A. (1997). Cranial MR ﬁndings in Wilson’s disease. Acta Radiol. 38, 250–258. Sarkar, B., Lingertat-Walsh, K., and Clarke, J. T. (1993). Copper-histidine therapy for Menkes disease. J. Pediatr. 123, 828–830. Schilsky, M. (1996). Wilson disease. Genetic basis of copper toxicity and natural history. Semin. Liver Dis. 16, 83–95. Sfaello, I., Castelnau, P., Blanc, N., Ogier, H., Evrard, P., and Arzimanoglou, A. (2000). Infantile spasms and Menkes disease. Epileptic Disord. 2, 227–230. Sokol, R. J., Devereaux, M., Mierau, G. W., Hambridge, K. M., and Shikes, R. H. (1990). Oxidant injury to hepatic mitochondrial lipids in rats with dietary copper overload: modiﬁcation by vitamin E deﬁciency. Gastroenterology 9, 1061–1071. Sokol, R. J., Devereaux, M. W., O’Brien, K., Khandwala, R. A., and Loehr, J. P. (1993). Abnormal hepatic mitochondrial respiration and cytochrome c oxidase activity in rats with long-term copper overload. Gastroenterology 105, 178–187. Sokol, R. J., Twedt, D., McKim, J. M., Devereaux, M. W., Karrer, F. M., Kam, I., et al. (1994). Oxidant injury to hepatic mitochondria in patients with Wilson’s disease and Bedlington terriers with copper toxicosis. Gastroenterology 107, 1788–1798. Steindl, P., Ferenci, P., Dienes, H. P., Grimm, G., Paninger, I, Madl, C., et al. (1998). Wilson’s disease in patients with liver disease: A diagnostic challenge. Gastroenterology 113, 212–218. Sternlieb, I. (1992). Fraternal concordance of types of abnormal hepatocellular mitochondria in Wilson’s disease. Hepatology 16, 728–732. Sternlieb, I. (2000). Wilson’s disease and pregnancy. Hepatology 31, 531–532. Sternlieb, I., Quintans, N., Volenberg, I., and Schilsky, M. L. (1995). An array of mitochondrial alterations in the hepatocytes of Long-Evans Cinnamon rats. Hepatology 22, 1782–1787. Tanzi, R. E., Petrukhin, K., Chernov, I., Pellequer, J. L., Wasco, W., Ross, B., et al. (1999). The Wilson disease gene is a copper transporting ATPase with homology to the Menkes disease gene. Nat. Genet. 5, 344–350. Terada, K., Kawarada, Y., Miura, N., Yasui, O., Koyama, K., and Sugiyama, T. (1995). Copper incorporation into caeroplasmin in rat livers. Biochem. Biophys. Acta 1270, 58–62. Terada, K., Nakako, T., Yang, X.-L., Iida, M., Aiba, N., Minamiya, Y., et al. (1998). Restoration of holoceruloplasmin synthesis in LEC rat after infusion of recombinant adenovirus bearing WND cDNA. J. Biol. Chem. 273, 1815–1820. Thomas, G. R., Jensson, O., Gudmundsson, G., Thorsteinsson, L., and Cox, D. W. (1995). Wilson Disease in Iceland: A clinical and genetic study. Am. J. Hum. Genet. 56, 1140–1146.

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Tumer, Z., and Horn, N. (1997). Menkes disease: Recent advances and new aspects. J. Med. Genet. 34, 265–274. Veltman, J. C., and Maines, M. D. (1986). Regulatory effect of copper on rat adrenal cytochrome P-450 and steroid metabolism. Biochem. Pharmacol. 35, 2903–2909. von Herbay, A., de Groot, H., Hegi, U., Stremmel, W., Strohmeyer, G., and Sies, H. (1994). Low vitamin E content in plasma of patients with alcoholic liver disease, hemochromatosis and Wilson’s disease. J. Hepatol. 20, 41–46. Wagner, G. S., and Tephly, T. R. (1975). A possible role of copper in the regulation of heme biosynthesis through ferrochelatase. Adv. Exp. Med. Biol. 58, 343–354. Walshe, J. M. (1989). Wilson’s disease presenting with hepatic dysfunction: A clinical analysis of eighty-seven patients. Q. J. Med. 70, 253–263. Walshe, J. M., and Munro, N. A. (1995). Zinc induced deterioration in Wilson’s disease aborted by treatment with penicillamine, dimercaprol, and a novel zero copper diet. J. Neurol. Neurosurg. Psychiat. 55, 692–696. Walshe, J. M., and Patston, V. (1965). Effect of penicillamine on serum iron. Arch. Dis. Child. 40, 651–653. Walshe, J. M., and Yealland, M. (1992). Wilson’s disease: The problem of delayed diagnosis. J. Neurol. Neurosurg. Psychiat. 55, 692–696. Walshe, J. M., and Yealland, M. (1993). Chelation treatment of neurological Wilson’s disease. Q. J. Med. 86, 197–204. White, A. R., Multhaup, G., Maher, F., Bellingham, S., Camakaris, J., Zheng, H., et al. (1999). The Alzheimer’s disease amyloid precursor protein modulates copper-induced toxicity and oxidative stress in primary neuronal cultures. J. Neurosci. 19, 9170–9179. Yamazaki, K., Ohyama, H., Kurata, K., and Wakabayashi, T. (1993). Effects of dietary vitamin E on clinical course and plasma glutamic, oxaloacetic, transaminase and glutamic pyruvic transaminase activities in hereditary hepatitis of LEC rats. Lab. Animal Sci. 43, 61–67.

HEREDITARY SPASTIC PARAPLEGIA

Christopher J. McDermott1 and Pamela J. Shaw Academic Neurology Unit University of Shefﬁeld Medical School Royal Hallamshire Hospital Shefﬁeld S10 2RX, United Kingdom

I. Introduction II. Clinical Features III. Genetics A. Autosomal-Recessive HSP (SPG7) B. Autosomal-Recessive HSP (SPG5, SPG11, and SPG14) C. Autosomal-Dominant HSP D. X-linked HSP E. Mitochondria, Neurodegeneration, and HSP References

I. Introduction

Hereditary spastic paraparesis (HSP) represents a group of inherited disorders in which the predominant clinical feature is progressive lower limb spasticity. However, these disorders are both clinically and genetically heterogeneous. Fourteen gene loci (SPG1–14) have been associated with a HSP phenotype, with X-linked, autosomal-recessive, and autosomal dominant inheritance all described (Fontaine et al., 2000; McDermott et al., 2000; Reid et al., 2000; Vazza et al., 2000). The gene at one of the autosomal recessive loci SPG7 has been identiﬁed as paraplegin (Casari et al., 1998). The identiﬁcation of paraplegin as a nuclear-encoded mitochondrial metalloprotease and the association of paraplegin mutation with impairment of oxidative phosphorylation was the ﬁrst evidence that mitochondrial disturbance may play a role in the development of the HSP phenotype. In this chapter we review the hereditary spastic paraplegias with particular reference to mitochondrial dysfunction. 1

Author to whom corespondence should be addressed.

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II. Clinical Features

Hereditary spastic paraparesis can be classiﬁed clinically into either pure or complicated forms (Harding, 1981). In pure HSP the spastic paraparesis is the dominant ﬁnding. Patients can present at any age with marked intraand interfamilial variation occurring in age at onset of symptoms. The ﬁrst complaints are most commonly difﬁculty running, stiffness, premature wear on shoes, or other people’s comments on a gait abnormality. As the disease progresses, lower limb stiffness and the resultant gait disturbance are the

TABLE I CLINICAL FEATURES THAT MAY BE OBSERVED IN ADDITION TO SPASTIC PARAPARESIS IN COMPLICATED HEREDITARY SPASTIC PARAPARESIS Clinical feature Amyotrophy

Cardiac defects Cerebellar signs Deafness Dementia

Epilepsy Extrapyramidal signs Sj¨ogren-Larsson syndrome Sensory neuropathy

Retinal changes Kallmann’s syndrome Hyperekplexia MASA syndrome Mast syndrome Kjellin syndrome SPG9

Description Muscle involvement can be limited to severe wasting of the small muscles of the hand, or be more generalized. In the Troyer syndrome amyotrophy is associated with delayed development, spastic quadraparesis, pseudobulbar palsy, choreathetosis, and short stature. Associated with mental retardation Dysarthria with a mild upper limb ataxia Sensorineural Dementia can occur in isolation with HSP, when it tends to be of the subcortical type, or be part of a much more complex phenotype. Linkage to SPG4 locus in a number of families. Myoclonic, simple/complex partial, absence, and grand mal seizure types are all described. Choreoathetosis, dystonia, and rigidity have been described. Icthyosis also with mental retardation and occasionally a pigmentary macular degeneration. If childhood onset, tends to be associated with painless ulcers and deformities secondary to neuropathic bone resorption. In adult onset trophic skin changes and foot ulcers are seen. The neuropathy may also be mild and asymptomatic. Optic atrophy and retinal degeneration. Hypogonadotrophic hypogonadism and anosmia. Neonatal hypertonia and an exaggerated startle response. Mental retardation, aphasia, a shufﬂing gait, and adducted thumbs. Caused by mutations in L1CAM gene (X-linked). Dementia, dysarthria, and athetosis in Amish people with onset in second decade. Dysarthria, upper limb ataxia, dementia, retinal degeneration ± amyotrophy Bilateral cataracts, gastroesophageal reﬂux, and amyotrophy.

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major symptoms. However, urinary symptoms are also frequently reported. Lower limb spasticity is the prominent ﬁnding on examination. The pattern of hypertonicity is responsible for the classical gait with the affected person demonstrating circumduction and toe walking. Weakness is not a major feature of HSP, and indeed the ﬁnding of marked spasticity in the presence of little or no muscle weakness is characteristic of HSP. Distal muscle wasting can be observed in long-standing cases (>10 years) but prominent wasting occurring earlier in the disease course or in a more generalized pattern would represent a complicated HSP phenotype. Signs of mild dorsal column dysfunction are common in the lower limbs, with impairment of vibration sensation distally. This sensory feature seems to occur more commonly, but not exclusively, in those with long-standing disease. Other features observed in pure HSP include absent ankle jerks, pes cavus, and mild cognitive impairment. Until recently, cases of HSP and cognitive impairment were felt to be examples of complicated HSP phenotypes often occurring with further additional features such as ataxia, cardiac defects, extrapyramidal features, or epilepsy (McDermott et al., 2000). However, it is now becoming apparent that mild cognitive impairment is a feature of pure HSP and may actually predate the onset of the spastic paraparesis in at-risk family members (Byrne et al., 2000). Whereas in pure HSP the progressive spastic paraparesis is the prominent feature, in complicated HSP it is merely one component of a much more diverse phenotype. HSP has been associated with an array of additional clinical features including amyotrophy, cardiac defects, sensorineural deafness, dementia, epilepsy, extrapyramidal disease, icthyosis, sensory neuropathy, retinal changes, hyperekplexia, Kallman’s syndrome, and gastrooesophageal reﬂux (Table I). In some instances the association is extremely rare with only one pedigree described. In others it may be that more than one inherited disorder is contributing to the overall phenotype. III. Genetics

Fourteen SPG loci have been identiﬁed showing association with HSP (Table II). The genes at four of these loci have been identiﬁed. A. AUTOSOMAL-RECESSIVE HSP (SPG7) Of the four loci for autosomal-recessive HSP the gene at one, SPG7, has been identiﬁed as paraplegin (Casari et al., 1998). The paraplegin gene contains 17 exons and encodes an 88-kDa protein. The paraplegin protein

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TABLE II GENETIC CLASSIFICATION OF HSPa Genome database designation

Chromosome

Inheritance

Phenotype

Genetic defect

SPG1 SPG2 SPG3 SPG4 SPG5 SPG6 SPG7 SPG8 SPG9 SPG10 SPG11 SPG12 SPG13 SPG14

Xq28 Xq22 14q11.2–24.3 2p22–21 8p12–q13 15q11.1 16q24.3 8q24 10q23.3–24.2 12q13 15q13-15 19q13 2q24–q34 3q27–q28

X-linked X-linked AD AD AR AD AR AD AD AD AR AD AD AR

Complicated Both Pure Both Pure Pure Both Pure Complicated Pure Both Pure Pure Complicated

L1CAM PLP Unknown Spastin Unknown Unknown Paraplegin Unknown Unknown Unknown Unknown Unknown Unknown Unknown

a L1CAM: L1 cell adhesion molecule; PLP: proteolipid protein; AD: autosomal dominant; AR: autosomal recessive.

is a nuclear-encoded mitochondrial metalloprotease and is a member of a group of proteins known as ATPases Associated with diverse cellular Activities (AAA). The AAA proteins play a role in various cell functions including cell cycle regulation, protein transportation, protein degradation, and organelle biogenesis. These functions require the assembly and function of protein complexes, and it has been proposed that AAA proteins act as chaperone proteins in these complexes. Members of the AAA protein family contain an area of homology known as the AAA cassette. Outside of this 230 amino acid domain, little homology is seen except among members of the same subgroup. Whereas little is known about paraplegin, other than the fact that it appears to be ubiquitously expressed, much more is known about the function of similar proteins in yeast. The Afg3p, Rca1p, and Yme1p are AAA mitochondrial metalloproteases found in yeast, and they share, 55, 55, and 52% amino acid homology with paraplegin, respectively. As well as the ATPase domain, this homology includes a conserved zinc binding domain. In yeast, Afg3p and Rca1p form a high molecular weight (850-kD) heterooligomeric complex in the inner mitochondrial membrane that is essential for mitochondrial biogenesis. These proteins are involved in ATP synthase assembly, respiratory chain complex formation, and degradation of incompletely synthesized mitochondrial polypeptides (Tauer et al., 1994; Tzagoloff et al., 1994; Paul and Tzagoloff, 1995; Langer and Neupert, 1996; Rep and Grivell, 1996). Deletion or mutation of the conserved proteolytic site of

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either Afg3p and Rca1p leads to dysfunction of respiratory chain activity and an impaired ability to degrade incompletely synthesized mitochondrial polypeptides. In yeast strains with proteolytically inactive Afg3p and Rca1p, deﬁciencies of both cytochrome oxidase subunit 1 (Cox1p) and cytochrome b (Cobp) have been demonstrated (Arlt et al., 1998). Interestingly, the respiratory chain deﬁciency associated with the deletion of Afg3p and Rca1p can be partially compensated for by the overexpression of proteins Pim1p and Oxa1p involved in the assembly of respiratory complexes and degradation of selective mitochondrial proteins. A further protein Mba1p has been identiﬁed speciﬁcally as a suppresser of the respiratory chain deﬁciency resulting from Afg3p and Rca1p deletion (Rep et al., 1996). The Yme1p forms a 850-kD homo-oligomeric complex on the mitochondrial inner membrane (Leonhard et al., 1996). The Yme1p and has been demonstrated to be involved in the degradation of partially formed intermembrane proteins, such as unassembled cox2p, and to possess chaperonelike properties binding unfolded polypeptides and suppressing their aggregation (Nakai et al., 1995; Pearce and Shermann, 1995; Leonhard et al., 1999). Mutation of Yme1p leads to a decrease in respiratory chain complexes and consequent respiratory deﬁciency. Deletion of Yme1p is extremely detrimental to cell viability (Thorsness et al., 1993; Nakai et al., 1995). These studies show the essential role the paraplegin-like genes Afg3p, Rca1p, and Yme1p play in normal mitochondrial biogenesis in yeast. Human homologues for Afg3p and Yme1p have been identiﬁed as AFG3L2 and YME1L1, respectively (Banﬁ et al., 1999; Coppola et al., 2000). These share domain homology with the yeast genes as well as a mitochondrial subcellular localization. Although their chromosomal locations 18p11 and 10p14 are not as yet identiﬁed as HSP loci, it is reasonable to speculate that that mutations in these genes would be candidates worth investigating as alternative causes of HSP. The exact role of paraplegin or related genes, such as AFG3L2 and YME1L1, in humans is unknown. From the studies of yeast homologues, it seems likely they function by forming multimeric complexes that have proteolytic and chaperone-like functions in the mitochondria, essential for the normal assembly and turnover of respiratory chain complexes. It is clear however, that mutation in the paraplegin gene causes impairment of oxidative phosphorylation. If, as in yeast, there are genes that can partially overcome the effects of paraplegin defects, one possible explanation for the heterogeneity seen in paraplegin-related HSP may be the status of these “compensator” genes. Hereditary spastic paraparesis associated with paraplegin mutation is clinically heterogeneous both with pure and with complicated families having been described (Casari et al., 1998; McDermott et al., 2001). The pure

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HSP pedigrees described have an adult onset with slow progression. In one large Italian pedigree, additional features in keeping with a diagnosis of pure HSP included diminished vibration sense and pes cavus. Clinically, pure HSP due to paraplegin mutation is indistinguishable from pure HSP at other SPG loci, either dominant or recessive forms. Pure HSP at the SPG7 locus is genetically heterogeneous with both a homozygous 9.5 kilobase (kb) deletion of the last ﬁve exons and a homozygous two base-pair deletion (784–785del) resulting in a frameshift that abolishes 60% of the paraplegin protein, having been described. The SPG7-complicated HSP pedigrees described have included a French family with optic atrophy, cortical and cerebellar atrophy cosegregating with the spastic paraparesis. In this French family the genetic defect was reported as an insertion (2228 ins A) that leads to a frameshift causing truncation of the paraplegin protein prior to the terminal 57 amino acids. A further complicated HSP family from the northeast of England was both phenotypically and genetically different, demonstrating the heterogeneity of complicated HSP at the SPG7 locus. In this English family the spastic paraparesis was complicated by amyotrophy, raised creatine kinase, and a sensorimotor peripheral neuropathy. This English family was unusual in that the proband was a compound heterozygote with both a nine base-pair deletion(1450–1458del) and a missense change (1529C → T) in paraplegin. The paraplegin missense change was inherited from a clinically normal mother. The deletion was inherited from a father who was reported to be mildly affected with spastic paraparesis. The authors postulated that the father either represents a manifesting heterozygote or that the deletion he carries is behaving in a dominant-negative manner (McDermott et al., 2001). In the latter case, it may be that the deletion is affecting the ability of the translated paraplegin protein to form multimeric complexes, which in the yeast homologues appear to be necessary for normal function. Muscle tissue analyzed from both pure and complicated patients described with paraplegin mutations conﬁrms mitochondrial involvement in SPG7-related HSP. The histological ﬁndings include the presence of raggedred ﬁbers, which stain intensely for succinate dehydrogenase and which stain negative for cytochrome oxidase c, the hallmark changes of oxidative phosphorylation impairment (Fig. 1). In one family described, the severity of the clinical phenotype matched the degree of abnormality seen in the muscle histology (Casari et al., 1998). Detailed assessment of respiratory chain complex activity in muscle from SPG7 patients has not been published. Spectrophotometric analysis of respiratory complexes in muscle homogenate from the complicated English pedigree was normal (unpublished data). This is not unexpected given the scattered pattern of muscle ﬁbers with impaired oxidative phosphorylation seen histologically.

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FIG. 1. Dual cytochrome c oxidase (COX)/succinate dehydrogenase (SDH) staining of muscle tissue from HSP patient with paraplegin mutation, demonstrating the presence of a COX-negative/SDH-positive ﬁber (∗ ), and mitochondrial proliferation (arrow).

B. AUTOSOMAL-RECESSIVE HSP (SPG5, SPG11, AND SPG14) The genes at the remaining three autosomal-recessive HSP loci SPG5, SPG11, and SPG14 are unknown. Four Tunisian families have been linked to the SPG5 locus. All the SPG5 families have a pure phenotype with a mean age at onset ranging from 1 to 20 years (Hentati et al., 1994a). Both pure and complicated families have been described at the SPG11 locus (Murillo et al., 1999). The complicated families at this locus demonstrate clinical heterogeneity. The commonest complicating feature is atrophy of the corpus callosum associated with mental retardation. This combination occurred in all ten of the Japanese pedigrees linked to this locus, but was less common in the European and American families. Other associations with spastic paraparesis at this locus were dysarthria, mixed motor sensory neuropathy, and high signal periventricular white matter changes demonstrated on magnetic resonance imaging (MRI). Only one complicated autosomal-recessive family has been linked to the SPG14 locus (Vazza et al., 2000). In this family, three siblings were affected by spastic paraparesis, distal motor neuropathy, and mild cognitive impairment.

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C. AUTOSOMAL-DOMINANT HSP Hereditary spastic paraparesis is most commonly inherited as an autosomal-dominant trait. There are at present eight identiﬁed autosomaldominant HSP loci. There are families with autosomal-dominant HSP for whom linkage to the known loci has been excluded, suggesting at least one further autosomal HSP locus (Ashley-Koch et al., 2001). The gene at one of these loci, SPG4, has been identiﬁed as spastin (Hazan et al., 1999). Spastin is a large 17 exon gene encoding a 67-kDa protein. Interestingly spastin, like paraplegin, is a member of the AAA group of proteins. However, the two proteins belong to different subgroups of the AAA protein family and share little homology outside the AAA consensus region. Spastin shares greatest homology with a subgroup of AAA proteins that include the 26s proteasome subunits, and therefore it has been postulated that spastin may play a role in cellular activities such as gene regulation, inducing proteolytic activation or the degradation of transcription factors involved in protein regulation. Spastin is predicted to have a nuclear localization due to the presence of a nuclear targeting motif within the amino acid sequence, although this has not yet been conﬁrmed. The mutations in the spastin gene associated with HSP are dispersed through the whole length of the gene with no particular “hot spot” for mutation (Burger et al., 2000; Fonknechten et al., 2000; Hentati et al., 2000; Lindsey et al., 2000; Santorelli et al., 2000). The majority of the mutations detected are predicted to result in either a truncated protein or a severely altered protein, implying that haploinsufﬁciency is the likely cause of the abnormal phenotype. The type or location of the mutation does not alter the phenotype observed. The majority of families at the SPG4 locus are thought to be of the pure phenotype. Within these families, there is marked inter- and intrafamilial variation in the age at onset and severity of symptoms. There is also a small but signiﬁcant number of families in which the spastic paraparesis is complicated by dementia. However, there is growing evidence that blurs the boundaries of the deﬁnitions of the pure and complicated forms of HSP at the SPG4 locus. It appears that subclinical cognitive decline can be detected in SPG4 patients and that this decline may even predate the onset of the spastic paraparesis (Byrne et al., 2000; McMonagle et al., 2000). This suggests that, rather than two distinct groups of pure HSP and HSP complicated with dementia, there is in fact a broad spectrum of cognitive decline associated with HSP at the SPG4 locus. Cognitive changes are not common in cases of HSP linked to other AD SPG loci, and it may be that they are a result of the abnormal SPG4 product. White et al. (2000) described the pathology from an individual with HSP complicated by dementia, associated with mutation in the spastin gene. In addition to the usual ﬁndings in

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the spinal cord of corticospinal tract degeneration, they observed unusual brain pathology that had not previously been demonstrated in HSP or in any previously described form of dementia. In the hippocampus there was gross depletion of neurones within the medial part of the CA1 region. There was also severe neuronal loss in the entorhinal cortex with attrition of the perforant pathway. Surviving neurones in these regions showed frequent τ -immunoreactive neuroﬁbrilliary tangles. In the limbic cortex and neocortex, prominent ballooned neurones were seen showing irregular cytoplasmic immunoreactivity for τ . These τ -related cortical changes do not correspond to the characteristic pathological changes in other deﬁned forms of dementia, and they were not accompanied by senile plaque formation. Neuropathological examination of further cases of spastin-related HSP will be required to determine whether these unusual τ -related cortical changes are a direct result of the cellular expression of mutant spastin. The genes at the remaining autosomal dominant loci have yet to be identiﬁed. The phenotypes observed in the families linked to SPG3, -6, -8, -10, -12, and -13 appeared to be pure rather than complicated (Hazan et al., 1993, 1994; Hentati et al., 1994b; Fink et al., 1995; Gispert et al., 1995; Lennon et al., 1995; Huang et al., 1997; Hedera et al., 1999; Reid et al., 1999; Seri et al., 1999; Fontaine et al., 2000; Reid et al., 2000). It is difﬁcult to make genotype–phenotype correlation as often as only a small number of families have been described at a particular locus. However, families linked to SPG3, -10 and -12 tend to have an earlier onset within the ﬁrst two decades, whereas families linked to SPG6 and -8 have a more severe phenotype. The phenotype observed in families linked to SPG9 is of complicated HSP, in which congenital cataracts, gastroesophageal reﬂux with persistent vomiting, and axonal motor neuropathy cosegregate with the spastic paraparesis.

D. X-LINKED HSP The two X-linked genes, leucocyte 1 cell adhesion molecule (L1CAM) and proteolipid protein gene (PLP), have been known for some time ( Jouet et al., 1994; Kobayashi et al., 1994; Saugier-Veber et al., 1994). They play a role in nervous system development and myelin maintenance, respectively. Mutations in L1CAM lead to the clinical syndrome referred to as CRASH (corpus callosum hypoplasia, retardation, adducted thumbs, spastic paraparesis, and hydrocephalus). Mutations in the PLP gene can lead to pure HSP, complicated HSP, or a severe dysmyelinating disorder, Pelizaeus-Merzbacher disease (PMD). There is no suggestion of a role for mitochondrial dysfunction in X-linked HSP.

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E. MITOCHONDRIA, NEURODEGENERATION, AND HSP The involvement of mitochondrial dysfunction in the pathogenesis of a number of neurodegenerative diseases is well recognized. Diseases such as MELAS (mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes), MERFF (myoclonic epilepsy and ragged-red ﬁbers), and KearnsSayre syndrome are due to mitochondrial DNA-encoded mitochondrial defects. In Freidriech’s ataxia (Campuzano, 1996; Bradley et al., 2000) and Wilson’s disease (Sternlieb, 1992; Lutsenko and Cooper, 1998), as in paraplegin-related HSP, the mitochondrial dysfunction is due to defects in nuclear DNA-encoded mitochondrial proteins. There is also mounting evidence for impaired oxidative phosphorylation and increased free radical production in other neurodegenerative diseases such as Parkinson’s disease and Huntington’s disease. In motor neurone disease (MND), a degenerative disorder of the motor system involving both upper motor neurone pathways (as in HSP) and lower motor neurones, there are several lines of evidence to support a role for mitochondrial dysfunction. Studies in human postmortem CNS tissue have been reported to show ultrastructural changes in mitochondrial morphology (Sasaki and Iwata, 1996), alterations in the activities of respiratory chain complexes (Fujita et al., 1996; Borthwick et al., 1999), and increased levels of the mitochondrial DNA “common deletion” in MND cases compared to controls (Dhaliwal et al., 2000). Studies of cellular and animal experimental models of MND have provided further evidence for the role of mitochondrial dysfunction in motor neurone injury. In a study of a neuronal cell line transfected with mutant SOD1, a loss of mitochondrial membrane potential was observed, together with an increase in cytosolic calcium, suggesting a reduction in the ability of mitochondria in these cells to sequester calcium (Carri et al., 1997). In a study of a mouse model of SOD1-related MND, morphological abnormalities of mitochondria, including swelling and vacuolation, were observed early in the disease process, before the onset of clinical signs in the mice (Kong and Xu, 1998). The demonstration of involvement of mitochondrial dysfunction in the development of the HSP phenotype should not be a surprise. One of the hallmarks of mitochondrial disease is diffuse involvement of the nervous system as seen in HSP. In pure HSP, although the predominant clinical ﬁnding is a progressive spastic paraparesis, there is often involvement outside the motor system with sensory and cognitive changes. The suspicion of mitochondrial involvement is perhaps even higher in cases of complicated HSP, where features such as epilepsy, optic atrophy, and peripheral neuropathies can cosegregate with the spastic paraparesis. Multifocal involvement of the neuroaxis in HSP is conﬁrmed pathologically with degeneration

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changes observed in the dorsal columns and spinocerebellar tracts in addition to corticospinal tracts. The degeneration occurs initially in the distal portions of these long axons and then proceeds toward the cell body, and has been described as a “dying back” phenomenon. The mechanism by which mitochondrial dysfunction causes the axonal degeneration in parapleginrelated HSP is unknown. The cell bodies of the upper motor neurones (Betz cells) have to support very long axonal processes, and this is a feature that may be predicted to place high metabolic demands on the cell. Therefore, it could be the motor neurones may be sensitive to any disturbance in mitochondrial energy production, because of their large size and their extremely long axonal processes. One of the key functions of mitochondria is the generation of ATP via the activity of the respiratory chain complexes. Intracellular energy deﬁcits will occur when the activities of the mitochondrial respiratory chain complexes are disturbed. Other detrimental intracellular consequences of mitochondrial dysfunction include increased generation of reactive oxygen species, oxidative stress, and impaired intracellular calcium homeostasis. Oxidative stress resulting from altered free radical homeostasis has been implicated in the pathogenesis of a number of neurodegenerative disorders and may play a role in paraplegin-related HSP. Whether mitochondrial dysfunction is a common feature in the pathogenesis of the HSP phenotype at the autosomal loci remains to be seen. There have been no studies as yet investigating mitochondrial function in the non-SPG7 autosomal-recessive families. In the dominant families at the SPG3, -4, -6, -8, and -9 loci, only a small number of muscle biopsies have been performed (mostly only one individual for each locus) for histochemical and biochemical analysis of mitochondrial function (Seri et al., 1999; Hedera et al., 2000). These preliminary results suggest there is no primary role for mitochondrial dysfunction in the development of the HSP phenotype linked to these loci. Further studies are now required to conﬁrm these ﬁndings and also to investigate families linked to the more recently discovered dominant loci SPG10, -12, and -13.

References

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Banﬁ, S., Bassi, M. T., Andolﬁ, G., et al. (1999). Identiﬁcation and characterization of AFG3L2, a novel paraplegin-related gene. Genomics 59, 51–58. Borthwick, G. M., Johnson, M. A., Ince, P. G., et al. (1999). Mitochondrial enzyme activity in amyotrophic lateral sclerosis: Implications for the role of mitochondria in neuronal cell death. Ann. Neurol. 46, 787–790. Bradley, J. L., Blake, J. C., Chamberlain, S., et al. (2000). Clinical, biochemical and molecular genetic correlations in Friedriech’s ataxia. Mol. Genet. 9, 275–282. Burger, J., Fonknechten, N., Hoeltzenbein, M., et al. (2000). Hereditary spastic paraplegia caused by mutations in the SPG4 gene. Eur. J. Hum. Genet. 8, 771–776. Byrne, P. C., Mc Monagle, P., Webb, S., et al. (2000). Age-related cognitive decline in hereditary spastic paraparesis linked to chromosome 2p. Neurology 54, 1510–1517. Campuzano, V. (1996). Freidreich’s ataxia: Autosomal recessive disease caused by an intronic GAA triplet repeat expansion. Science 271, 1423–1427. Carri, M. T., Ferri, A., and Battistoni, A. (1997). Expression of Cu/Zn superoxide dismutase typical of familial amyotrophic lateral sclerosis induces mitochondrial alteration and increase of cytosolic Ca2+ concentration in transfected neuroblastoma SH-SY5Y cells. FEBS Lett. 414, 365–368. Casari, G., De Fusco, M., Ciarmatori, S., et al. (1998). Spastic paraplegia and OXPHOS impairment caused by mutations in paraplegin, a nuclear-encoded mitochondrial metalloprotease. Cell 93, 973–983. Coppola, M., Pizzigoni, A., Banﬁ, S., et al. (2000). Identiﬁcation and characterization of YME1L1, a novel paraplegin-related gene. Genomics 66, 48–54. Dhaliwal, G. K., and and Grewal, R. P. (2000). Mitochondrial DNA deletion mutation levels are elevated in ALS brains. Neuroreport. 11, 2507–2509. Fink, J. K., Wu, C.-T. B., Jones, S. M., et al. (1995). Autosomal dominant familial spastic paraplegia: Tight linkage to chromosome 15q. Am. J. Hum. Genet. 56, 188–192. Fonknechten, N., Mavel, D., Byrne, P., et al. (2000). Spectrum of SPG4 mutations in autosomal dominant spastic paraplegia. Hum. Mol. Genet. 9, 637–644. Fontaine, B., Davoine, C.-S., Durr, A., et al. (2000). A new locus for autosomal dominant pure spastic paraplegia, on chromosome 2q24–q34. Am. J. Hum. Genet. 66, 702–707. Fujita, K., Yamauchi, M., and Shibayama, K. (1996). Decreased cytochrome c oxidase activity but unchanged superoxide dismutase and glutathione peroxidase activities in the spinal cords of patients with amyotrophic lateral sclerosis. J. Neurosci. Res. 45, 276–281. Gispert, S., Santos, N., Damen, R., et al. (1995). Autosomal dominant familial spastic paraplegia: Reduction of the FSP1 candidate region on chromosome 14q to 7 cM and locus heterogeneity. Am. J. Hum. Genet. 56, 183–187. Harding, A. E. (1981). Hereditary “pure” spastic paraplegia: A clinical and genetic study of 22 families. J. Neurol. Neurosurg. Psychiat. 44, 871–883. Hazan, J., Fonknechten, N., Mavel, D., et al. (1999). Spastin, a novel AAA protein, is altered in the most frequent form of autosomal dominant spastic paraplegia. Nat. Genet. 23, 296–303. Hazan, J., Fontaine, B., Bruyn, R. P. M., et al. (1994). Linkage of a new locus for autosomal dominant familial spastic paraplegia to chromosome 2p. Hum. Mol. Genet. 3, 1569–1573. Hazan, J., Lamy, C., Melki, J., et al. (1993). Autosomal dominant familial spastic paraplegia is genetically heterogeneous and one locus maps to chromosome 14q. Nat. Genet. 5, 163–167. Hedera, P., DiMauro, S., Bonilla, E., et al. (2000). Mitochondrial analysis in autosomal dominant hereditary spastic paraplegia. Neurology 55, 1591–1592. Hedera, P., Rainer, S., Alvarado, D., et al. (1999). Novel locus for autosomal dominant hereditary spastic paraplegia, on chromosome 8q. Am. J. Hum. Genet. 64, 563–569. Hentati, A., Deng, H.-X., Zhai, H., et al. (2000). Novel mutations in spastin gene and absence of correlation with age at onset of symptoms. Neurology 55, 1388–1390.

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Hentati, A., Pericak-Vance, M. A., Hung, W.-Y., et al. (1994a). Linkage of “pure” autosomal recessive familial spastic paraplegia to chromosome 8 markers and evidence of genetic locus heterogeneity. Hum. Mol. Genet. 3, 1263–1267. Hentati, A., Pericak-Vance, M. A., Lennon, F., et al. (1994b). Linkage of a locus for autosomal dominant spastic paraplegia to chromosome 2p markers. Hum. Mol. Genet. 3, 1867–1871. Huang, S., Zhuyu,, Li, H., et al. (1997). Another pedigree with pure autosomal dominant spastic paraplegia (AD-FSP) from Tibet mapping to 14q11.2– q24.3. Hum. Genet. 100, 620–623. Jouet, M., Rosenthal, A., Armstrong, G., et al. (1994). X-linked spastic paraplegia (SPG1), MASA syndrome and X-linked hydrocephalus result from mutations in the L1CAM gene. Nat. Genet. 7, 402–407. Kobayashi, H., Hoffman, E. P., and Marks, H. G. (1994). The rumpshaker mutation in spastic paraplegia. Nat. Genet. 7, 351–352. Kong, J., and Xu, Z. (1998). Massive mitochondrial degeneration in motor neurones triggers the onset of amyotrophic lateral sclerosis in mice expressing a mutant SOD1. J. Neurosci. 18, 3241–3250. Langer, T., and Neupert, W. (1996). Regulated protein degradation in mitochondria Experimentia 52, 1069–1076. Lennon, F., Gaskell, P. C., Wolpert, C., et al. (1995). Linkage and heterogeneity in hereditary spastic paraparesis. Am. J. Hum. Genet. 57, A217. Leonhard, K., Hermann, J. M., Stuart, R. A., et al. (1996). AAA proteases with catalytic sites on opposite membrane surfaces comprise a proteolytic system for the ATP-dependent degradation of inner membrame proteins in mitochondria. EMBO J. 15, 4218–4229. Leonhard, K., Stiegler, A., and Langer, T. (1999). Chaperone-like activity of the AAA domain of the yeast Yme 1 AAA protease. Nature 398, 348–351. Lindsey, J. C., Lusher, M. E., McDermott, C. J., et al. (2000). Mutation analysis of the spastin gene (SPG4) in patients with hereditary spastic paraparesis. J. Med. Genet. 37, 759–765. Lutsenko, S., and Cooper, M. J. (1998). Localization of the Wilson’s disease protein product to mitochondria. Proc. Natl. Acad. Sci. USA 95, 6004–6009. McDermott, C. J., White, K., Bushby, K., et al. (2000). Hereditary spastic paraparesis: A review of new developments. J. Neurol. Neurosurg. Psychiatry 69, 150–160. McDermott, C. J., Dayaratne, R. K., Tomkins, J., et al. (2001). Paraplegin gene analysis in hereditary spastic paraparesis (HSP) pedigrees in northeast England. Neurology 56, 467– 471. McMonagle, P., Byrne, P. C., Fitzgerald, B., et al. (2000). Phenotype of AD-HSP due to mutations in the SPAST gene. Comparison with AD-HSP without mutations. Neurology 55, 1794–1800. Murillo, F., Kobayashi, H., Pegoraro, E., et al. (1999). Genetic localization of a new locus for recessive familial spastic paraparesis to 15q13–15. Neurology 53, 50–56. Nakai, T., Yasuhara, T., Fujiki, Y., et al. (1995). Multiple genes, including a memeber of the AAA family, are essential for the degradation of unassembled subunit 2 of cytochrome oxidase in yeast mitochondria. Mol. Cell. Biol. 15, 4441–4452. Paul, M.-F., and Tzagoloff, A. (1995). Mutations in RCA1 and AFG3 inhibit F1-ATPase assembly in Saccharomyces cerevisiae. FEBS Lett. 373, 66–70. Pearce, D. A., and Shermann, F. (1995). Degradation of cytochrome oxidase subunits in mutants of yeast lacking cytochrome c and suppression of the degradation by yme1. J. Biol. Chem. 270, 20879–20882. Reid, E., Dearlove, A. M., Osborn, O., et al. (2000). A locus for autosomal dominant “pure” hereditary spastic paraplegia maps to chromosome 19q13. Am. J. Hum. Genet. 66, 728–732. Reid, E., Dearlove, M., Rhodes, M., et al. (1999). A new locus for autosomal dominant “pure” hereditary spastic paraplegia mapping to chromosome 12q13, and evidence for further genetic heterogeneity. Am. J. Hum. Genet. 65, 757–763.

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Rep, M., and Grivell, L. A. (1996). The role of protein degradation in mitochondrial function and biogenesis. Curr. Genet. 30, 367–380. Rep, M., Nooy, J., Guelin, E., et al. (1996). Three genes for mitochondrial proteins supress null mutations in both Afg3 and Rca1 when overexpressed. Curr. Genet. 30, 206–211. Santorelli, F. M., Patrono, C., Fortini, D., et al. (2000). Intrafamilial variability in hereditary spastic paraplegia associated with an SPG4 gene mutation. Neurology 55, 702–705. Sasaki, S., and Iwata, M. (1996). Ultrastructural study of the synapses in the anterior horn of neurones of patients with amyotrophic lateral sclerosis. Neurosci. Lett. 204, 53–56. Saugier-Veber, P., Munnich, A., Bonneau, D., et al. (1994). X-linked spastic paraplegia and Pelizaeus-Merzbacher disease are allelic disorders at the proteolipid protein locus. Nat. Genet. 6, 257–261. Seri, M., Cusano, R., Forabosco, P., et al. (1999). Genetic mapping to 10q23.3-q24.2, in a large Italian pedigree, of a new syndrome showing bilateral cataracts, gastroesophageal reﬂux, and spastic paraparesis with amyotrophy. Am. J. Hum. Genet. 64, 586–593. Sternlieb, I. (1992). Fraternal concordance of types of abnormal hepatocellular mitochondria in Wilson’s disease. Hepatology 16, 728–732. Tauer, R., Mannhaupt, G., Schnall, R., et al. (1994). Yta10p, a member of a novel ATPase family in yeast, is essential for mitochondrial function. FEBS Lett. 353, 197–200. Thorsness, P. E., White, K. H., and Fox, T. D. (1993). Inactivation of YME1, a putative member of the FtsH-SEC18–PAS1–CDC48 family of putative ATPase-encoding genes, causes increased escape of DNA from mitochondria in Saccharomyces cerevisiae. Mol. Cell. Biol. 13, 5418–5426. Tzagoloff, A., Yue, J., Jang, J., et al. (1994). A new member of a family of ATPases is essential for assembly of mitochondrial respiratory chain and ATP synthetase complexes in Saccharomyces cerevisiae. Mol. Cell. Biol. 13, 5418–5426. Vazza, G., Zortea, M., Boaretto, F., et al. (2000). A new locus for autosomal recessive spastic paraplegia associated with mental retardation and distal motor neuropathy, SPG14, maps to chromosome 3q27–q28. Am. J. Hum. Genet. 67, 504–509. White, K. D., Ince, P. G., Cookson, M., et al. (2000). Clinical and pathological ﬁndings in hereditary spastic paraparesis with spastin mutation. Neurology 55, 89–94.

CYTOCHROME c OXIDASE DEFICIENCY

Giacomo P. Comi,∗ Sandra Strazzer,∗ ,† Sara Galbiati,∗ and Nereo Bresolin∗ ,† ,1 ∗ Centro Dino Ferrari Dipartimento di Science Neurologiche Universita` degli Studi di Milano IRCCS Ospedale Maggiore Policlinico Milano, Italy † IRCCS E. Medea, Associazione La Nostra Famiglia Bosisio Parini, Italy

I. Cytochrome c Oxidase Biogenesis II. Nuclear Genes Affecting COX Assembly and Stability A. COX-Negative Leigh Syndrome B. SCO1 C. SCO2 D. COX10 E. Infantile Autosomal-Recessive Muscle COX Deficiencies F. Infantile Autosomal-Recessive Mitochondrial Encephalomyopathies with COX Deficiency III. Nuclear Genes Affecting mtDNA Level and/or Stability A. mtDNA Depletion Syndromes B. Autosomal-Dominant Progressive External Ophthalmoplegia (PEO) and Autosomal-Recessive PEO C. Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE) IV. mtDNA Defects A. C O I Mutations B. C O I I Mutations C. C O I I I Mutations D. Mutations Affecting mtDNA Translation (Transfer RNA Point Mutations, Deletions, and Duplications) References

I. Cytochrome c Oxidase Biogenesis

Cytochrome c oxidase (COX) is the terminal component (complex IV) of the mitochondrial respiratory chain, the main energy-generating system of eukaryotic cells. The enzyme, embedded in the mitochondrial inner 1

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membrane, is a complex metalloprotein, catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen, and preserves the free energy released in this exergonic reaction by maintaining a transmembrane proton gradient that is used to drive the syntesis of ATP or ion transport across the membrane (Capaldi, 1990). The humane enzyme is composed of 13 polypeptide subunits, the three largest subunits (I–III) are encoded by mitochondrial DNA (mtDNA) and are synthesized inside the mitochondrion. Subunit I spans the membrane 12 times and lacks any large extramembrane parts. Subunits II and III are associated with the transmembrane region of subunit I, and there is no direct contact between them. Subunits I and II bear the prosthetic groups required for the electron transfer reaction. There are two heme A molecules, both of which are located in the hydrophobic interior of subunit I, and are denoted hemes a and and a3 based on their spectral properties. In addition, there are two copper atoms that form the binuclear CuA site on subunit II and a single copper atom (designed CuB) that is located adjacent to the heme a3 site in subunit I. The provision of these prosthetic groups is an essential part of the biosynthetic pathway for COX. The mtDNA-encoded subunits are thought to be crucial for the catalytic functions of the enzyme. The remaining subunits (IV, Va, Vb, VIa, VIb, VIIa, VIIb, VIIc, and VIII) are encoded by nuclear DNA (nDNA), synthesized on cytosolic ribosomes, and imported into the mitochondria. They are associated with the surface of the three core subunits but leave many areas uncovered. All ten nuclear-encoded subunits of the human enzyme have been sequenced at the complementary DNA (cDNA) level, but remarkably little is known about their function. The characterization of their electrophoretic mobility, amino acid sequences, and antibody speciﬁcity suggest possible regulatory roles. A considerable amount of information regarding COX synthetic pathways has been derived from biochemical and genetic studies of respiratory defective petite mutants of Saccharomyces cerevisiae (Goffeau et al., 1996). Indeed in yeast, about three dozen complementation groups have been reported to consist of mutants displaying a selective deﬁciency in COX (McEwen et al., 1985; Tzagoloff and Dieckmann, 1990). In addition to mutations in the structural genes, these strains are also affected in: (1) processing of the mitochondrial COX pre-messenger RNAs (pre-mRNAs) (McEwen et al., 1986; Seraphin et al., 1988; Pel et al., 1992), (2) translation of the resultant mRNAs (Costanzo et al., 1986; Poutre and Fox, 1987), (3) heme a biosynthesis (Tzagoloff et al., 1993), (4) copper import and transfer to the apoenzyme (Glerum et al., 1996a,b), and (5) as yet poorly understood events in the pathway leading to the functional enzyme (ten Berge et al., 1974; McEwen et al., 1993; Bonnefoy et al., 1994). While the ﬁrst

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mechanism is speciﬁc to the intron-containing transcripts of simple eukaryotes, it is becoming clear that the other genetic controls found in yeast are also present in humans. Only the data that are now relevant for human disorders is brieﬂy described. One of the best characterized pathway to COX biosynthesis is that related to the copper transport from the cytoplasm to the mitochondria and to copper insertion into the active site of COX. Cytochrome c oxidase 17 is the copper chaperone that is responsible for recruiting copper to the mitochondrial intermembrane space (Glerum et al., 1996a). Mutations in the COX17 gene of S. cerevisiae cause a respiratory deﬁciency due to a block in the production of a functional COX complex, which may be rescued by copper-supplemented media. Recent data indicate that Sco1p (the protein product of the SCO1 gene—from s ynthesis of COX) provides copper, imported by COX17, to the CuA site of the COX subunit II at a late step of the assembling pathway (Dickinson, 2000). Mutation analysis of the yeast SCO1 gene has shown that the CxxxC binding motif is essential for protein, since mutant proteins failed to restore the respiratory competence of the Sco1 null mutant (Rentzsch et al., 1999). Another SCO-like protein, SCO2, has been identiﬁed in both humans and yeast. The SCO1 and SCO2 are highly homologous, since the proteins share a 40% identity, especially in the core region of the protein. Although the respective roles of the two proteins remain to be fully established, several lines of evidence suggest that both are involved in mitochondrial copper trafﬁcking from the intermembrane space to the inner mitochondrial membrane. Overexpression of both proteins partially rescue a Cox17-null mutant, suggesting a partial redundancy of the two genes (Glerum et al., 1996a). The SCO1 and SCO2 genes have a largely similar pattern of expression in human tissues (Papadopoulou et al., 1999). Another nuclear gene of S. cerevisiae, COX10, has been proved to be involved in human pathology (Valnot et al., 2000a). In human and yeast, the COX10 protein product is localized in mitochondria and is necessary for the synthesis of COX. The COX10 shares some of the properties of COX11, a farnesyl transferase that converts protoheme to heme O. The Escherichia coli COX10 ortholog has been shown to code for the enzyme that transfers a farnesyl group to the vinyl at position 9 of the porphirin ring system in the conversion of protoheme to heme O (Saiki et al., 1992). Yeast COX10 is a hemeA:farnesyltransferase. (Tzagoloff et al., 1993). Once the prosthetic groups are correctly synthesized and copper molecules are properly inserted into the COX subunits, other chaperone proteins cooperate to further maturation. For instance, COX20, another protein of the mitochondrial inner membrane, is essential for the maturation and for

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subsequent assembly of the mitochondrially encoded pCox2p, the precursor of subunit 2 of COX. Cox20p binds directly to Cox2p during cytochrome oxidase assembly. The interaction with Cox20p occurs after pCox2p is inserted into the inner membrane but precedes the proteolytic cleavage by the peptidase Imp1p. Cox20p is supposed to hold pCox2p in a processing competent conformation. The ability of Cox20p to bind mature, unassembled Cox2p raises further the possibility that Cox20p may also chaperone the Cox2p during its subsequent assembly into the COX complex (Hell et al., 2000). A further step of COX assembly relies on the SURF-1 protein function. Also, this human protein has a yeast counterpart, SHY 1, which has been identiﬁed in a pet mutant. Both SHY 1 and SURF-1 proteins have a characteristic mitochondrial targeting sequence at their N-terminus. Mutations in SHY 1 produce a partial pleiotropic respiratory chain deﬁciency and an inability to grow on nonfermentable substrates, which is different from the isolated COX deﬁciency observed in humans. The transcription and translation of the structural subunits of COX complex appear to be normal in Leigh syndrome (LS) COX-deﬁcient patients, which indicates that either the assembly or the maintenance of the COX complex is impaired without functional Surf-1 (Yao and Shoubridge, 1999). Mature Surf-1 protein (Surf-1p) is a 30-kDa hydrophobic polypeptide whose function is still unknown. This protein is imported into mitochondria as a larger precursor, which is then processed into the mature product by cleaving off an N-terminal leader polypeptide of approximately 40 amino acids. By using Western blot analysis with speciﬁc antibodies, Tiranti et al. (1999a) showed that Surf-1p is localized in, and tightly bound to the mitochondrial inner membrane. The same analysis revealed that no protein is present in cell lines harboring loss-of-function mutations of SURF-1, regardless of their type and position. Northern blot analysis showed the virtual absence of speciﬁc SURF-1 transcripts in different mutant cell lines. This result suggests that several mutations of SURF-1 are associated with severe mRNA instability. Functional domain analysis showed that none of the truncated or partially deleted SURF-1 cDNAs, expressed into Surf-1p null mutant cells, are able to rescue the COX phenotype, suggesting that different regions of the protein are all essential for function. Finally, experiments based on blue native two-dimensional gel electrophoresis indicated that assembly of COX in Surf-1p null mutants is blocked at an early step, most likely before the incorporation of subunit II in the nascent intermediates composed of subunit I alone or subunit I plus subunit IV. However, detection of residual amounts of fully assembled complex suggests a certain degree of redundancy in this system.

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II. Nuclear Genes Affecting COX Assembly and Stability

A. COX-NEGATIVE LEIGH SYNDROME Subacute necrotizing encephalomyelopathy, or Leigh’s syndrome (LS), was ﬁrst recognized as a pathological entity in 1951 (Leigh). Leigh syndrome is an invariably fatal encephalopathy of infancy and childhood characterized by psychomotor retardation, usually with regression, brain-stem, and respiratory abnormalities, and seizure. Neuropathological ﬁndings include focal symmetric necrotic lesions, most prominent in the brain stem, and characterized by vascular proliferation, demyelination, astrocytosis, and cystic cavitations. Elevated lactate levels in both blood and cerebrospinal ﬂuid (CSF) are often present. Magnetic resonance imaging (MRI) scan shows symmetric lesions in the basal ganglia, midbrain, brain stem, and spinal cord. (see Table I.) In recent years several defects in enzyme systems involved in mitochondrial energy production, including the pyruvate dehydrogenase complex, respiratory chain complexes I, II, IV (COX), and the mitochondrial-encoded ATPase 6 subunit, have been identiﬁed. Leigh’s syndrome can be inherited as an X-linked, autosomal-recessive, or maternal trait. Different particular clinical features, including age of onset and progression course, have been described. Leigh’s syndrome due to COX deﬁciency is heterogeneous, too. Only in a few cases has a correlation been found: the Leigh phenotype seems to be determined by the degree of impairment of energy production in certain brain regions to a greater extent than by the speciﬁc gene involved (Rahman et al., 1996). 1. SURF-1 Two different groups using fusion of LS COX—cell lines with rodent/ human ρ 0 hybrids (Tiranti et al., 1998) or a microcell-mediated chromosome transfer (Zhu et al., 1988) to complement the respiratory chain deﬁciency in patient ﬁbroblasts—mapped the gene defect of COX LS to chromosome 9q34, a locus containing the surfeit locus. In humans this locus contains ﬁve juxtaposed clustered genes, numbered 1 through 5. These genes and their associated CpG-rich islands are conserved over the 600 million years of divergent evolution that separates birds and mammals (Williams et al., 1988; Colombo et al., 1992). One of these genes, SURF-1, harbors mutations in a proportion of COX-negative LS. To date, SURF-1 mutations have been reported in 38 cases (Zhu et al., 1988; Tiranti et al., 1999a,b; Teraoka et al., 1999; Yao and Shoubridge, 1999;

TABLE I NUCLEAR GENE DEFECTS ASSOCIATED WITH COX DEFICIENCY DNA defect/disease locus Nuclear genes affecting COX assembly and stability

Disease phenotype

COX deﬁciency

Leigh syndrome

Severe decrease of COX (<20%) is present in muscle and in ﬁbroblasts and in other tissue

Chromosome 2p16 Lac-Saint-Jean COX deﬁciency

Leigh syndrome. Moderate development delay, hypotonia, ataxia, strabismus, and mild facial dysmorphism were frequent. The children had elevated blood and CSF lactate levels. Most of patients die during episodes of fulminant metabolic acidosis Leigh syndrome

Many tissues from these patients are deﬁcient in COX activity. Speciﬁcally, brain and liver tissues have 10–20% of the normal COX activity; ﬁbroblast and skeletal muscle have 50% of normal activity; kidney and heart tissues have almost 100% of normal activity Severe decrease of COX (<20%) is present in muscle and in ﬁbroblasts and in other tissues A severe isolated COX deﬁciency in postmortem liver of one of the affected newborn. COX deﬁciency was also found in lymphocytes, fetus chorionic villi, and amnionic cell ﬂuid Muscle COX deﬁciency was more severe in these patients than in patients with SURF1 mutations and the mtDNA-encoded subunits are

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SURF-1 (chromosome 9q34)

Leigh SURF1 negative

SCO1 (locus D17S1852 on chromosome 17)

Neonatal hepatic failure and ketoacidotic comas as the onset symptoms

SCO2 (chromosome 22q13)

Cardioencephalomyopathy. Age at onset within the ﬁrst 3 months of life and the death no later than 12 months for cardiac failure.

COX10 (chromosome 17p13.1—q11.1)

The patients presented respiratory difﬁculties and severe rapidly progressive cardiomyopathy. Neurological signs were psychomotor retardation, nystagmus, exotropia, severe hypotonia, occasional limb dystonia, and seizure Kidney tubulopathy and leukodystrophy

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Infantile autosomal recessive muscle COX deﬁciency Fatal infantile myopathy

Profound neonatal hypotonia and weakness, with severe lactic acidosis. Death before one year of age (respiaratory failure)

Benign infantile myopathy

Severe generalized weakness, requiring assisted ventilation, but improve spontaneously and appear normal by two or three years of age

more severely involved. Moreover, in SCO2 patients COX deﬁciency is less severe in ﬁbroblasts

Isolated COX deﬁciency in muscle, lymphocytes, and ﬁbroblast cultures. Western blot analysis of patient ﬁbroblasts revealed almost undetectable levels of COX subunit II (3%), marked reduction of subunits III and VIc (50% of normal levels) COX deﬁciency in these patients is conﬁned in skeletal muscle (absence of the nuclear DNA (nDNA)-encoded subunit VIIa,b of COX) Both VIIa,b and the mtDNA-encoded subunit II were absent

(continues)

TABLE I (Continued ) DNA defect/disease locus Infantile autosomal recessive mitochondrial encephalomyopathies with COX deﬁciency

Nuclear genes affecting mtDNA level and/or stability

Disease phenotype

COX deﬁciency

Leigh-like syndromes

Leigh syndrome with atypical neuropathology, or normal or atypical CT scan, or typical neuroradiology with normal lactate levels.

Decrease of COX in muscle and in ﬁbroblasts and in other tissues

Alpers’ disease (AR) (Progressive sclerosing poliodystrophy)

Intractable epilepsy, myoclonic jerks, progressive spasticity, optic atrophy with associated liver disease Heterogeneous group: most patients presented with muscle weakness; in others liver was the most or the only affected organ; other clinical picture was characterized by coexistence of myopathy and hepatopathy, of myopathy and renal tubulopathy. Encephalomyopathies, severe hypertrophic cardiomyopathy, or dilated cardiomyopathy are also described Ptosis, PEO, progressive muscle weakness and/or variably craniosomatic abnormalities, mental retardation, cardiomyopathy, hypogonadism, neuropathy, ataxia and tremor Gastrointestinal dysmobility, ophthalmoparesis, peripheral neuropathy, mitochondrial myopathy, and leukoencephalopathy

Deﬁciency of COX in the muscle biopsy in few cases

mtDNA depletion

212 PEO (AD and AR) (AD-PEO 1: chromosome 10q 23.3–10q 25.2 AD-PEO2: adenin-nucleotide translocator 1, chromosome 3p14.1–21.2) MNGIE (AR) (Chromosome 22q12.23, the gene encodes thymidine phosphorylase)

COX activity was less than 10% in affected tissues

Focal COX deﬁciency in the muscle biopsy

Muscle biopsy with ﬁbers COX negative and COX deﬁcient

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Poyau et al., 2000; Sue et al., 2000; Santoro et al., 2000; Darin et al., 2001). These patients are usually normal at birth; they present the ﬁrst symptoms in early infancy: the average age of onset was 11 months for Tiranti et al. (1999a) (18 patients) and 13.4 months for Sue et al. (2000) (6 patients). The average age at death was 60 months for Tiranti et al. (1999a) (15 patients) and 42.2 months for Sue et al. (2000) (5 patients). Rahman and colleagues (1996) proposed stringent inclusion criteria to select LS patients and to study the correlation between distinct phenotype and mutations. According to Tiranti et al. (1999a) and Sue et al. (2000), SURF-1 patients fulﬁlled Rahman criteria for LS. These criteria include (1) progressive neurological disease with motor and intellectual development delay, (2) signs and symptoms of brainstem and/or basal ganglia disease, (3) raised lactate levels in blood and CSF, and (4) one or more of the following: (a) characteristic features of LS on neuroradioimaging (symmetric hypodensities in the basal ganglia on computed tomography or hyperintense lesions on T2-weighted magnetic resonance imaging), (b) typical neuropathological changes at postmortem, or (c) typical neuropathology in a similar affected sibling. Seizure was only occasionally present in patients with SURF-1 mutations (Sue et al., 2000). In one case the authors reported peripheral neuropathy (Santoro et al., 2000). Cardiomyopathy has been described in a girl by Poyau et al. (2000); these authors reported a case without lactic acidosis in another girl. Severe decrease in COX (<20%) was present in muscle, in ﬁbroblasts, and in other tissue. Most of cases did not show ragged-red ﬁbers (RRF) at the muscle biopsy. The incidence of SURF-1 mutations, in Leigh patients, were different in various studies (from 75% of Tiranti et al., 1999, and 26% of Sue et al., 2000) this may reﬂect regional differences between the populations or the homogeneity of patients in Tiranti’s study that belonged to a single complementation group. Different mutations were found, including frameshift mutations, nonsense mutations, splice mutations (Tiranti et al., 1999a), and 2 base-pair (bp) deletion (Teraoka et al., 1999). A disomy of chromosome 9, close to SURF-1 locus, has been reported in monozygotic LS-cox deﬁciency female twins (Tiranti et al., 1999b). All the earlier reported patients had null mutations; however, missense mutations have been also more recently reported (Teraoka et al., 1999; Poyau et al., 2000). Missense mutations do not seem to modify SURF-1 mRNA expression but are able to prevent its function. Poyau and colleagues found that the G385A mutation in a patient changed the Gly124 into a Glu. It was not observed in 120 controls and is therefore not likely to be a common polymorphism. In addition, Gly124 is strictly conserved throughout evolution. Finally, infection of the patient ﬁbroblasts with a retroviral vector containing the normal SURF-1 restores their COX activity, demonstrating that the deﬁcit in COX assembly is caused

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by the mutated SURF-1. The Gly124 located in a highly conserved region of SURF-1 must therefore be crucial to the structure and/or the function of the protein. The second missense mutation, found by Poyau et al. (2000), changed the Ile246 into a Thr that removed the hydrophobic amino acid and the predicted β-sheet secondary structure. Both these structures could play an essential role in SURF-1 function. 2. Chromosome 2p16 Lac-Saint-Jean Cytochrome c Oxidase Deﬁciency The French-Canadian-type Leigh’s syndrome, an autosomal recessive form of congenital lactic acidosis, must be due to a mutation in a gene other than SURF-1 or SCO2 because the clinical phenotype maps to chromosome 2. The incidence was estimated at 1/2063 live births between 1979 and 1990, and the carrier rate was estimated at 1/23 inhabitants in Saguenay Lac-Saint-Jean region (Morin et al., 1993). Moderate developmental delay, hypotonia, ataxia, strabismus, and mild facial dysmorphism were frequent. The children had elevated blood and cerebral ﬂuid lactate levels. Most of the patients died in episodes of fulminant metabolic acidosis (Morin et al., 1993). Many tissues from these patients were deﬁcient in COX activity. Speciﬁcally, brain and liver tissues had 10–20% of the normal COX activity; ﬁbroblast and skeletal muscle had 50% of normal activity; kidney and heart tissues have almost 100% of normal activity (Merante et al., 1993). The cDNA sequence of the liver-speciﬁc COX subunits VIa and VIIa were determined in samples from patient liver and ﬁbroblasts, and showed normal coding sequence (Merante et al., 1993). Using high-resolution genetic mapping with additional microsatellite markers, Lee et al. (2001) were able to limit the critical region to ∼2 cM, between D2S119 and D2S2174. In this region COX7AR, a gene encoding a COX7a-related protein, has previously been mapped but the authors found no functional mutations (Lee et al., 2001). 3. Leigh SURF-1 Negative A proportion of cases of LS are not associated with SURF-1 mutations (Tiranti et al., 1999a; Sue et al., 2000; Darin et al., 2001). This also is conﬁrmed by complementation assay in cell culture, suggesting genetic heterogeneity of this condition (Tiranti et al., 1999a).

B. SCO1 Valnot et al. (2000b) described a family with two affected male siblings and two male fetuses that featured COX deﬁciency and SCO1 mutations. The parents were not consanguineous and the mother had six pregnancies with two boys affected, a healthy girl, two male-affected fetuses, and

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a healthy male fetus. The two born boys were found to be hypotonic and lethargic, and they required immediate assistance for respiratory distress. They presented with a severe metabolic acidosis. The ﬁrst boy had pH 7.19, plasma bicarbonate 7.7, and lactate 12.7 mM (normal is <2.5), severe axial hypotonia, hypoglycemia, and liver enlargement. He presented several episodes of apnea and bradycardia. They died at age two months and ﬁve days, respectively. An histopathological examination of the liver showed swollen hepatocytes, with microvesicular lipid vacuoles and panlobular steatosis. Histological study of a muscle biopsy sample revealed an accumulation of lipid droplets. A severe isolated COX deﬁciency with markedly altered activity ratios was found in postmortem liver biopsy of one of the affected newborns. Cytochrome c oxidase deﬁciency was also found in lymphocytes, fetus chorionic villi, and amnionic cell ﬂuid. A linkage study revealed that the four affected individuals carried an identical genotype at locus D17S1852 on chromosome 17, whereas the two healthy sibslings carried different haplotypes at this locus. Cytochrome c oxidase 10 and SCO1, two COX assembly genes, mapped in this region. All affected individuals were compound heterozygotes for SCO1 mutations. A 2 base pair (bp) deletion (GA; nucleotides 363–364) resulted in both a frameshift and a premature stop codon in exon 2. A second allelic mutation was found in exon 3. This mutation, a C → T transition at nt 520, changed a highly conserved proline into a leucine in the protein. The authors investigated the reason why the clinical consequences of the SCO1 mutations are markedly different from those caused by mutations in the functionally related SCO2 gene. Indeed, SCO1 mutations caused neonatal hepatic failure and ketoacidotic comas as onset symptoms, whereas SCO2 mutations developed hypertrophic cardiomyopathy and hypotonia during the ﬁrst weeks of life. Whether the clinical discrepancies between closely related gene mutations are signiﬁcant, fortuitous, or related to the small number of reported cases is still debatable.

C. SCO2 The clinical phenotype caused by mutations in human SCO2, another COX assembly gene located on chromosome 22q13, differs from that caused by mutations in SURF-1 Leigh syndrome. Only six patients (Papadopoulou et al., 1999; Sue et al., 2000; Jaksch et al., 2000) have been described, and they cannot be included in LS Rhaman criteria because they had atypical clinical, radiological, and laboratory features. Age of onset was within the ﬁrst 3 months of life and death no later than 12 months owing to cardiac

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failure. They presented respiratory difﬁculties, which required ventilatory support, and severe rapidly progressive cardiomyopathy. Neurological signs were psychomotor retardation, nystagmus, exotropia, severe hypotonia, occasional limb dystonia, and seizure. One of these children had dysmorphic facial features and electromyographic evidence of myopathy and peripheral neuropathy. Acid lactate was increased in blood and in CSF. Brain-stem auditory-evoked responses and somatosensory-evoked potentials revealed increased central conduction time. Brain MRI results were normal in one case, and it revealed cerebellar atrophy with alterations in frontal brain regions pointing to maturation disturbances and an infratentorial arachnoid cyst in another case ( Jaksch et al., 2000). In two cases, at the autopsy, the authors described brain atrophy, bilateral necrosis of the globus pallidus or of the medulla, and gliosis and moderate patchy loss of motor neurons. These features were more compatible with LS than with ischemic injury (Papadopoulou et al., 1999; Sue et al., 2000). In the third case the authors observed brain edema with focal lesions in hippocampal regions, brain atrophic cells, and increased gliosis ( Jaksch et al., 2000). In a further case atrophy and abnormal gyral pattern of the cerebral hemisphere have been observed (Papadopoulou et al., 1999). In the last two reported cases no cerebral and cerebellar abnormalities were seen on macroscopic examination ( Jaksch et al., 2000). Most organs showed hyperemia with an enlarged liver (Papadopoulou et al., 1999; Jaksch et al., 2000). Muscle COX deﬁciency was more severe in these patients than in patients with SURF-1 mutations and the mtDNA-encoded subunits were more severely involved. Moreover, in SCO2 patients COX deﬁciency is less severe in ﬁbroblasts. All the patients with mutations in SCO2 were compound heterozygotes for nonsense or missense mutations: G1541A mutation (resulting in E140K substitution) has been found in all six patients, but not in control subjects, suggesting that it is either an ancient mutation or a mutational hot spot. The SCO2 mutations are associated with a distinct phenotype of cardioencephalomyopathy and are absent in 42 patients with isolated COX deﬁciency and 45 sudden infant death syndrome (SIDS) infants ( Jaksch et al., 2000). Jaksch et al. (2000) sequenced the SCO2 gene in ten patients with infantile cardioencephalomyopathy due to COX deﬁciency in nine families and found mutations in three individuals. The COX deﬁciency in one patient’s ﬁbroblasts (approximately 50%) did not result in a measurable decrease in the steady-state levels of COX complex polypeptide subunits and could be rescued by microcell-mediated transfer of chromosome 22, but not other chromosomes. The authors speculated that another gene in the copper delivery pathway might contribute to the genetic heterogeneity of this disorder, although they cannot exclude a mutation in the promoter region

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of SCO2. The role of two SCO2 missense mutations (E140K and S225F) in COX deﬁciency was evaluated in yeast homologues: The yeast E155K Sco1p (equivalent to E140K) conferred respiratory competence in a haploid yeast strain, while the yeast S240F (equivalent to S225F ) mutant lacks COX activity, although COX was partially assembled. Therefore, the human E140K might become pathogenic only when a second null allele is present (Dickinson, 2000). D. COX10 Cytochrome c oxidase 10 is another nuclear gene responsible for complex IV deﬁciency in human disease. A boy born to ﬁrst cousin parents has been described (Valnot et al., 2000a). He was the second of three siblings; the two sisters died at ﬁve and three years of age respectively of a mitochondrial encephalopathy owing to COX deﬁciency. The patient had a normal development until the age of 18 months, when he developed ataxia and his neurological conditions gradually worsened. At two years of age, he presented with poor eye contact, severe muscle weakness, hypotonia, ataxia, ptosis, and pyramidal syndrome and status epilepticus. Brain MRI resulted in leukodystrophy-like changes. Heart ultrasound was normal. Lactate in blood and cerebral ﬂuid was high. Increased urinary amino acids were suggestive of a proximal tubulopathy. The proband presented with isolated COX deﬁciency in muscle, lymphocytes, and ﬁbroblast cultures. Western blot analysis of patient ﬁbroblasts revealed almost undetectable levels of COX subunit II (3%), and marked reduction of subunits III and VIc (50% of normal levels). He died at two years of age. No mtDNA mutations compatible with the patient disease have been found. A conventional linkage analysis in his large African consanguineous family allowed mapping of the disease gene chromosome 17p13.1-q11.1, to ﬁnd a homozygous C-to-A transversion in exon 4 of the COX10 gene, which resulted in an asparagine to lysine substitution (N204K) at a conserved position in the protein. The inability of the mutant gene to complement the yeast cox10 null strain, when expressed in low copy, conﬁrms that this substitution is the disease-causing mutation. This substitution is in the essential region for catalytic function of heme A:farnesyltransferase (Mogi et al., 1994). E. INFANTILE AUTOSOMAL-RECESSIVE MUSCLE COX DEFICIENCIES Two infantile forms of myopathy have been described with similarly severe presentation, but different prognosis, making differential diagnosis of

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vital importance. Soon after birth, affected children exhibit severe generalized weakness, respiratory distress, and lactic acidosis. 1. Fatal Infantile Myopathy Children with fatal infantile myopathy have a profound neonatal hypotonia and weakness, with severe lactic acidosis, and die of respiratory failure before one year of age (HeimanPetterson et al., 1982; Minchom et al., 1983; Bresolin et al., 1985). Many patients have proteinuria, glycosuria, phosphaturia, and generalized aminoaciduria (The Toni Fanconi syndrome) (Van Biervliet et al., 1977; DiMauro et al., 1980; Zeviani et al., 1985; Nonaka et al., 1988; Eshel et al., 1991). Different clinical phenotypes, probably not related genetically to the fatal infantile myopathy, are characterized by the association of myopathy and cardiomypathy in the same patients (Hart and Chang, 1988; Zeviani et al., 1986; Majander et al., 1995). Family history is usually noninformative, although there have been reports of affected siblings (Nonaka et al., 1988; Eshel et al., 1991). Parents were asymptomatic, and there was no consanguinity in any of the reported cases. Muscle biopsy showed large clumps of granules positive with oxidative enzyme stains and increased lipid droplets. Ultrastructural studies showed large aggregates of mitochondria, many of which were greatly enlarged. The COX deﬁciency in these patients is conﬁned in skeletal muscle. The tissue speciﬁcity of the disease suggests that the defect may involve one of the nuclear-encoded subunits. 2. Benign Infantile Myopathy Children with benign infantile myopathy also present with severe generalized weakness; they require assisted ventilation, but improve spontaneously and appear normal by two or three years of age (DiMauro et al., 1983; Zeviani et al., 1987; Servidei et al., 1988; Nonaka et al., 1988; Salo et al., 1992). Lactic acidosis, which is initially even more severe than in the fatal form, also remits spontaneously. At early stages, the clinical course fails to provide useful clues for differential diagnosis between the two forms of the disease. It is only the subsequent course and eventual outcome that distinguishes the patients with benign myopathy from those with fatal myopathy. Early differential diagnosis between fatal and benign COX-deﬁcient myopathies is of critical importance for prognosis and management of these infants, because the benign form is initially life-threatening but ultimately reversible. Benign infantile myopathy correlates with a gradual return of COX activity in muscle, which can be demonstrated both histochemically and biochemically.

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Tritschler et al. (1991) studied muscle biopsies from four patients with fatal myopathy and four with benign myopathy using biochemical, histochemical, and immunohistochemical techniques to differentiate the two forms of myopathy of infancy due to COX deﬁciency. The authors demonstrated that immunohistochemistry with antibodies directed against individual subunits of COX differentiated the two phenotypes: fatal infantile myopathy was characterized by absence of the nuclear DNA (nDNA)-encoded subunit VIIa,b of COX, while in benign myopathy both VIIa,b and the mtDNA-encoded subunit II were absent. F. INFANTILE AUTOSOMAL-RECESSIVE MITOCHONDRIAL ENCEPHALOMYOPATHIES WITH COX DEFICIENCY 1. Leigh-Like Syndromes According to the ﬁndings of Rahman and colleagues (1996), nearly half of the patients with a progressive mitochondrial encephalomyopathy could not be included in the stringently deﬁned group of LS because of atypical neuropathology (6/67 Rahman’s patients), lack of computed tomography (CT) or MRI scan (4 patients), normal CT scan (8 patients), atypical CT ﬁndings (11 patients), or typical neuroradiology with normal lactate levels and still living (3 patients). These data are conﬁrmed by Tiranti et al. (1999a), who studied 46 patients all characterized by reduced COX activity in muscle tissue or ﬁbroblast cells. Twenty-four patients were classiﬁed as COX-LS, 6 others as Leigh-like patients (with typical neuroradiology or neuropathology, but atypical laboratory or clinical features), and a third group as “non COX-LS” who lacked the typical MRI or neuropathological lesions consistent with LS. Most of these non LS patients were characterized by a progressive encephalomyopathy with impairment of multiple neurological system. In all patients the onset was in infancy. Sue et al. (2000) found 23 COX-LS patients and 18 patients with Leigh-like clinical presentation but without typical brain lesions. Darin and colleagues (2001) described a case of a child with clinical picture of LS and with COX deﬁciency, but MRI ﬁndings consisted of leukodystrophy without changes in brain stem or basal ganglia. Other single cases of variants of COX deﬁciency suggesting an autosomalrecessive transmission were reported by several authors in these years. Angelini et al. (1986) described the case of the male child of third cousins who was normal until age two years, when he had difﬁculty walking. At age eight he showed limb weakness, ataxia, loss of tendon reﬂexes, dyslalia, and mild retardation. During fasting, urinary organic acid excretion was abnormally high. The COX activity in muscle was 7% of the normal mean.

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Ogier et al. (1988) described a patient with severe muscle COX deﬁciency who had de Toni-Fanconi-Debr´e syndrome and acute neurologic deterioration resembling LS, without clear evidence of muscle abnormality. Metabolic investigations revealed elevated CSF lactate values contrasting with normal blood lactate. Markedly reduced COX activity was found by Saunier et al. (1995) in cultured skin ﬁbroblasts of an infant with recurrent episodes of acute myoglobinuria, hypertonia, muscle stiffness, and elevated plasma levels of sarcoplasmic enzymes (creatine kinase 96950 U/L, normal below 150) since the age of three weeks. A child with a spinal muscular atrophy (SMA)-like picture, cardiomyopathy, and COX deﬁciency was reported by Rubio-Gozalbo et al. (1999). Electromyography and muscle biopsy showed ﬁndings typical of SMA. However, COX staining of the muscle was negative. DNA analysis did not detect deletions in the survival motor neuron (SMN) gene. The lactate and lactateto-pyruvate ratios were increased in blood and CSF. Cytochrome c oxidase activity was reduced in muscle and ﬁbroblasts. Western blot analysis showed reduced contents for all COX subunits. 2. Alpers’ Disease (Progressive Sclerosing Poliodystrophy) Alpers’ syndrome is a rare autosomal recessive neurodegenerative disorder of uncertain aetiology characterized by untreatable epilepsy, myoclonic jerks, progressive spasticity, optic atrophy with associated liver disease. (Montine et al., 1995). The syndrome onset typically occurs in infancy, rarely after ﬁve years of age, and is rapidly progressive with death mostly before the age of three years. Cerebral imaging shows focal abnormalities in the occipital or parietal lobes, progressing to localized atrophy, but some patients shows generalized atrophy. Neuropathology shows thinning and discoloration of the cerebral cortex with neuron loss, gliosis, and spongiosis, progressing from superﬁcial to deep layers. Changes are focal, but the striate cortex is almost always severely affected. Cerebral spinal ﬂuid CSF protein levels and cell count may be normal or elevated. Some infants exhibit increased levels of lactic acid in the CSF, and in some cases, in the blood. Liver biopsy, performed at an early stage of the hepatic disease, revealed centrolobular necrosis, loss of hepatocytes, bile duct proliferation, and extended microvescicular fatty changes (Sokol et al., 1999). Several different mitochondrial enzyme deﬁciences affecting pyruvate metabolism have been reported in brain, liver, muscle, or ﬁbroblasts in some but not all affected children. In some cases deﬁciency of COX in the muscle biopsy was found (Worle et al., 1998).

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III. Nuclear Genes Affecting mtDNA Level and/or Stability

A. mtDNA DEPLETION SYNDROMES In 1983, Boustany et al. described a family in which the proband developed a fatal mitochondrial myopathy with marked hypotonia, ptosis, ophthalmoplegia, and high serum lactate. Muscle biopsy showed ragged-red ﬁbers and ultrastructurally abnormal mitochondria. Biochemical analysis of isolated muscle mitochondria showed that cytochromes aa3 and b were not detectable, but cytochrome cc was found to be normal by spectroscopy. The COX activity was less than 1% of normal in muscle, but it was normal in the liver. Mitochondria from kidney, liver, heart, lung, and brain examined postmortem had normal cytochromes and preserved COX activity. Her second cousin (related through the maternal grandfather) died at nine months of hepatic failure with generalized aminoaciduria but no lactic acidosis. Liver biopsy showed hepatocytes packed with large rounded mitochondria. Postmortem liver mitochondria showed deﬁcient cytochromes aa3 and b, and COX activity was about 10% of normal. Kidney mitochondria showed normal cytochromes. Muscle was not studied. In an addendum (Moraes et al., 1991), the authors noted that a sister of the proband presented at two months of age with an identical clinical, pathological, and biochemical phenotype, and died of respiratory failure at 21/2 months of age. Moraes et al. (1991) studied the probands of Boustany et al. (1983), and the authors found a severe depletion of the mtDNA in affected tissues. They detected the same genetic abnormalities in the muscle of an unrelated infant with myopathy, and in the muscle and kidney of a fourth child with myopathy and nephropathy. Tritschler et al. (1992) described two other infants with mitochondrial myopathy and severe reduction in mtDNA levels in muscle. In both patients the onset was at birth, and there was severe COX deﬁciency in muscle. Three other children, with later onset (about one year of age), have been reported; they showed a slower course and a less severe reduction in mtDNA content. Morphological studies of muscle biopsies of these patients showed that COX deﬁciency and mtDNA depletion were present in a mosaic and segmental pattern in skeletal muscle. Mazziotta et al. (1992) described an infant with severe lactic acidosis and progressive hepatopathy leading to death at age 4 months. Two half-siblings (different mothers) died at three months of an undiagnosed myopathy, and a third half-sibling was healthy. The liver from the infant with hepatopathy showed severe mtDNA depletion (10%); muscle was mildly depleted (53%). The two other half-siblings could not be studied.

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In this way, Moraes et al. (1991), Mazziotta et al. (1992), and Tritschler et al. (1992) deﬁned a “new” class of mitochondrial disease that does not involve qualitative errors in mtDNA but rather quantitative depletion of mtDNA in affected tissues. In this disorder, individuals exhibit variable levels of mtDNA depletion (up to 98%) in affected tissues, while unaffected tissues have relatively normal levels of mtDNA. In addition, different tissues may be involved in related patients (Moraes et al., 1991; Tritschler et al., 1992); thus, syndromes involving depletion of mtDNA can be diagnosed only when the activity of the respiratory chain enzymes and the content of mtDNA are investigated in the most affected tissues (Maaswinkel-Mooij et al., 1996). The patients with mtDNA depletion may be a heterogeneous group (Poulton et al., 1995), and some authors (Vu et al., 1998a) distinguished two subtypes: the congenital form, where symptoms begin at birth or during neonatal period, and most patients survive for less than one year; and the infantile form, which may begin in infancy or childhood, and survival is longer (range four months to the second decade). Some authors found a correlation between age of onset and clinical severity and relative mtDNA depletion (Vu et al., 1998a), while others authors did not (Campos et al., 1998). Moraes et al. (1991) found no evidence of a mtDNA mutation in the areas surrounding the origin of replication of the heavy strand (H-strand) or light strand (L-strand) of mtDNA. There was also no evidence of maternal inheritance: complementation with nuclear DNA from a normal cell line (Bodnar et al., 1993; Taanman et al., 1997) indicated involvement of the nuclear genome. Moraes et al. (1991) suggested that mtDNA depletion may be inherited as autosomal recessive; Mazziotta et al. (1992) proposed that it is an autosomal dominant trait with incomplete penetrance. Alternatively, Moraes et al. (1991) also suggested that this phenotype is the result of a dominant nuclear mutation expressed only when combined with a certain mitochondrial genotype; however, as the molecular basis of mtDNA depletions is not known, it is possible that different families may have different genetic defects and different modes of inheritance. Poulton et al. (1994) were prompted to investigate the human mitochondrial transcription factor A (TFAM), which is a 25-kD protein that may be an important regulator of both transcription and replication of mtDNA. Deﬁciency of the yeast homologue, ABF2, is associated with loss of mtDNA. They found that the ratio of mtDNA to nuclear DNA in skeletal muscle was low in the muscle of the three patients. Thus, they suggested that deﬁciency of TFAM might be a marker of, or possibly a cause of, mtDNA depletion in some patients with this condition. However, a causal relationship has not been established, and the low level of TFAM may be a secondary phenomenon Vu et al. (1998a).

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Until today, mitochondrial DNA depletion syndrome has been documented in 62 children. In the 9 cases of mtDNA depletion that have been reported, the liver was the most or the only affected organ, and the children usually died before nine months of age (Moraes et al., 1991; FigarellaBranger et al., 1992; Ricci et al., 1992; Bakker et al., 1996; Blake et al., 1999); Ducluzeau et al. (1999) described the case of a 28-month-old child who presented with a transient liver cholestasis, beginning at the age of 2 months, complicated by progressive ﬁbrosis. Most patients (26 cases) presented with muscle weakness associated with a variable value of depletion of mtDNA (50–99%) that appears limited to muscle (Moraes et al., 1991; Mazziotta et al., 1992; Telerman-Toppet et al., 1992; Tritschler et al., 1992; Poulton et al., 1995; Paquis-Flucklinger et al., 1995; Campos et al., 1998; Vu et al., 1998a). Few children with myopathy presented a mosaic pattern of depletion in muscle ﬁbers with a less severe clinical course (Tritschler et al., 1992; Vu et al., 1998a). Furthermore, mtDNA depletion may mimic other conditions, such as Duchenne dystrophy (Vu et al., 1998a) or spinal muscular atrophy (Pons et al., 1996). The clinical picture of other eight children was characterized by coexistence of myopathy and hepatopathy (Mazziotta et al., 1992; Bodnar et al., 1993; Maaswinkel-Mooij et al., 1996; Taanman et al., 1997; Tsao et al., 2000). Encephalomyopathies have been described in nine cases of mtDNA depletion by Poulton et al. (1995), Mariotti et al. (1995), Vu et al. (1998a), Campos et al. (1998), and Absalon et al. (2001) that reported a case of Leigh syndrome. A depletion of mtDNA (60–75%) has been reported by MarinGarcia et al. (1997, 2000) in two cases of severe hypertrophic cardiomyopathy and in three cases of dilated cardiomyopathy in which speciﬁc cardiac mitochondrial complex IV enzyme activity defects were found. Moraes et al. (1991) and Vu et al. (1998a) have described the coexistence of myopathy and renal tubulopathy. Severe mtDNA depletion may present with nonspeciﬁc symptoms such as vomiting, failure to thrive, and developmental delay; multiorgan involvement such as hepatomegaly, pancreatitis (Tsao et al., 2000), and myopathy occurs later (Poggi et al., 2000; Tsao et al., 2000).

B. AUTOSOMAL-DOMINANT PROGRESSIVE EXTERNAL OPHTHALMOPLEGIA (PEO) AND AUTOSOMAL-RECESSIVE PEO Progressive external ophthalmoplegia is a mitochondrial myopathy characterized by ptosis, PEO progressive muscle weakness, and/or craniosomatic abnormalities. Other associated features include mental retardation, cardiomyopathy, hypogonadism, neuropathy, ataxia, and tremor.

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Morphological mitochondrial abnormalities have been found in some ﬁbers: subsarcolemmal accumulation of mithocondria or ragged-red ﬁbers. The total COX activity and cytochrome aa3 concentration in muscle mitochondrial fractions is usually approximately 40% of normal. The disease occurs in both sporadic and familial forms, and in the familial form both autosomal-dominant (AD-PEO) and autosomal-recessive (AR-PEO) transmission have been observed. In AD-PEO/AR-PEO, the structural integrity of mtDNA is affected, suggesting that the underlying nuclear defect predispose to somatic mutations of mtDNA with accumulation of multiple mtDNA deletions. (Li et al., 1999). At least three nuclear loci are believed to be responsible for AD-PEO. By linkage analysis the ﬁrst disease locus was assigned to chromosome 10q23.3– 10q25.2 in a single Finnish family and conﬁrmed in a Pakistan family. A second locus was mapped to chromosome 3p14.1–21.2 in three Italian families, although the more informative family in this study was subsequently shown to be linked to chromosome 4q34; therefore chromosome 3 p linkage awaits further conﬁrmation (Kaukonen et al., 2000). In another Italian family, the disorder is linked to chromosome 4q34– q 35. The critical region of the 4q PEO locus includes the gene encoding the heart and skeletal muscle speciﬁc isoform of the adenine nucleotide translocator (ANT1). Adenine nucleotide translator or ADP/ATP translocator, is the most abundant protein in the inner mitochondrial membrane, and it mediates signals of nucleocytoplasmic energy consumption to the mitochondrial respiratory chain. Two heterozygous missense mutations in the nuclear gene-encoding ANT1 in ﬁve families and one sporadic patient have been identiﬁed (Kaukonen et al., 2000). The familial mutation substitutes a proline for a highly conserved alanine at position 114 in the ANT1 protein. The analogous mutation in yeast caused a respiratory defect. Carrozzo et al. (1998) studied three patients with AD-PEO and three patients with autosomal recessive PEO to identify differences in the clinical, histological, and molecular genetic features of patients. Clinically in the AD form, weakness affected extraocular and proximal limb muscles starting in early adulthood (ages 24–39 years). Patients with AR-PEO were younger at onset (ages 8–24 years). The differences between the two groups are the following: the AD patients present with a predominant myopathy, whereas AR patients show multisystem disorders. Muscle biopsy from all six patients show RRF and COX-negative ﬁbers; however, patients with AD-PEO had signiﬁcantly higher numbers of abnormal muscle ﬁbers than patients with AR-PEO. In both groups of patients the molecular genetic analysis show only deleted and not duplicated mtDNA molecules, in contrast with sporadic form of PEO. Patients with AD-PEO harbored a higher proportion of deleted relative to normal mtDNA in skeletal muscle than patients with AR-PEO.

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C. MITOCHONDRIAL NEUROGASTROINTESTINAL ENCEPHALOMYOPATHY (MNGIE) MNGIE is an autosomal recessive multisystemic disorder characterized by onset between the ﬁrst to ﬁfth decades, gastrointestinal dysmobility, ophthalmoparesis, peripheral neuropathy, mitochondrial myopathy, and leukoencephalopathy. Analysis of mitochondrial DNA in skeletal muscle shows partial depletion, multiple deletions, or both. Muscle biopsy shows mitochondrial abnormalities such as ragged-red ﬁbers and focal COX deﬁciency. We studied (Papadimitriou et al., 1998) three affected siblings (two were monozygotic twins) born to nonconsanguineous parents. All patients were of short stature and cachectic. The ﬁrst symptom in the twins was childhood diarrhea, whereas the elder brother ﬁrst showed progressive hearing loss at the age of 34 years. Age at onset of skeletal muscle symptoms was 40 years for the twins and 48 years for the elder brother. Although our patients did not show any sign of CNS involvement, brain MRI studies demonstrated a diffuse abnormally high signal in the white matter of both cerebral hemispheres that may be the result of myelin edema secondary to mitochondrial impairment. Morphologic and biochemical analysis of muscle showed RRF, COX-negative, and COX-deﬁcient ﬁbers. A defect of NADH-DH and NADH cytochrome c (cyt-c) R revealed an additional partial defect in complex I of the respiratory chain enzymes. The MNGIE locus was mapped to the chromosome 22q12.23–qter; the gene encodes thymidine phosphorylase (TP), an enzyme that catalyzes the phosphorolysis of thymidine to thymine, rather than a factor directly associated with mtDNA replication or repair. In patients, thymidine is increased because of low TP catalytic activity. There are two distinct TPs in the cell: ctyoplasmic TP (TP1), which is only active in replicating cells; and mitochondrial TP (TP2), which is constitutively active even in quiescent cells. Thus, increased thymidine is salvaged predominantly in mitochondria, especially in quiescent cells, such as muscle and nerve cells, resulting in the nucleotide pool imbalance and the suppression of mtDNA replication resulting in multiple mtDNA deletions or mtDNA depletion, or both (Nishino et al., 2001).

IV. mtDNA Defects

A. COI MUTATIONS A heteroplasmic 5-bp microdeletion in the mitochondrial COX subunit I gene (COI ) has been described in association with severe isolated muscle

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TABLE II MITOCHONDRIAL ENCEPHALOMYOPATHIES WITH DEFECTS OF mtDNA Mithocondrial DNA mutation Point mutation

Gene location tRNA Lys A8344G

G8342A T8356C G8363A A8296G G8328A G8313A tRNA Leu A3243G

C3256T A3251G T12311C G12315A A3252G A3260G T3271C T3291C T3250C A3288G A3302G A12320G C3254G A3260G C3303T T3264C G12301A

Disease

MERRF, spinocerebellar syndrome, Leigh syndrome, bilateral striatal necrosis, multiple symmetric lipomatosis PEO/myoclonus MERRF, MERRF/MELAS MERRF, multisystem involvement with cardiomyopathy Hypertrophic cardiomyopathy, diabetes/deafness Mitochondrial encephalomyopathy Gastrointestinal s./encephaloneuropathy MELAS, PEO, myopathy/painful stiffness, myopathy/dystonia, diabetes/deafness, infantile encephalopathy Multysystem/PEO PEO, myopathy PEO PEO MELAS MELAS, MIMyCa MELAS MELAS Myopathy Myopathy Myopathy Myopathy MIMyCa MIMyCa MIMyCa Ataxia/diabetes/PEO Acquired idiopathic sideroblastic anemia

tRNA Ile T4274C T4285C G4309A G4298A A4295G A4300G A4269G C4320T

PEO PEO PEO PEO/multiple sclerosis Hypertrophic cardiomyopathy Hypertrophic cardiomyopathy Multisystem cardiomyopathy Multisystem cardiomyopathy

tRNA Asn A5692G G5703A

PEO PEO

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TABLE II (Continued ) Mithocondrial DNA mutation

Single large deletion Large-scale tandem duplication Microdeletion

Gene location

Disease

tRNA Ser T7512C

MERRF/MELAS

tRNA Cys A5814G

MELAS

tRNA Val G1642A G1606A

MELAS Mitochondrial encephalomyopathy

tRNA Phe G583A T618C A606G

MELAS Myopathy Exercise intolerance/myoglobinuria KSS, PEO

COX I

Motor neuron disease-like

COX I G6930A T6721C T6742C

Mytochondrial encephalomyopathy Acquired idiopathic sideroblastic anemia Acquired idiopathic sideroblastic anemia

Point mutation

COXII T7587C

Severe encephalomyopathy

Insertion

COXIII C9537

Leigh-like syndrome

Point mutation

COX III T9957C G9952A

Point mutation

MELAS Encephalopathy, exercise intolerance proximal myopathy

COX deﬁciency in a patient with atypical motor neuron disease. The mutation is a heteroplasmic 5-bp microdeletion located in the 5 of the COI gene, leading to premature termination of the corresponding translation product. Muscle COX subunits II–IV were shown to be decreased as well both by immunohistochemistry and Western blot analysis, suggesting a defective assembly of COX holoenzyme (Comi et al., 1998). A stop-codon mutation in COI subunit gene was then identiﬁed in a young woman with a multisystem mitochondrial disorder. Histochemical analysis of a muscle-biopsy sample showed virtually absent COX stain, and biochemical studies conﬁrmed an isolated reduction of COX activity. (see Table II.) Sequence analysis of the mitochondrial-encoded COX-subunit genes identiﬁed a heteroplasmic G:A transition at nucleotide position 6390 in the

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COI gene that causes a disruption in the assembly of the respiratory-chain complex IV (Bruno et al., 1999). In line with a proposed role of COX I as scaffold for the other subunits during COX assembling, both the described mutations result in a decreased steady state level of COX II–IV. Other nucletide changes in COI sequence have been described: two missense mutations in patients with acquired idiopathic sideroblastic anemia (Gattermann et al., 1997) and a homoplasmic somatic COI stop-codon mutation in colorectal-cancer cell lines (Polyak et al., 1998).

B. COII MUTATIONS A mutation in the initiation codon of the COX subunit II (COII ) gene, in which a T to C substitution is predicted to change methionine to threonine, was found in a 57-year-old woman with fatigue and unsteadiness of gait, and in her 34-year-old son affected with a severe encephalomyopathy (Clark et al., 1999). The mutation load was present at 67% in the muscle of the mother and at 91% in the muscle of the patient’s clinically affected son. Muscle biopsy revealed isolated COX deﬁciency and mitochondrial proliferation. The COX II polypeptide was nearly absent and the COX II mRNA markedly reduced in patient’s ﬁbroblasts nearly homoplasmic for the T7587C mutation. A 50% decrease of COX IV immunoreactivity seems to support a defective assembling of COX holoenzyme.

C. COIII MUTATIONS More mutations are reported in the COX subunit III (COIII ) gene. A 15-bp microdeletion in a highly conserved region of the gene was associated with recurrent myoglobinuria and myopathy in a 15-year-old girl (Keightley et al., 1996); a missense mutation at position 9957 was found in an adult with MELAS (Manfredi et al., 1995); a stop mutation at nucleotide 9952 was described in a 36-year-old woman suffering from episodes of encephalopathy associated with lactic acidemia, exercise intolerance, and proximal myopathy (Hanna et al., 1998); and virtually homoplasmic insertion of an extra C at nucleotide position 9537 in skin ﬁbroblasts and in the muscle of a child affected by a progressive neurological disorder (Tiranti et al., 2000). The ﬁrst stop-codon mutation in mtDNA to be reported was in association with a case of a 36-year-old woman who experienced episodes of encephalopathy accompanied by lactic acidemia, and who had exercise intolerance and proximal myopathy. Histochemical analysis showed that 90% of muscle ﬁbers exhibited decreased or absent COX activity. Sequences

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analysis of mtDNA identiﬁed a novel heteroplasmic G to A point mutation at position 9952, located in the 3 end of the gene for subunit III of COX (Hanna et al., 1998). The ﬁrst case of a Leigh-like syndrome due to a mutation in a structural COX gene has been reported (Tiranti et al., 2000). A virtually homoplasmic frameshift mutation in the mtDNA COIII gene was found in skin ﬁbroblasts and in the muscle of a child affected by a progressive neurological disorder. This mutation, due to the insertion of an extra C at nucleotide position 9537 of mtDNA, creates a stop codon leading to the synthesis of prematurely truncated polypeptide. The child, a 11-year-old girl, was born at term after an uneventful pregnancy. The family history was negative and psychomotor development was normal during the ﬁrst year of life. She suffered multiple episodes of metabolic coma associated with severe metabolic acidosis, hyperchetonuria, and hypoglycemia at the age of one, two, and four years. She was subsequently affected by severe progressive tetraparesis, associated with ophthalmoparesis, convergent strabismus, reduced visul acuity, and moderate mental retardation. Severe metabolic acidosis and high levels of blood lactate were detected. Nuclear MRI examination of the brain revealed the presence of bilateral lesions in the putamina and mild atrophy of the brain and cerebellum, whereas the brain-stem structures appeared normal. The authors found a virtual absence of reaction to COX by histochemical and biochemical analysis in muscle and skin. RRF were absent. The relatively mild course of the disease is in contrast with the marked biochemical defect of COX activity and with the severity of the genetic lesion. Probably this mutation, homoplasmic in muscle and ﬁbroblasts, may be heteroplasmic in brain and other tissues.

D. MUTATIONS AFFECTING mtDNA TRANSLATION (TRANSFER RNA POINT MUTATIONS, DELETIONS, AND DUPLICATIONS) An almost unlimited list of disorders share as common muscle pathological hallmarks focal COX deﬁciency and mitochondrial proliferation with the aspects of RRF at the modiﬁed Gomori’s stain. They are generally due to heterogeneous heteroplasmic mtDNA mutations, either sporadic or maternally inherited, and they include both transfer RNA (tRNA) point mutations and macrorearrangements (such as deletions and duplications). The peculiar clinical and sometimes histopathological features of the associated syndromes require a separate description, although formal evidence for their correlation with speciﬁc phenotypes is still lacking.

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1. Myoclonic Epilepsy with RRF (MERRF) MERRF is characterized by myoclonus, ataxia, tonic-clonic generalized seizures and myopathy with RRF. Dementia, heart block, short stature, hearing loss, neuropathy, and optic atrophy are common additional features (Silvestri et al., 1993). Onset is usually in childhood, but onset in adult age has also been described. Muscle biopsy reveals a high percentage of RRFs on Gomori trichrome staining, most of which are also COX negative. Biochemistry shows complex I and IV deﬁciency, although other respiratory chain enzymes may also be affected. (Schapira and Cock, 1999). Pathological studies have revealed neuronal loss in the inferior olivary nucleus and dentate nucleus, diffuse gliosis of cerebellar and brainstem white matter, and degeneration of the spinal cord posterior columns. The “classical” MERRF mutation is an A–G transition at nt 8344 in the tRNALys gene of mitochondrial DNA (Shoffner et al., 1990). The relationship between the percentage of mutated mtDNA and clinical severity is unclear. This mutation can be associated with other phenotypes, including Leigh’s syndrome, myoclonus or myopathy with truncal lipomas and proximal myopathy. Although the A8344G mutation seems to be the most frequent, it does not appear to be responsible for all the MERRF cases because several A8344G negative families with typical maternal-inherited MERRF have been reported. Two other MERRF mutations are also located in the tRNALys gene, at nt-8356 and nt-8363 (Zeviani et al., 1993; Ozawa et al., 1997). Other mutations were observed in the tRNALeu (UUR) and tRNASer(UCN) (Moraes et al., 1993). All these mutations were heteroplasmic. 2. Mitochondrial Encephalomyopathy with Lactic Acidosis and Stroke-Like Episodes (MELAS) MELAS is the common maternally inherited mitochondrial disease. It is deﬁned by stroke typically before age 40, encephalopathy characterized by dementia, seizures, evidence of mitochondrial dysfunction with RRF, and lactic acidosis (Hirano et al., 1992). Other frequent clinical features include recurrent vomiting, migrainelike headaches, limb weakness, exercise intolerance, and short stature. Deﬁciences of complexes I, III, and IV of the respiratory chain have been documented, most commonly complex I (Ciafaloni et al., 1992). Typically it is characterized by early normal development and childhoodonset recurrent neurologic deﬁcits. It is usually, but not invariably, associated with the A3243G point mutation in the mitochondrial DNA tRNA Leu (UUR) gene. (Goto et al., 1990). However, severe psychomotor delay in early infancy was also reported in three unrelated children with the A3243G mutation (Sue et al., 1999). In addition to early onset, other atypical features

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were absence of an acute or focal neurologic deﬁcit, variable serum and CSF lactate levels, and lack of RRF in muscle biopsy specimens. The proportion of mutant mtDNA in available tissue was relatively low. This study documented an association of the A3243G mutation with PEO phenotypes, suggesting that other factors contribute to the phenotypic expression of this mutation. It is known that the tRNA Leu (UUR) gene has at least two functions: not only it is necessary to produce a tRNA for translation, but it is also crucial in regulating the amount of ribosomial RNAs that the mitochondrion produces. The rRNA region of the mitochondrial genome is transcribed at a much higher rate than the rest of the genome, in order to produce the large amounts of 12 S and 16 S rRNA required to translate the mRNAs encoding the 13 polypeptides. The three other tRNALeu(UUR) mutations are nt 3252 (Morten et al., 1993), nt 3271, and nt 3291 (Goto et al., 1991). One mutation was also reported in tRNAVal (Taylor et al., 1996). All these mutations were heteroplasmic. Other tRNA mutations have been found in syndromes overlapping with MERRF. The cause of the stroke, which commonly causes hemianopsia and cortical blindness, is not known. Imaging shows that the brain lesions do not conform to the distribution of major cerebral arteries (Matthews et al., 1991). Excessive accumulations of mitochondria were found in the walls of small arteries and capillaries in muscle and brain (Ohama et al., 1987). These small vessel abnormalities—called strongly SDH-reactive blood vessels, or SSVs (the angiopathic analogue of RRFs) are not found in the majority of other mitochondrial disorders (with the exception of MERRF—Hasegawa et al., 1991). It is, however, unclear whether the strokes are due to “small vessel angiopathy” or are, in fact, “metabolic strokes” independent of any vascular pathology. 3. Maternally Inherited PEO Progressive external ophthalmoplegia is characterized by ptosis and weakness of the extraocular muscles, often accompanied by limb weakness. Progressive external ophthalmoplegia has been associated with mtDNA single deletions, which can arise sporadically, or multiple deletions inherited as a Mendelian trait. The remaining patients inherit PEO as a maternal trait—for example, the A3243G tRNA Leu (UUR). Other mutations were described in tRNAIle (Silvestri et al., 1996), tRNA (Asn), and in the hot spot of the tRNA Leu (UUR) gene. The mutation in tRNA (Asn) is associated with isolated ophthalmoplegia, whereas the mutations in tRNA Leu (UUR) cause neurological syndromes resembling MERRF plus optic neuropathy, retinopathy, and diabetes. (Moraes et al., 1993). Many of the described patients harbor the nt-3243 mutation, which is more commonly associated with MELAS (Goto et al., 1990).

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4. Maternally Inherited Myopathy and Cardiomyopathy (MIMYCA) Maternally inherited myopathy and cardiomyopathy has been described as maternally inherited adult-onset syndrome, clinically characterized by the variable combination of skeletal and heart muscle failure, and at the molecular level by the presence of a heteroplasmic point mutation, consisting of an A–G transition at nucleotide 3260 in the mtDNA gene speciﬁng tRNA Leu (UUR). Signiﬁcant correlations were found between the proportion of the mtDNA mutation and complexes I and IV deﬁciency (Zeviani et al., 1995). 5. Kearns-Sayre Syndrome (KSS) As opposed to the maternally inherited point mutations, a group of mtDNA mutations occurs spontaneously, with no obvious genetic component. These mutations fall into a completely different genetic category— namely, the large-scale partial deletions and duplications of mtDNA. Since they were ﬁrst discovered in 1988 (Holt et al., 1988), almost 200 species of rearranged mtDNAs have been reported. Kearns-Sayre Syndrome consists of progressive external ophthalmoplegia, ptosis, atypical pigment retinopathy and cardiac conduction defects. It is one of a diverse group of mitochondrial disorders that may be predominantly myopathic or involve multiple-organ systems (Provenzale et al., 1996). Other features of KSS include hearing loss, endocrinopathies (particularly hypoparathyroidism and diabetes mellitus), proximal limb weakness, and renal tubular dysfunction. Blood and CSF have elevated levels of lactate, and muscle biopsies show RRF. The course is relentlessly downhill and death usually occurs at a young age. Partial mtDNA deletions were ﬁrst described in 1988 in patients with KSS (Holt et al., 1988). The deletions ranged in size from 2 to 7 kilobases (kb), and did not localize to any single region of the mitochondrial genome. There was no correlation between the size or site of the deletion, biochemical abnormality of mitochondrial enzymes, or clinical severity. (Zeviani et al., 1988). In 1989, Poulton’s group ﬁrst reported the association of KSS with heteroplasmic mtDNA tandem duplications. It was later demonstrated that duplications and deletions coexisted in a subset of KSS patients (Poulton et al., 1994). It has been postulated that mtDNA deletions impair mitochondrial protein synthesis due to the loss of tRNA genes (Nakase et al., 1990). By contrast, the pathogenic signiﬁcance of mtDNA duplications is still uncertain. For instance, large-scale heteroplasmic mitochondrial DNA rearrangements were found in a 50-year-old woman with an adult-onset progressive myopathy. The predominant mtDNA abnormality was a 21.2 kb duplicated molecule. In addition, a small population of the corresponding partially deleted 4.6 kb molecule was detected. Muscle biopsy showed ﬁbers negative for COX activity (Manfredi et al., 1997).

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Valnot, I., von Kleist-Retzow, J.-C., Barrientos, A., Gorbatyuk, M., Taanman, J.-W., Mehaye, B., Rustin, P., Tzagoloff, A., Munnich, A., and Rotig, A. (2000a). A mutation in the human heme A:farnesyltransferase gene (COX10) causes cytochrome c oxidase deﬁciency. Hum. Mol. Genet. 9, 1245–1249. Valnot, I., Osmond, S., Gigarel, N., Mehaye, B., Amiel, J., Cormier-Daire, V., Munnich, A., Bonnefont, J. P., Rustin, P., and Rotig, A. (2000b). Mutations of the SCO1 gene in mitochondrial cytochrome c oxidase deﬁciency with neonatal-onset hepatic failure and encephalopathy. Am. J. Hum. Genet. 67, 1104–1109. Van Biervliet, J. P. A. M., Bruinvis, L., Ketting, D., DeBree, P. K., Heiden, E. V., Wadman, S. K., Willems, J. L., Bookelman, H., VanHaelst, U., and Monnens, L. A. (1977). Hereditary mitochondrial myopathy with lactic acidemia, a DeToni-Fanconi-Debre syndrome and a defective respiratory chain in voluntary striated muscles. Pedriatr. Res. 11, 1088–1093. Vu, T. H., Sciacco, M., Tanji, K., Nichter, C., Bonilla, E., Chatkupt, S., Maertens, P., Shanske, S., Mendell, J., Koenigsberger, M. R., Sharer, L., Schon, E. A., DiMauro, S., and DeVivo, D. C. (1998a). Clinical manifestations of mitochondrial DNA depletion. Neurology 50, 1783–1790. Vu, T. H., Tanji, K., Valsamis, H., Di Mauro, S., and Bonilla, E. (1998b). Mitochondrial DNA depletion in a patient with long survival. Neurology 51, 1190–1193. Williams, T., Yon, J., Huxley, C., and Fried, M. (1988). The mouse surfeit locus contains a very tight cluster of four “housekeeping” genes that is conserved through evolution. Proc. Natl. Acad. Sci. USA 85, 3527–3530. Worle, H., Kohler, B., Schlote, W., Wikkler, P., and Bastanier, C. K. (1998). Progressive cerebral degeneration of childhood with liver disease (Alpers’ Huttenlocher disease) with cytochrome oxidase deﬁciency presenting with epilepsia partialis continua as the ﬁrst clinical manifestation. Clin. Neuropathol. 17, 63–68. Yao, J., and Shoubridge, E. A. (1999). Expression and functional analysis of SURF1 in Leigh syndrome patients with cytochrome c oxidase deﬁciency. Hum. Mol. Genet. 8, 2541–2549. Zeviani, M., Nonaka, I., Bonilla, E., Okino, E., Moggio, M., Jones, S., and DiMauro, S. (1985). Fatal infantile mitochondrial myopathy and renal dysfunction caused by cytochrome c oxidase deﬁciency: Immunological studies in a new patient. Ann. Neurol. 17, 414–417. Zeviani, M., Van Dyke, D. H., Servedei, S., Bauserman, S. C., Bonilla, E., Beaumont, E. T., Sharda, J., Vanderlaan, K., and DiMauro, S. (1986). Myopathy and fatal cardiopathy due to cytochrome c oxidase deﬁciency. Arch. Neurol. 43, 1198–1202. Zeviani, M., Peterson, P., Servidei, S., Bonilla, E., and DiMauro, S. (1987). Benign reversible muscle cytochrome c oxidase deﬁciency: A second case. Neurology 37, 64–67. Zeviani, M., Moraes, C. T., DiMauro, S., Nakase, H., Bonilla, E., Schon, E. A., and Rowland, L. P. (1988). Deletions of mitochondrial DNA in Kearns-Sayre syndrome. Neurology 38, 1339–1346. Zeviani, M., Muntoni, F., Savarese, N., Serra, G., Tiranti, V., Carrara, F., Mariotti, C., and DiDonato, S. (1993). A MERRF/MELAS overlap syndrome associated with a new point mutation in the mitochondrial DNA tRNA (Lys) gene. Eur. J. Hum. Genet. 1, 80–87. Zeviani, M., Mariotti, C., Antozzi, C., Fratta, G. M., Rustin, P., and Prelle, A. (1995). OXPHOS defects and mitochondrial DNA mutations in cardiomyopathy. Muscle Nerve 3, 170–174. Zeviani, M., Moraes, C. T., DiMauro, S., Nakase, H., Bonilla, E., Schon, E., and Rowland, L. (1998). Deletions of mitochondrial DNA in Kearns-Sayre syndrome. Neurology 51, 1525– 1534. Zhu, Z., Yao, J., Johns, T., Fu, K., De Bie, I., Macmillan, C., Cuthbert, A. P., Newbold, R. F., Wang, J., Chevrette, M., Brown, G. K., Brown, R. M., and Shoubridge, E. A. (1988). SURF1, encoding a factor involved in the biogenesis of cytochrome c oxidase, is mutated in Leigh syndrome. Nat. Genet. 20, 337–343.

SECTION IV TOXIN-INDUCED MITOCHONDRIAL DYSFUNCTION

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TOXIN-INDUCED MITOCHONDRIAL DYSFUNCTION

Susan E. Browne1 and M. Flint Beal Department of Neurology and Neuroscience Weill Medical College of Cornell University New York, New York 10021

I. Introduction II. Inhibitors of Mitochondrial Complex I: NADH Ubiquinine Oxidoreductase A. MPTP/MPP+ B. Rotenone C. Neurolathyrism III. Inhibitors of Mitochondrial Complex II: Succinate Ubiquinol Oxidoreductase A. 3 -Nitropropionic Acid (3 -NP) B. Malonate IV. Inhibitors of Mitochondrial Complex IV: Cytochrome c Oxidase A. Cyanide B. Hydrogen Sulfide C. Sodium Azide V. Manganese VI. 3-Acetylpyridine VII. Myopathies and Myotoxic Agents VIII. Discussion: What Determines the Regional and Cellular Speciﬁcity of Mitochondrial Toxins? References

I. Introduction

Mitochondria constitute the cellular powerhouse in the body, providing the bulk of energy generated from aerobic catabolism, and are therefore critical for maintaining cellular function and homeostasis. Within the CNS, where functional activity depends entirely on oxidative glucose metabolism under normal conditions, the mitochondria represent not only a fundamental organelle for neuronal and glial function, but also a target for damage within cells with potentially catastrophic consequences for the cell, organ, and organism. Mitochondrial dysfunction is well characterized in a number of human disorders, sometimes stemming from inherited or spontaneous mitochondrial or nuclear DNA mutations, from disease related perturbations in 1

To whom correspondence should be addressed.

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cell homeostasis, or alternatively from direct damage arising from extrinsic sources. Mitochondrial toxins play important roles in the study of mitochondrial function and dysfunction. They not only serve as useful tools for the study of mechanistic pathways involved in mitochondrial damage, consequences on cellular function, and for assays of putative neuroprotective therapies, but in some cases they may also be directly involved in the pathogenesis of mitochondrial disorders in humans. Here we review the mode of action of several well characterized mitochondrial toxins, and, where relevant, contrast their effects with mechanisms of cell death in various degenerative disorders. The majority of known toxins speciﬁcally target components of mitochondrial metabolic pathways. Here we have grouped toxic agents in terms of their principal target site within the mitochondria, often reﬂecting effects on components of the mitochondrial respiratory system. In several cases, however, these agents have multiple sites of action due to their afﬁnity for certain chemical moieties present in multiple mitochondrial proteins, demonstrated by cyanide’s propensity for Fe3+ containing enzymes which include both complexes II and IV of the electron transport chain.

II. Inhibitors of Mitochondrial Complex I: NADH Ubiquinine Oxidoreductase

A. MPTP/MPP+ 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication in humans, nonhuman primates, and rodents induces a motor disorder clinically and pathologically mimicking Parkinson’s disease (PD), due to direct actions on mitochondrial function via complex I inhibition (Gerlach and Riederer, 1996; Olanow and Tatton, 1999; Blum et al., 2001). Interestingly, the pathophysiological effects of MPTP only came to light as a result of poor chemistry in the illicit drug trade. In 1978–1982 several heroin addicts in California developed multiple irreversible, L-DOPA-responsive [DOPA: 3-(3,4-dihydroxyphenyl)alanine] PD symptoms as a result of self-administration of a synthetic fentanyl heroin derivative (Davis et al., 1979). The cause was found to be a slight contamination (∼3%) with MPTP, a by-product generated accidentally during the synthetic process (Langston et al., 1983). The toxicity of MPTP is actually conferred by its metabolite 1-methyl-4phenylpyridinium ion, MPP+, which accumulates in mitochondria selectively within dopaminergic neurons in the CNS.The cascade of cell-damaging events caused by MPP+ appears to be triggered by its potent inhibition of complex I activity, resulting in deﬁcient oxidation of NAD+ substrates,

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impaired α-ketoglutarate dehydrogenase activity, and subsequent disruption of the respiratory electron transport chain (Niklas et al., 1985; Mizuno et al., 1989; Sayre, 1989). Reduced oxidative phosphorylation and ATP synthesis ensue, along with disruption of the mitochondrial membrane potential. The ﬁnding that MPTP induces a complex I inhibition associated with a PD-like phenotype is in keeping with multiple reports from studies in PD postmortem brain tissue, platelets, and cybrid cell lines that a mitochondrial deﬁciency of complex I activity underlies the pathogenesis of PD (Parker et al., 1989; Menegon et al., 1998; Swerdlow et al., 1998). The typical symptoms of MPTP/MPP+ intoxication—rigidity, akinesia, resting tremor, posture, and gait abnormalities—stem from the speciﬁc cellular localization of MPP+’s toxicity within the CNS. The MPTP causes dopamine depletion in the neostriatum due to selective loss of dopaminergic neurons in the substantia nigra that mimics the pathology seen in PD (Burns et al., 1983; Beal, 2001). In primates treated with MPTP, neurons within the substantia nigra pars compacta (SNpc) and the locus coeruleus are particularly vulnerable, while neurons in the ventral tegmental area (a dopamine nucleus projecting predominantly to the cortex) are relatively resistant. Chronic treatment with low doses of MPTP in monkeys preferentially targets dopaminergic terminals in the putamen, similar to the typical pathology seen in PD brain. In fact, a chronic subacute MPTP dosing regimen in animal models appears to recapitulate the slow evolution of parkinsonian traits seen in humans far better than acute dosing paradigms, inducing uneven striatal dopamine ﬁbre loss and more selective depletion of substantia nigra dopamine neurons in primates (Albanese et al., 1993; Varastet et al., 1994). Another valuable model system in primates involves unilateral internal carotid artery infusion of MPTP to induce a hemiparkinsonian state (Bankiewicz et al., 1986). Typical experimental approaches to evaluate the extent of MPTP toxicity include histologic Nissl staining, biochemical assays of regional content of dopamine and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and immunocytochemical localization of tyrosine hydroxylase activity (the enzyme that catalyzes the conversion of tyrosine to L-DOPA, the rate-limiting step in dopamine synthesis). The regional and cellular selectivity of MPTP toxicity is conferred by properties of the agent itself. A highly lipophilic molecule, MPTP readily crosses the blood–brain barrier in humans, primates and mice (but not in rats). Within the CNS it is taken up into glial cells, where it is oxidatively deaminated by monoamine oxidase B (MAOB ), ﬁrst to the unstable intermediate 1-methyl-4-phenyl-2,3-dihydropyridinium ion (MPDP+), and then to MPP+ (see Fig. 1). The MPP+ is a polar molecule, and when released from glial cells it is selectively taken up into dopaminergic neuron terminals and cell bodies via the plasma membrane dopamine transporter for which it

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FIG. 1. Dopamine neuron-selective toxicity of MPTP. MPP+, generated by the catalytic conversion of MPTP in glial cells, is selectively taken up into dopaminergic neurons due to its high afﬁnity for the dopamine transporter. Within cells, MPP+ is concentrated in mitochondria where it binds to and inhibits activity of complex I (“I”). Increased superoxide radical (O2− ), and hydroxyl radical (OH−) generation may occur as a consequence of the ensuing disruption of the electron transport chain and impaired ATP production. Abbreviations: DA, dopamine; MPT, mitochondrial permeability transition; SOD, superoxide dismutase; TCA, tricarboxylic acid cycle.

has high afﬁnity (Irwin and Langston, 1995; Santiago et al., 1996). In neurons, MPP+ is concentrated within mitochondria via a carrier-independent mechanism, apparently due to its high lipophilicity, where it binds to and irreversibly inhibits complex I at the inner mitochondrial membrane. Both active and passive mechanisms appear to be involved in MPP+ uptake into mitochondria, but enzyme inhibition appears to depend on steric rather than electrostatic properties of MPP+, since related isoquinoline derivatives are less potent inhibitors of NADH reductase activity in intact mitochondria (Aiuchi et al., 1995; McNaught et al., 1998). Metabolic inhibition has been shown to precede cell death in in vitro assays (Storch et al., 1999). The selective involvement of the dopaminergic system in the motor disorder resulting from MPTP toxicity is reﬂected by the fact that MPTP-treated primates

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show excellent phenotypic responses to treatment with the dopamine precursor L-DOPA, in addition to other dopamine receptor agonists (Treseder et al., 2000; Tahar et al., 2001). Furthermore, lack of the plasma membrane dopamine transporter in knockout mice confers protection against the toxic effects of MPTP (Bezard et al., 1999), as does inhibition of the human dopamine transporter (hDAT), demonstrated using GBR12909 to inhibit dopamine re-uptake in a human embryonic kidney cell line (Storch et al., 1999). The MPP+ can also be sequestered by the brain vesicular monoamine transporter VMAT2, which uses energy from vesicular proton gradients to pump monoamine neurotransmitters and dopamine neurotoxins from the neuronal cytoplasm into synaptic vesicles. Consequently, VMAT2 function can also affect animals’ tolerance to MPP+. This is demonstrated by the observation that MPTP administration at a dose that reduces tyrosine hydroxylase (TH) immunoreactivity 13% in wild-type mice, was found to decrease SNpc TH levels 30% in VMAT2 heterozygous knockout mice (Takahashi et al., 1997). Indirect evidence that mitochondrial energetic defects underlie MPTPinduced damage comes from ﬁndings that pro-energetic agents ameliorate the damaging effects of MPTP, independent of any actions on MPTP metabolism or MPP+ uptake. One such compound is coenzyme Q10 , an essential co-factor for electron transport between complex I and complex III of the electron transport chain, and a potent antioxidant in mitochondria. Coenzyme Q10 markedly reduces indices of MPTP damage in aged mice (Beal et al., 1997; Shults et al., 1997; Ebadi et al., 2001). Another pro-energy agent is creatine, which putatively acts by increasing cytosolic energy stores. Creatine treatment results in increased brain levels of the high-energy substrate phosphocreatine, and has also been suggested to modulate toxicity via inhibitory effects on the mitochondrial permeability transition. Chronic oral creatine administration dose dependently protects against MPTP-induced neuronal damage in animal models, with optimal effects at a concentration of 2% creatine in the diet (Matthews et al., 1999). Another protective agent is acetyl-L-carnitine, involved in fatty acid oxidation in mitochondria and responsible for transporting accumulated acyl groups out of mitochondria, which protects against MPTP-induced parkinsonism in primates (Bodis-Wollner et al., 1991). Disruption of mitochondrial energy production also can instigate excitotoxic cascades within cells (Steiner et al., 1997). To explain brieﬂy: reduced ATP generation will disrupt vital ATPase pump activities and reduce the cell membrane potential. If extensive enough, this may facilitate membrane depolarization and activation of glutamate receptor subtypes (notably N-methyl-D-aspartate, NMDA) by normally inert extracellular levels of the excitatory neurotransmitter glutamate (Novelli et al., 1988; Zeevalk and Niklas, 1991). The consequent cellular inﬂux of calcium

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may be sufﬁcient to stimulate free radical generation and induce oxidative damage (Albin and Greenamyre, 1992; Beal, 2000). The involvement of this secondary excitotoxic phenomenon in MPTP toxicity is supported by observations that glutamate receptor antagonists, including the NMDA antagonist MK-801, protect against MPTP toxicity in mice (Brouillet and Beal, 1993), while the glutamate release inhibitor riluzole is partially protective in both mice and primates (Boireau et al., 1994; Benazzouz et al., 1995). In addition, the dopamine D2 receptor agonists bromocriptine and pramipexole, which also have antioxidant activity, attenuate MPTP-induced dopamine depletion (Muralikrishnan and Mohanakumar, 1998; Zou et al., 2000). Increased free radical generation and breakdown in calcium homeostasis also occur following MPTP administration, putatively as a direct consequence of the energetic disturbance induced by MPTP/MPP+ (although the mitochondrial source of free radical generation has yet to be unequivically proven) (Di Monte et al., 1986; Rossetti et al., 1988; Tipton and Singer, 1993). Mitochondrial MPP+ accumulation can lead to increased nitric oxide (NO) generation, which may exacerbate cellular damage by reacting with superoxide anion (O2− ) to generate the reactive free radical peroxynitrite (Beckman, 1996; Matthews et al., 1997; Przedborski and Jackson-Lewis, 1998). In support of this hypothesis, the involvement of peroxynitrite in MPTP-mediated damage has been demonstrated in both primates and mice by ﬁndings of increased 3-nitrotyrosine immunoreactivity following MPTP administration (Schulz et al., 1995a; Pennathur et al., 1999). This observation is reminiscent of similar alterations reported in PD brain (Good et al., 1998). In addition, MPTP toxicity is ameliorated in mice lacking the neuronal isoform of NO synthase (nNOS), or by treatment with nNOS inhibitors (Schulz et al., 1995b; Hantraye et al., 1996; Klivenyi et al., 2000a). Mice that lack the inducible form of NOS (iNOS) also appear to be resistant to the toxin, but protection is conﬁned to dopaminergic neuron cell bodies and not terminals (Liberatore et al., 1999; Dehmer et al., 2000). The importance of oxidative damage to the etiology of MPTP-mediated damage is further exempliﬁed by the demonstration that its toxicity in mouse brain is signiﬁcantly attenuated when free-radical scavenging enzymes, including Cu/Zn-superoxide dismutase (SOD1) and MnSOD (SOD2), are overexpressed (Przedborski et al., 1992; Klivenyi et al., 1998a). In contrast, mice deﬁcient in certain free-radical scavenging enzymes including glutathione peroxidase, SOD1 and SOD2, show exacerbated MPTP-mediated damage (Wullner ¨ et al., 1996; Andreassen et al., 2001). Further, ebselen, a glutathione peroxidase analogue, prevents both neuronal loss and clinical symptoms produced by MPTP in primates (Moussaoui et al., 2000), and several freeradical spin traps and scavengers including erythropoietin and 17-oestradiol show neuroprotective effects in mice (Matthews et al., 1999; Callier et al.,

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2000; Genc et al., 2001). An involvement of oxidative damage to DNA is also implied by ﬁndings that inhibiting or knocking-out poly-ADP-ribose polymerase (PARP), an enzyme involved in DNA repair that is activated by oxidative damage, signiﬁcantly attenuates MPTP neurotoxicity in mice (Cosi and Marien, 1998). This is of particular interest since activation of poly-ADP-ribose polymerase could itself be deleterious to cells under conditions of MPTP-induced oxidative stress, since enzyme activation depletes cellular ATP and NAD+ stores, potentially exacerbating the energetic dysfunction triggered by MPTP. These in vivo observations are supported by numerous in vitro studies demonstrating increased generation of reactive oxygen species following MPTP administration to cell lines (Kitamura et al., 1998). Interestingly, MPTP administration to striatal synaptosomes has also been reported to reduce cellular levels of the antioxidant glutathione in this preparation (Desole et al., 1993). However, Seyfried and colleagues (2000) demonstrated in PC12 cells that this is a biphasic dose-dependent effect of MPP+, since low doses (250 μM ) markedly increased cellular levels of glutathione (reduced form, GSH) while ten fold higher doses decreased GSH content and increased levels of the oxidized form (GSSG), apparently due to a redox shift in cells that enhanced glutathione oxidation. Another characteristic of PD is the deposition of Lewy bodies in affected brain regions. These dense ﬁbrillary structures contain proteins including α-synuclein and ubiquitin. Neuronal inclusions containing α-synuclein and ubiquitin, partially resembling Lewy bodies, have been reported in aged primates chronically treated with MPTP (Forno et al., 1986; Kowall et al., 2000), but they do not appear to be a typical feature of MPTP toxicity. However, MPTP does increase α-synuclein expression in both mice and primates (Kowall et al., 2000; Vila et al., 2000), a capability linked with the formation of neuronal inclusions, mitochondrial morphological abnormalities, mitochondrial dysfunction, and increased free-radical production in experiments overexpressing α-synuclein in a hypothalamic neuronal cell line (Hsu et al., 2000). Furthermore, MPTP induces nitration of α-synuclein in mice, reminiscent of the α-synuclein nitration seen in PD brain (Giasson et al., 2000; Przedborski et al., 2001). In addition, expression of mutant α-synuclein in NT2/D1 cells is associated with increased markers of oxidative damage and increased susceptibility to cell death in response to H2O2 or MPP+ exposure (Lee et al., 2001). These ﬁndings therefore provide a potential link between the mitochondrial dysfunction and oxidative damage evident following MPTP exposure and the deposition of α-synuclein, which is a typical feature of PD pathology. In addition to direct mitochondrial toxicity, it should be noted that cellular damage mechanisms originating outside the mitochondria have also been implicated in the etiology of MPTP/MPP+damage (Khan et al., 1997;

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Przedborski and Jackson-Lewis, 1998). Notably, Przedborski and JacksonLewis (1998) demonstrated that rho zero cells devoid of an electron transport system are still sensitive to MPP+ toxicity. Other pathogenic pathways include apoptotic processes, which may involve either mitochondrial and/or extramitochondrial components (reviewed by Nicotra and Parvez, 2000). Interestingly, the MPTP dosing regimen used in experimental models largely dictates whether cells follow apoptotic or necrotic pathways to death. In mice, a subacute dosing regime of MPTP daily over ﬁve days was found to induce cell death with terminal deoxynucleotidyl transferase (TUNEL) labeling and chromatin clumping typical of apoptosis (Tatton and Kish, 1997). This is consistent with reports of apoptotic markers in PD brain, including increased caspase-3 and Bax immunoreactivity, TUNEL staining, and chromatin condensation in melanin-containing nigral neurons (Hartmann et al., 2000; Tatton, 2000). Several groups have shown that mice overexpressing Bcl-2 are partially protected against MPTP following an acute dosing paradigm, but to a lesser extent after chronic dosing over ﬁve days (Offen et al., 1998; Yang et al., 1998). Similarly, mice with a dominant-negative inhibition of caspase-1 are protected against MPTP, as are mice deﬁcient in the pro-apoptotic protein Bax (Klivenyi et al., 1999; Vila et al., 2001). Further, adenoviral administration of a caspase inhibitor XIAP (X-chromosomelinked inhibitor of apoptosis) protects dopaminergic cell bodies but not terminals, while protection can be extended to the terminals when XIAP is given in combination with the growth factor GDNF (glial cell-derived neurotrophic factor) (Eberhardt et al., 2000). The p53 gene, which regulates growth and programmed cell death, also appears to be involved in the pathway of cell death, since p53 knockout mice are resistant to MPTP-induced death of dopamine neurons (Trimmer et al., 1996). Further, involvement of apoptotic processes is suggested by the activation of C-Jun N-terminal kinase ( JNK) by MPTP (Saporito et al., 1999, 2000), and the partial protection against MPTP-induced loss of nigral neurons afforded by the anti-apoptotic agent CGP3466 (Waldmeier et al., 2000). MPP+ exposure has also been shown to upregulate apoptotic markers in a number of different cell types in vitro, inducing increased DNA fragmentation and Bcl-2 expression in human neuroblastoma SH-SY5Y cell lines (Itano and Nomura, 1995; Sheehan et al., 1997). The Bcl-2 overexpression has also been found to attenuate MPP+-mediated damage in a dopaminergic neuronal cell line (Oh et al., 1995). Other evidence of a role for apoptosis include induction of apoptotic markers in ventral rat mesencephalic-striatal cultures, differentiated PC12 cell lines, cerebellar granule neurons (Mochizuki et al., 1994; Mutoh et al., 1994; Du et al., 1997), and inhibition of p21WAF/Cip1 -mediated cell proliferation by MPTP (Soldner et al., 1999). Inﬂammatory processes are also implicated by observations in both humans and in animal models. Neuropathological studies in three patients

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3–16 years after exposure to MPTP showed clustering of microglia around neurons along with active ongoing cell loss in the subatantia nigra (Langston et al., 1999). Similarly, both activated microglia and lymphocytes are evident in mice after MPTP administration, and inhibition of either cyclooxygenase-1 or -2 are markedly neuroprotective (selective inhibition of cyclooxygenase-2 by meloxicam; or both cyclooxygenase-1 and -2 by acetylsalicylic acid) (Kurkowska-Jastrzebska et al., 1999; Teismann and Ferger, 2001). Deﬁciency in cytoplasmic phospholipase A2 is also protective against MPTP toxicity, putatively due to blockade of production of the substrate for cyclooxygenase-2 (Klivenyi et al., 1998b), as is the anti-inﬂammatory agent sodium salicylate although this agent also has anti-oxidant effects (Ferger et al., 1999; Mohanakumar et al., 2000).

B. ROTENONE Rotenone is a common component of pesticides that induces mitochondrial toxicity via selective inhibition of the oxidative activity of complex I, and of oxidative free radical production. As might be anticipated given its mode of action, rotenone induces CNS lesions and motor impairments with Parkinsonian features in several in vivo models. It is a naturally occurring compound derived from the roots of certain plant species, whose toxic properties have been utilized for many years in farming practices, to regulate ﬁsh populations and as an insecticide. Several epidemiological studies have subsequently implicated accidental rotenone exposure as a pathogenetic risk factor in the etiology of PD (Gorrell et al., 1998; Kitada et al., 1998), although studies in animals suggest that its CNS toxicity is extremely low following oral exposure (Betarbet et al., 2000). This observation does not rule out the possibility of susceptibility to environmental exposure, especially when a chronically exposed subject also expresses genetic defects in complex I, or an impaired ability to metabolize xenobiotics (Parker et al., 1989; Menegon et al., 1998; Swerdlow et al., 1998). Rotenone’s toxic action is mediated by steric inhibition of complex I activity after binding at the PSST subunit (Ernster et al., 1963; Horgan et al., 1968; Gutman et al., 1970). This subunit is postulated to play a key role in electron transfer by functionally coupling the iron–sulfur cluster N2 to quinone, and hence metabolic impairment can be induced by blockade of its binding site by rotenone and other inhibitors including piericidin A, pyridaben, and bullatacin (Schuler et al., 1999). Direct stereotaxic injection of rotenone into the substantia nigra in rats lesions dopaminergic neurons in this region (Heikkila et al., 1985). However, in vivo studies of rotenone’s CNS actions after systemic administration are hindered by the agent’s hydrophobicity and insolubility in aqueous solvents. Consequently, recent

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studies have turned to a relatively extreme route of administration, employing chronic administration directly into the jugular vein via subcutaneously implanted minipumps. Ferrante and colleagues (1997) demonstrated that intrajugular administration of rotenone to rats (10–18 mg/kg/day for 7–9 days) induced a motor syndrome involving rigidity and akinesia. Histology revealed this was associated with neuronal degeneration and astrogliosis in the striatum and globus pallidus, but found no evidence of cell loss or alterations in tyrosine hydroxylase (TH) immunoreactivity within the substantia nigra, unlike the pattern seen in PD. More recently, Betarbet and colleagues (2000) have further characterized this PD model in rats, again using intrajugular infusions but employing a chronic low dose regimen, and incorporating both histological and biochemical assessments of lesion attributes. Administration of 2–3 mg/kg/day for up to ﬁve weeks was found to produce optimal lesion effects. Even using this administration method, the usefulness of this approach as a laboratory model is restricted by the variability of individual rats’ susceptibility to the toxin, with only 50% of treated animals developing detectable lesions. In those Lewis rats susceptible to toxicity, however, rotenone induced striatal lesions and a progressive degeneration of nigrostriatal neurons concomitant with a loss of immunoreactivity for tyrosine hydroxylase, the dopamine transporter, and the vesicular monoamine transporter VMAT2 (Betarbet et al., 2000). Furthermore, cytoplasmic α-synuclein- and ubiquitin-positive ﬁbrillary inclusions were observed in nigral neurons of affected rats, similar to Lewy bodies found in PD brain. Intoxicated rats exhibit bradykinesia, postural instability, unsteady gait, and some evidence of tremor. Signiﬁcantly, the extent of these deﬁcits improved after treatment with the dopamine receptor agonist apomorphine, supporting a dopaminergic selectivity of rotenone’s action. Unlike MPTP toxicity, where the toxin is selectively taken up into the neuronal population which is then targeted for destruction, peripheral rotenone administration results in uniform distribution of the toxin throughout the brain. Yet remarkably, Betarbet and colleagues (2000) reported that in their study rotenone’s toxic effects were restricted to dopaminergic neurons within the nigrostriatal system, independent of a requirement for the dopamine transporter (echoing the regional speciﬁcity of systemically administered 3-NP, discussed later). This implies that neurons within the SNpc are selectively vulnerable to complex I inhibition, and this is consistent with ﬁndings of decreased complex I activity in PD postmortem tissue and platelets (Parker et al., 1989; Schapira et al., 1990; Mann et al., 1992). However, Betarbet and colleagues suggest that rotenone’s toxicity might be mediated primarily by oxidative damage, since the brain rotenone concentration achieved in their study (20–30 nM ) was only enough to partially

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inhibit complex I, and not high enough to signiﬁcantly impair oxidative phosphorylation. In addition, several in vitro studies have demonstrated that rotenone-mediated complex I inhibition can induce free radical generation, result in damage to proteins and DNA, and chronic exposure may stimulate cytochrome c release from mitochondria (Seaton et al., 1997; Hensley et al., 1998; Sherer et al., 2001).

C. NEUROLATHYRISM Another CNS motor syndrome involving a mitochondrial component is the spastic paraplegia that occurs as a result of ingestion of certain strains of chick peas found in Europe and India (Lathyrus sativus L.). This form of toxicity is also implicated in the etiology of amyotrophic lateral sclerosis and PD dementia of Guam. The toxicity of the chick pea in humans apparently derives from the actions of three excitotoxins identiﬁed in these toxic strains— namely amino-β-oxalylaminopropionic acid, amino-oxalylaminobutyric acid, and β-N-oxalylamino-L-alanine (L-BOAA) (Spencer, 1995). Of these, most is understood about the actions of L-BOAA, whose neurodegenerative effects are mediated via actions at AMPA glutamate receptor subtypes, and results in selective inhibition of mitochondrial complex I (Pai and Ravindranath, 1993; Kunig et al., 1994). Toxicity can be prevented by pretreatment with α-amino-3-hydroxy-5-methyl-4-isoxazole(AMPA)/kainate(but not NMDA) glutamate receptor antagonists, including 1,2,3,4,-tetrahydro-6nitro-2,3-dioxo-benzo(f)quinoxaline-7-sulfonamide disodium (NBQX) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)(Pai and Ravindranath, 1993; Willis et al., 1993; Zeevalk and Nicklas, 1994). In vitro, neuron exposure to micromolar concentrations of L-BOAA produces morphologic changes to cell bodies including postsynaptic vacuolization, followed by degeneration (Ross and Spencer, 1987). The involvement of oxidative damage processes in L-BOAA toxicity are suggested by observations that toxicity is reversed by thiol treatment (Sriram et al., 1998). Furthermore, focal administration of putative free radical scavenging agents (including dimethylformamide, DMF; dimethyl sulfoxide, DMSO; dimethylthiourea, DMTU; and mannitol) protected against L-BOAA-induced neurotoxicity induced by intra-hippocampal injections in rats (Willis et al., 1994). The toxicity of L-BOAA following systemic administration is restricted to speciﬁc regions of the CNS, primarily affecting the thoracolumbar spinal cord where myelin loss and eventually axon degeneration occur following anterolateral sclerosis (Spencer et al., 1986, 1987). There is also evidence of amyloid deposition and neuroﬁbrillary tangle formation in Ammon’s horn, although a direct link with Lathyrus has not been proven (Denny-Brown,

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1947). Clinical symptoms vary greatly between patients in onset and priming dose, but are characterized initially by lumbar pain, and weakness with stiffness of the lower extremities (Haque et al., 1996). Weakness is progressive and eventually leg spasticity and clonic seizures occur, leading to gait disturbances. Upper extremities may become involved in severe cases. Muscle atrophy ensues, with sensory deﬁcits, pain, and parasthesias (the latter two lasting one to two weeks). The patient is left with a spastic gait. Interestingly, in both humans and in animal models males have increased risk of developing pathology after exposure to L-BOAA, a circumstance suggested to be due to anti-oxidant properties of female hormones.

III. Inhibitors of Mitochondrial Complex II: Succinate Ubiquinol Oxidoreductase

A. 3-NITROPROPIONIC ACID (3-NP) The mitochondrial toxin 3-nitropropionic acid (3-NP) irreversibly inhibits the activity of succinate dehydrogenase, a metabolic enzyme that participates in both the tricarboxylic acid (TCA) cycle and in complexes II–III of the electron transport chain. Systemic administration of this agent to humans, nonhuman primates, and rodents results in CNS lesions that selectively target subpopulations of striatal neurons, closely replicating the nature and regional speciﬁcity of pathological events seen in Huntington’s disease (HD). The induction of striatal lesions by 3-NP is accompanied by multiple cognitive and motor symptoms, including acute encephalopathies and coma, following initial gastrointestinal disturbances. In fact,3-NP was ﬁrst identiﬁed following reports of motor disturbances in livestock in the United States that were exposed to fungal-contaminated diet, and observations of symptoms resembling HD with concomitant lesions of the basal ganglia in children in China after ingestion of fungal-contaminated sugar cane. The fungus in question, Arthrinium, was subsequently found to contain the 3-nitopropanol metabolite 3-NP (for review, see Ludolph et al., 1991). Of the human cases studied, some comatose patients eventually recovered fully, but most were left with irreversible motor impairments including dystonia, jerky movements, torsion spasms, and facial grimaces. Approximately 10% of affected individuals died. Intoxication generally produces basal ganglia lesions visible by computed tomography (CT) scans, localized principally to the putamen but sometimes extending to the caudate (Ludolph et al., 1991; He et al., 1995). The spatial, pathologic and (to a limited extent) behavioral

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features of 3-NP intoxication can be replicated by systemic administration in experimental animals (Gould and Gustine, 1982; Hamilton and Gould, 1987). Hence, this toxin has become a widely used experimental tool to try to model aspects of HD pathology in primates, rats, and mice. However, the extent of lesions and the nature of the pathologic sequelae are minutely dependent on the dosing regimen used, with low-dose chronic treatment paradigms yielding cerebral effects most closely mimicking those seen in HD (for review, see Brouillet et al., 1999). Systemic administration of 3-NP to both rats and primates produces age-dependent striatal lesions that are strikingly similar to those seen in HD (Brouillet et al., 1995, 1998). In primates, chronic 3-NP administration produces selective striatal lesions characterized by a depletion of calbindin neurons with sparing of NADPH-d neurons, and by proliferative changes in the dendrites of spiny neurons. Animals also show both spontaneous and apomorphine-inducible choreiform movement disorders resembling HD (Brouillet et al., 1995). The 3-NP basal ganglia lesions in rats are associated with elevated lactate levels, similar to the increased lactate production seen in HD patients ( Jenkins et al., 1993; Matthews et al., 1998). Systemic administration of 3-NP results in increased binding of tritiated MK-801, consistent with activation of NMDA receptors as a secondary consequence of energy depletion (Wullner et al., 1994). Consequently, 3-NP lesions can be prevented by prior removal of glutamatergic excitatory corticostriatal inputs by decortication, by glutamate release inhibitors, and by glutamate receptor antagonists, suggesting that 3-NP toxicity is mediated by secondary excitotoxic mechanisms (Beal, 1994; Schulz et al., 1996a). Succinate dehydrogenase (SDH) catalyzes the oxidation of succinate to fumarate. Due to its joint roles in the TCA cycle and electron transport chain, it is located on the surface of the inner mitochondrial membrane (Fig. 2). The biochemical mechanism of SDH inhibition by 3-NP has not yet been unequivically proven, but the close similarity in structures of 3-NP (C3H5NO4) and succinic acid (C4H6O4) may be the key to 3-NP’s potency. Currently, two theories predominate. One hypothesis proposes that the dianion form of 3-NP can generate carbanions that preferentially react with the ﬂavin moiety in SDH, forming a covalent adduct that alters the substrate speciﬁcity of the enzyme (Alston et al., 1977). Alternatively, Coles and colleagues (1979) suggest that the substrate binding site of SDH is speciﬁcally targeted by 3-NP. They demonstrated that the dianion form of 3-NP can bind to SDH, generating nitroacrylate. This nitroacrylate group can then react with and irreversibly bind a thiol group within SDH to block access of succinate to the enzyme. In vitro this inhibition of succinate catabolism by 3-NP occurs extremely rapidly (kobs 1.2 min−1 ), and is dose dependent.

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FIG. 2. Sites of metabolic inhibition by mitochondrial toxins. A schematic representation of the enzyme complexes that constitute the electron transport chain, illustrating the principal sites of respiratory disruption by mitochondrial metabolic toxins. Metabolic substrates generated by glycolysis and the TCA cycle donate electrons to complexes I and II, which then transfer electrons to complex III via coenzyme Q10 (coQ10). Cytochrome c (cyt c) modulates electron donation on to complex IV. This process promotes the pumping of protons across the inner mitochondrial membrane by complexes I, III, and IV, with the resultant generation of a potential energy gradient. This is converted into stored energy in the high-energy phosphate bond of ATP, by the terminal electron acceptor ATP synthase (not shown). To facilitate this process, complexes I, III, and IV occur in organized supramolecular clusters (respirasomes), situated between the inner and outer mitochondrial membranes (IMM and OMM, respectively). Succinate dehydrogenase (complex II) is located on the surface of the IMM. Disruption of the electon transport chain by direct inhibition of any of the complex subunits may therefore have catastrophic effects on both energy generation, and on the homeostatic regulation of free radical generation during oxidative phosphorylation. Abbreviations: 3-AP, 3-acetylpyridine; − L-BOAA, β-N- oxalylamino-L-alanine; e , electron; H2S, hydrogen sulphide; MPTP, 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine; 3-NP, 3-nitropropionic acid.

By inhibiting components of both the TCA cycle and oxidative phosphorylation, 3-NP’s toxicity is doubly detrimental to mitochondrial metabolism. The 3-NP impairs both the delivery of NADH from the TCA cycle to complex I of the oxidative phosphorylation pathway, and inhibits the passage of reducing equivalents along the electron transfer chain, thus reducing ATP generation by a two-pronged attack on mitochondrial function. Inhibition of cerebral SDH activity by 3-NP has been demonstrated in vivo in a number of studies. Interestingly, the neurodegenerative sequelae of systemic administration of the toxin are largely restricted to the striatum despite a relatively homogeneous distribution of the toxin, resulting in a uniform reduction in SDH activity throughout the brain (Gould and Gustine, 1982; Gould et al., 1985; Brouillet et al., 1998; Browne and Beal, unpublished observations).

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This observation appears to once again underscore the vulnerability of striatal neurons to metabolic stress. In fact, 3-NP markedly inhibits SDH activity within 2 h of intraperitoneal administration in rodents, producing 50– 70% reductions in SDH activity throughout the brain (Brouillet et al., 1998; Browne and Beal, unpublished observations), consistent with the degree of complex II and III deﬁciency reported in HD striatum in postmortem studies (Gu et al., 1996; Browne et al., 1997). The energetic component of 3-NPmediated toxicity is further reﬂected by observations that the agent found to produce the most profound protection against 3-NP acts by stimulating energy generation within cells. Creatine administration in rats markedly reduces neuronal cell loss induced by 3-NP, as well as attenuating increases in cerebral lactate levels and decreases in levels of high-energy phosphate compounds (including ATP) seen in the striata of 3-NP-treated rats (Matthews et al., 1998). The 3-NP toxicity in animals is also associated with increased oxidative damage in the CNS. Hydroxyl (OH−) free radical production is elevated in the striatum following systemic 3-NP administration, as are levels of the DNA damage marker 8-hydroxy-deoxyguanosine (OH8dG) and 3-nitrotyrosine (a marker for peroxynitrite-mediated oxidative damage) (Schulz et al., 1996b). Findings that 3-NP-induced lesions and concomitant increases in oxidative damage markers are markedly attenuated in mice overexpressing the superoxide free radical scavenger Cu/Zn superoxide dismutase (SOD1) imply that oxidative free radicals contribute to lesion formation (Beal et al., 1995). Furthermore, 3-NP striatal lesions are attenuated by free radical spin traps and nitric oxide synthase (NOS) inhibitors (Schulz et al., 1995c). In addition, lack of the free radical scavenging enzyme glutathione peroxidase (GSHPx) in knockout mice exacerbates striatal damage and 3-nitrotyrosine elevations casued by systemic administration of 3-NP (Klivenyi et al., 2000b). These results indicate that a knockout of GSHPx may be adequately compensated under normal conditions, but following toxin-induced mitochondrial dysfunction GSHPx plays an important role in detoxifying oxygen radicals.

B. MALONATE Malonate is another selective inhibitor of succinate dehydrogenase activity that induces motor impairment following intrastriatal administration in rodents. Lesion pathology reveals neuronal selectivity similar to the pattern of neuronal vulnerability in HD brain. In contrast to 3-NP toxicity, systemic administration of this agent is ineffective as it is unable to cross the blood–brain barrier. However malonate is still a useful tool for modeling

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the effects of complex II inhibition in vitro and in vivo. Intrastriatal injection of malonate produces age-dependent striatal lesions that can be signiﬁcantly attenuated by treatment with the NMDA receptor antagonist MK-801. Further indirect evidence that energetic defects contribute to malonateinduced neurodegeneration come from observations that pro-energy compounds protect against cell death. Coenzyme Q10 (CoQ10), a co-factor for electron transfer during oxidative phosphorylation in the mitochondria and a potent antioxidant, attenuates malonate neurotoxicity in animal models (Beal et al., 1994; Schulz et al., 1996b). Interestingly, oral administration of CoQ10 has also been shown to ameliorate elevated lactate levels seen in the cerebral cortex of HD patients (Koroshetz et al., 1997). Coenzyme Q10 is reported to also improve symptoms in some other mitochondrial-associated disorders including MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) and Kearns-Sayre syndrome, where it additionally reduces CSF and serum lactate and pyruvate levels, and enhances mitochondrial enzyme activities in platelets (Bresolin et al., 1988; Ihara et al., 1989). In addition, oral supplementation with either creatine or cyclocreatine produced signiﬁcant protection against malonate lesions, and ameliorated malonate-induced increases in hydroxyl radical generation (Matthews et al., 1998). The magnitude of protection against malonate toxicity, in terms of lesion volume, was exacerbated by combining nicotinamide with creatine treatment (Malcon et al., 2000). Malonate-induced increases in conversion of salicylate to 2,3- and 2,5dihydroxybenzoic acid, an index of hydroxyl radical generation, were greater in GSHPx knockout mice than in wild-type control animals, suggesting an involvement of oxidative damage mechanisms in malonate toxicity (Klivenyi et al., 2000b). This is supported by ﬁndings that impaired nitric oxide (NO) generation in mice lacking the gene for the neuronal isoform of NOS (nNOS) reduced the volume of malonate lesions (Schulz et al., 1996b). Further, elevated 3-nitrotyrosine concentrations are reported after intrastriatal malonate injection, and malonate lesions are attenuated by free radical spin traps and nitric oxide synthase (NOS) inhibitors. Hence there is substantial evidence that NO-mediated oxidative damage is involved in cell death processes following energetic disruption induced by both 3-NP and malonate.

IV. Inhibitors of Mitochondrial Complex IV: Cytochrome c Oxidase

A. CYANIDE Cyanide is an extremely potent neurotoxin principally targeting complex IV of the electron transport chain, although its actions within

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the mitochondria are not limited to this site. Ingestion of an average dose of only 200 mg of potassium cyanide by humans proves fatal. Although most cyanide-related deaths in the United States result from homicide or suicide attempts, intoxication may also occur from accidental exposure to cyanide compounds used in industrial processes, cyanide gas from burning certain compounds including polyurethanes, nitrocellulose, and wool, or in vivo biotransformation of cyanogenic compounds including nitriles (Albin, 2000). Cassava and some fruit stones (for example, apricot and peach) contain cyanogenic compounds, and sodium nitroprusside treatment can lead to cyanide generation in vivo. Cyanide inhibits ferric ion (Fe3+ ) containing enzymes, including cytochrome oxidase (a vital component of complex IV), succinate dehydrogenase, and mitochondrial and cytoplasmic SOD. Hence its actions are principally inhibition of mitochondrial aerobic metabolism and increased free radical generation. The CNS is particularly vulnerable to the toxic effects of cyanide, putatively because of its high metabolic rate and dependence on oxidative phosphorylation to supply functional energy demands. Death is caused by respiratory arrest thought to result from paralysis of brain-stem neurons that control respiratory muscles. Initial symptoms are headache, delirium, seizures, agitation, and swift onset of coma, and fatality often occurs within minutes of exposure. In the few survivors of cyanide exposure, motor neurologic symptoms appear days or weeks after dosing. The principle symptoms are parkinsonism and dystonia, with the possibility of dysarthria, ataxia, and eye movement abnormalities (Uitti et al., 1985; Borgohain et al., 1995; Rosenow et al., 1995). Lesions occur in the putamen and globus pallidus, while magnetic resonance (MR) studies also reveal changes in the cortex, cerebellum, subthalamus, and substantia nigra (Uitti et al., 1985). Positron emission tomography (PET) studies have also shown presynaptic dopaminergic terminal loss after cyanide exposure. Treatment paradigms are limited, but include thiosulfate administration to induce cyanide conversion to thiocyanate, administration of nitrites to promote methemoglobin formation (which has a high afﬁnity for cyanide and thus draws it from the tissues), and respiratory support.

B. HYDROGEN SULFIDE Hydrogen sulﬁde is an environmental toxin that is thought to induce its toxic effects in a similar manner to cyanide by inhibiting Fe3+ containing enzymes, particularly cytochrome oxidase in complex IV (Tvedt et al., 1991; Snyder et al., 1995; Kerns and Kirk, 1998). In contrast to cyanide, its effects are generally reversible. Acute symptoms include vertigo, seizures, delirium, coma, and respiratory paralysis, thought to arise from inhibition

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of brain-stem neurons modulating respiratory mechanics. Again, the CNS is particularly vulnerable to the toxic effects of hydrogen sulﬁde, and putaminal and pallidal degeneration may occur in severe cases.

C. SODIUM AZIDE Sodium azide (NaN3) is another compound selectively toxic to mitochondria that targets Fe3+ containing proteins, in particular cytochrome oxidase (see Fig. 2). Azide exposure induces striatal-speciﬁc degeneration in animal models following systemic administration (Hicks, 1950a; K¨oryney, 1963; Miyoshi, 1968; Shibasaki, 1969). Observations in rats (following a dosing regimen of 3 mg/kg NaN3 i.p., four times per day for four days) reported initial slight motor effects, typically sluggishness, followed a few days later by the rapid onset of marked motor abnormalities including akinesis and paretic gait, with forelimb tremor (Miyoshi, 1967). Animals generally survived but showed sustained gait abnormalities despite dosing cessation. After this treatment paradigm distinct bilateral striatal necrosis was evident, with variable involvement of other brain regions including the globus pallidus in some animals, and occasional degeneration in the cerebral cortex and demyelination of the optic tract. Systemic toxicity was also evident, affecting lung and heart muscle predominantly. Other studies also show axon damage in the corpus callosum (Hicks, 1950a,b; K¨oryney, 1963). Systemic NaN3 administration produces reductions in cytochrome oxidase activity diffusely throughout the brain, including both the striatum and regions spared by damage (such as cerebral cortex, cerebellum, and choroid plexus), again underscoring the peculiar sensitivity of striatal neurons to impaired energy metabolism in vivo.

V. Manganese

Chronic manganese exposure induces a movement disorder characterized by bradykinesia and rigidity. Onset is slow but insidious, with several subjects experiencing severe psychiatric symptoms before onset of an akineticrigid parkinsonian state. Manganese toxicity was ﬁrst observed in manganese ore miners, millworkers, and smelter workers in 1837 (Couper, 1837), but also results from accidental exposure to or ingestion of potassium permanganate, fungicides, and gasoline additives containing manganese (Albin, 2000). The symptom phenotype of manganese toxicity results from the fact that systemically administered manganese accumulates within the CNS in

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the basal ganglia—in particular, in the globus pallidus, caudate, and putamen (Newland et al., 1989). The exact mechanism of manganese toxicity is still ill-deﬁned, but the fact that within cells manganese accumulates in mitochondria suggests that mitochondrial dysfunction plays an intrinsic role. One hypothesis is that manganese increases oxidative damage due to catecholamine oxidation (Sloot et al., 1994). More recently, manganese has been shown to stimulate astrocytic and microglial NOS production in vitro, potentially increasing nitric oxide production leading to free radical damage (Spranger et al., 1998; Chang and Liu, 1999). Further, manganese has been shown to directly inhibit mitochondrial oxidative phosphorylation, inhibiting activity of the TCA cycle enzyme aconitase, located in the mitochondrial matrix (Zheng et al., 1998). Zheng and colleagues suggest that a potential mechanism of enzyme impairment might be by the substitution of manganese for iron in enzymes that require iron as a co-factor. The involvement of manganese in mitochondrial dysfunction is supported by PET studies that demonstrate reductions in cerebral glucose utilization rates in a number of subjects with mild manganese toxicity (Wolters et al., 1989). Calne et al. (1994) describe a movement disorder closely resembling idiopathic PD following chronic manganese exposure, with early signs of gait disturbances and dysarthria. Sufferers may show axial and extremity dystonia and a postural tremor, but resting tremor does not occur. Dementia and cerebellar dysfunction may also occur. Symptoms can still progress after discontinued exposure to manganese, and manganese clears only very slowly from mitochondria and the brain (Huang et al., 1998; Pal and Calne, 1999). It has been suggested that this progressive feature of the disorder might reﬂect a vicious cycle of oxidative damage as a result of mitochondrial impairment, leading to mitochondrial DNA damage and perpetuated mitochondrial dysfunction (Brouillet et al., 1993; Desole et al., 1997). Although clinical symptoms of manganese toxicity partially resemble Parkinsonism, the pathological alterations are very different. In the few manganeseexposure victims studied, neuronal degeneration and reactive gliosis were prevalent in the globus pallidus, particularly in the internal segment, and to a lesser extent in the striatum and substantia nigra pars reticulata, SNr (Yamada et al., 1986). This pathology contrasts with the primary loss of dopaminergic neurons within the SNpc in PD. Animal models of manganese exposure, in nonhuman primates and rodents, mimic this pattern of pallidal and SNr vulnerability, and show some degeneration within the subthalamic nucleus (Pentschew et al., 1962; Brouillet et al., 1993; Olanow et al., 1996). Dopamine depletion has been demonstrated in experimental animals after direct intrastriatal or intranigral manganese injection in primates and in rats (Lista et al., 1986; Newland et al., 1989; Daniels and Abarca, 1991), and after chronic systemic exposure in rabbits and squirrel monkeys (Neff et al., 1969; Mustafa and Chandra, 1971). Manganese application also has been shown

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to decrease dopamine release in striatal slice preparations (Daniels et al., 1981). Moreover, a study in rat striatal tissue slices reported that manganese exposure reduced dopamine biosynthesis by inhibiting tyrosine hydroxylase activity (Hirata et al., 2001). This effect was associated with increased lactate production in the slices, suggesting abnormal aerobic metabolism following manganese exposure. In contrast, ﬂuorodopa PET imaging studies showing that the dopaminergic nigrostriatal pathway remains intact in manganese exposure patients, and observations that dopaminergic treatment is ineffective in the disorder, argue against an involvement of the dopaminergic system in the toxicity phenotype (Calne et al., 1994; Lu et al., 1994).

VI. 3-Acetylpyridine

Another neurotoxin that selectively targets mitochondrial components is the nicotinamide antagonist 3-acetylpyridine (3-AP). Systemic administration of 3-AP in animals induces selective degeneration of the inferior olivary nucleus and cerebellum, resulting in a motor disorder resembling olivopontocerebellar atrophy in humans (Deutsch et al., 1989). Toxicity is mediated via incorporation of 3-AP into nicotinamide nucleotides within the cell, resulting in inhibition of both NADH and NADPH-dependent enzymes (Herken, 1968). Consequently, hydrogen ion transfer between enzymes and substrates using NADH and NADPH as co-factors is impaired, potentially leading to dysfunction of both the TCA cycle and the electron transport chain within mitochondria. Dehydrogenase enzymes are particularly affected, including the mitochondrial enzymes lactate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, and glutathione reductase. Cytosolic glycolytic enzymes may also be vulnerable, including glucose-6-phosphate dehydrogenase. Thus, 3-AP toxicity results in energy depletion, as has been demonstrated by ﬁndings of reduced ATP levels following systemic 3-AP administration (Sethy et al., 1996), and after intrastriatal 3-AP injections in rats (Schulz et al., 1994). Findings that intrastriatal 3-AP injections did not induce acute lactate generation are consistent with a putative impairment of lactate dehydrogenase activity by 3-AP. However, the involvement of metabolic inhibition in 3-AP-mediated toxicity is supported by the observation that enhancing glycolytic ATP production by preadministration of fructose-1,6-biphosphate partially attenuated 3-AP lesions (Schulz et al., 1994). The 3-AP toxicity, both in vitro and in vivo, is antagonized by nicotinamide (Desclin and Escubi, 1974; Weller et al., 1992).

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Consistent with the involvement of a secondary excitotoxic mechanism resulting from impaired mitochondrial energy metabolism, cell loss following stereotactic injections of 3-AP into both striatum and hippocampus can be attenuated by competitive and noncompetitive NMDA receptor antagonists, including 2-amino-5-phosphonovaleric acid (APV), LY274614, and MK-801 (Armanini et al., 1990; Schulz et al., 1994). Schulz and colleagues (1994) went on to show that the glutamate release inhibitor lamotrigine also reduced lesion volume after intrastriatal 3-AP injection, and noted that the pattern of cell vulnerability following 3-AP resembles that following excitotoxic lesions induced by NMDA. Both lesion paradigms show relative sparing of NADPH-diaphorase-positive neurons that contain NOS within the striatum (Beal et al., 1991). However, MK-801 did not protect cultured cerebellar granule cells against 3-AP toxicity, whereas nicotinamide was protective in this in vitro model (Weller et al., 1992). The 3-AP also induces increased oxidative stress, as shown by reports that striatal injection of 3-AP increased hydroxyl (OH−) radical generation, and that administration of the spin trap N-tert-butyl-α-(2-sulphophenyl)-nitrone (S-PBN) markedly reduced striatal lesion volume (Schulz et al., 1995c). In fact, spin traps were found to be more effective against 3-AP lesions than against other mitochondrial toxins. This phenomenon is potentially due to 3-AP’s propensity to deplete NADPH, since NADPH is necessary for glutathione reductase-mediated regeneration of the free-radical scavenger glutathione (GSH) from its oxidized form (GSSG). In addition, pyrrolopyramidine antioxidants including U-104067F markedly attenuate the reduction in cerebellar ATP production induced by systemic 3-AP administration in rats, and restore motor impairments (Sethy et al., 1996). This effect is putatively due to inhibition of OH−-mediated lipid peroxidation. Dopaminergic agonists and GABAA receptor partial agonists have also been reported to protect against 3-AP-mediated cerebellar ATP depletions and loss of inferior olivary neurons (Sethy et al., 1997a,b). Systemic administration of 3-AP primarily targets the climbing ﬁber projection from the inferior olivary nucleus that innervates cerebellar purkinje cells (Desclin and Escubi, 1974; Balaban, 1985; Sethy et al., 1996). Other areas affected include the SNpc, hippocampal formation, diagonal band of Broca horizontal limb, dorsal motor nucleus of the vagus, interpeduncular nucleus, nucleus ambiguus, hypolassal nucleus, and supraoptic and paraventricular nuclei. Although 3-AP does not cause degeneration of the basis pontis, the neurotoxin induces motor impairment closely resembling OPCA with a phenotype including ataxia, forelimb tremor, hyperkinesia, and tonic cramps (Herken, 1968). Concomitant depletion of dopamine levels and TH immunoreactivity in rat striatum, and of nigral TH immunoreactivity in mice, led to the proposal that 3-AP toxicity may model OPCA

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with Parkinsonism, or multiple system atrophy (Deutsch et al., 1989; Takada and Kono, 1993). As with the majority of mitochondrial toxins, the reason for the regional selectivity of 3-AP toxicity is unclear. However, selective vulnerability may be explained by regional impairments or mutations in one or more NAD(P)-dependent oxidoreductases. One of the enzymes particularly susceptible to 3-AP is glutamate dehydrogenase (GDH). Inhibition of GDH activity by 3-AP could lead to increased extracellular glutamate levels, and thus increase the risk of excitotoxic injury. Overall, observations to date strongly imply that both secondary excitotoxicity and free-radical mediated cellular damage play roles in 3-AP-mediated toxicity, concomitant to impaired mitochondrial energy metabolism.

VII. Myopathies and Myotoxic Agents

A number of mitochondrial toxins are also implicated in toxin-induced myopathies. Some antiviral nucleoside drugs used in HIV therapy including zalcitabine (ddI) and didanosine (ddC) produce axonal neuropathies, thought to be caused by disrupted mitochondrial function ( Jay et al., 1994; Pedrol et al., 1996; Benbrick et al., 1997). Thallium toxicity also interferes with mitochondrial oxidative activity, and both nucleoside therapy and thallium intoxication are associated with painful peripheral neuropathies. Zidovudine (AZT), a thymidine analogue also used in HIV treatment, produces a different sequelae of myotoxic events, but still mitochondrial dysfunction is involved. Zidovudine inhibits reverse transcriptase and mitochondrial DNA polymerase, hence blocking DNA replication and leading to depletion of mitochondrial DNA (Semino-Mora et al., 1994). Mitochondrial DNA levels in patients have been reported to be reduced reversibly by up to 78% following treatment doses of AZT, inducing impaired mitochondrial protein synthesis (Love and Miller, 1993). In addition, AZT produces partial cytochrome c oxidase deﬁciency, reducing mitochondrial energy production. Accumulations of mitochondria are visible in muscle biopsies, where “ragged-red ﬁbers” and mitochondrial proliferation are seen. There is also evidence of increased lipid and reduced carnitine levels in affected ﬁbers, which respond to carnitine therapy. Myalgia generally occurs 6–11 months after commencing AZT treatment, and gradually dissipates on cessation of treatment. Muscle’s primary energy source when at rest or during mild exercise (provided blood supply is adequate) is lipid metabolism utilizing cytoplasmic long-chain fatty acids (LCFAs). This process employs L-carnitine to

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transport these LCFAs across the inner mitochondrial membrane and into the mitochondrial matrix. The reversible binding of the LCFAs to carnitine to form acylcarnitine esters is catalyzed by carnitine palmitoyl transferase. Within the matrix, β oxidation of the acylcarnitine esters yields acetylcoenzyme A (acetyl-coA) and high-energy molecules including ATP, along with the reduced forms of ﬂavin adenine dinucleotide (FADH2) and nicotinamide adenine dinucleotide (NADH) which can take part in the electron trasport chain. Acetyl-coA enters the TCA cycle in the matrix to yield further ATP, NADH, and FADH2. Myopathy can be produced by the inhibition of this pathway—for example, by agents that induce carnitine deﬁciency, leading to impaired energy production in the muscle. Such agents include the medication valproic acid, which can also partially inhibit β oxidation. Valproate toxicity is characterized by increased intracellular lipid levels, abnormal mitochondrial morphology, and a mild myopathy (Melegh and Trombitas, 1997). Other agents affecting carnitine function to induce mitochondrial toxicity include pivampicillin, which inhibits absorption of dietary carnitine (Love and Miller, 1993), and chloramphenicol, which inhibits protein synthesis and induces myogenic abnormalities in animal models (Korohoda et al., 1993).

VIII. Discussion: What Determines the Regional and Cellular Speciﬁcity of Mitochondrial Toxins?

Agents that disrupt the mitochondrial electron transport chain have a propensity to induce selective neuronal damage within the CNS, often targeting the basal ganglia. In some instances the reason for regional selectivity of a given substance is well understood. For example, in the case of MPTP it is the afﬁnity of the dopamine reuptake transporter that localizes the toxic moiety, MPP+, to dopaminergic nerve terminals in the SNpc, resulting in selective neuronal degeneration in the nigrostriatal pathway. In the cases of other toxins, the cause of the regional vulnerability is less clear. 3-NP, for example, when administered systemically in various animal species, induces neuronal damage primarily in the basal ganglia, despite a universal and more or less equal distribution throughout the CNS. In this case, its regional selectivity may be related more to the susceptibility of basal ganglia neurons than to metabolic inhibition. Complex II and III activity is equally impaired throughout the brains of rodents and primates following systemic 3-NP administration, but striatal neurons are preferentially damaged. Potential explanations for this phenomenon include the suggestion

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that striatal neurons have a high energy demand and may be less capable of buffering energetic stress than neurons in other brain regions. The striatum also receives a profound excitatory glutamatergic innervation from cortical regions, increasing the probability of excitotoxic processes being invoked in times of metabolic compromise. These suppositions are supported by observations that other metabolic toxins including cyanide and azide also selectively target striatal neurons, despite widespread complex IV inhibition throughout the CNS, and observations that NMDA receptor antagonist treatment or decortication protect striatal neurons against toxin-induced damage. Further, other hypoxic conditions also preferentially target the striatum, including insulin hypoglycemia, carbon disulﬁde, and malononitrile toxicity (disregarding ischemic lesions that are dependent on vascular zones for lesion localization) (Hicks, 1950a,b; Kristian et al., 1995; Huang et al., 1996; Hageman et al., 1999). The underlying reasons for the metabolic vulnerability of certain neuronal populations within the brain, however, have yet to be elucidated. One potentially important factor in the phenomenon of neuronal vulnerability is the role that differences in mitochondrial composition may play. For instance, rotenone binding to complex I differs between brain regions, suggesting variability in complex I subunit composition (Higgins and Greenamyre, 1996). It is also known that the relative expression levels of different isoforms of complex IV subunits vary between CNS regions. Other factors likely to be important in differential vulnerability are numbers and types of glutamate receptors, calcium binding proteins, presence of nitric oxide synthase, and levels of antioxidant enzymes. For instance, levels of the mitochondrial free radical scavenger MnSOD are particularly high in striatal NADPH-diaphorase-positive neurons, which show relative resistance to mitochondrial toxins including 3-NP and 3-AP (Inagaki et al., 1991; Gonzalez-Zulueta et al., 1998). In conclusion, mitochondrial toxin models provide a wealth of information on the contributions of different functional components of mitochondria to overall cell and system function, and mechanisms of dysfunction in degenerative disorders. They have also given new insight into potential therapeutic approaches in models of several neurodegenerative diseases. In particular, a number of studies in mitochondrial toxin models suggest that CoQ10 and creatine may be useful for the treatment of PD, HD, and Friedrich’s ataxia (Lodi et al., 2001; Tarnapolsky and Beal, 2001). Consequently, ﬁndings of neuroprotection in toxin model systems have been extrapolated to transgenic mouse models of HD (Ferrante et al., 2000; Andreassen et al., 2001), and candidate therapeutic agents originally identiﬁed using mitochondrial toxin models are beginning to reach clinical trials for human degenerative diseases.

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SECTION V NEURODEGENERATIVE DISORDERS

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PARKINSON’S DISEASE

L. V. P. Korlipara∗ and A. H. V. Schapira∗ ,† ∗ University Department of Clinical Neurosciences Royal Free and University College Medical School London, United Kingdom, NW3 2PF † Institute of Neurology London, United Kingdom, WC1N 3BG

I. Introduction II. Mitochondrial Dysfunction in Parkinson’s Disease A. MPTP and Parkinsonism B. Complex I Deficiency in Idiopathic Parkinson’s Disease III. Etiology of Parkinson’s Disease A. Parkinson’s Disease and Mitochondrial Toxins B. Mitochondria and Genetic Etiologies of Parkinson’s Disease IV. Mitochondrial Dysfunction and the Pathophysiology of Parkinson’s Disease A. Oxidative Stress B. Excitotoxicity and NO C. Apoptosis and Mitochondria D. Protein Aggregation V. Concluding Remarks References

I. Introduction

Parkinson’s disease (PD) is a hypokinetic movement disorder, ﬁrst described by James Parkinson in 1839. The features of classical idiopathic PD are typiﬁed by bradykinesia, tremor, muscular rigidity, and postural instability. More recently, levodopa responsiveness has been considered to be a diagnostic feature. A variety of other clinical symptoms, including dementia, depression, and sleep disorders, may be seen, and these are generally not related to dopamine depletion. Current therapies are effective for the control of motor features but levodopa is associated with long-term side effects, including wearing off phenomena and dyskinesias. Neuropathologically, the striking feature is the loss of pigmented dopaminergic neurons in the substantia nigra pars compacta. Other areas and other neurotransmitter systems, including the noradrenergic locus ceruleus and cholinergic substantia innominata may also be affected in the INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 53

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disease. Nigral dopaminergic neurons project onto the caudate nucleus and putamen of the corpus striatum and therefore the neurochemical effects of such neuronal loss is a depletion of striatal dopamine (Hornykiewicz et al., 1975). Eosinophilic intracytoplasmic Lewy bodies, immunoreactive for ubiquitin and alpha synuclein, are present in a proportion of the surviving neurons and are considered to be the neuropathological hallmark of the disease. Mitochondria are ubiquitous intracellular organelles that play a pivotal role in the provision of cellular energy by oxidative phosphorylation. Therefore tissues with high metabolic activity, including neurons and skeletal muscles carry high mitochondrial mass. Correspondingly, disorders of mitochondrial function often cause clinical manifestations in these systems. Mitochondria have been implicated in the pathogenesis of a variety of neurodegenerative diseases. In PD, interest in the role of mitochondria initially revolved around respiratory chain dysfunction. The biochemical changes observed in PD would suggest that mitochondria may be involved in other diverse but interrelated ways, including increased oxidative stress, excitotoxicity, and apoptosis. The description of rare mutations in the alpha synuclein and parkin genes (Kitada et al., 1998; Polymeropoulos et al., 1997) conﬁrmed the impression that PD may have a variety of causes in different patients. However, there may be a common neurotoxic pathway resulting in the same clinicopathological end point. Correspondingly, the nature and extent of mitochondrial involvement may differ between patients.

II. Mitochondrial Dysfunction in Parkinson’s Disease

The respiratory chain is located on the inner mitochondrial membrane and is composed of ﬁve protein enzyme complexes, termed complexes I–V. A series of redox reactions mediate the transfer of electrons along the respiratory chain resulting in the reduction of oxygen to water and the conversion of ADP to ATP. Dysfunction of the respiratory chain, in particular complex I, has been observed in both animal models of PD and in idiopathic PD. It is not clear whether in the majority of cases this is a primary or secondary defect. However, in either case, mitochondrial dysfunction may contribute to the toxic process in a number of ways that will be discussed in Section IV. There is much debate as to the relative contributions of environmental factors and genetic factors in PD pathogenesis generally. In this respect, mitochondrial involvement mirrors this discussion and the evidence for mitochondrial toxins and mitochondrial DNA (mtDNA) abnormalities will be discussed.

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A. MPTP AND PARKINSONISM In the 1970s, a parkinsonian syndrome was described in intravenous drugs users of a newly designed synthetic meperidine analog (Davis et al., 1979). The causative agent was subsequently discovered to be the contaminant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Langston et al., 1983). This discovery was interesting from a number of perspectives. The clinical syndrome induced by MPTP was strikingly similar, albeit not identical, with those of idiopathic PD, and responded to levodopa treatment (Langston et al., 1984a). Pathological study of a brain from a patient with MPTP-induced parkinsonism revealed dopaminergic neuronal loss in the substantia nigra although Lewy bodies were not observed, while 18-ﬂuorodopa PET scans indicated nigrostriatal cell loss at a rate faster than aging long after the initial exposure of the drug (Vingerhoets et al., 1994). Moreover, in mice and monkeys, systemic MPTP reproduced the clinical, neurochemical, and pathological features of idiopathic PD and therefore lent itself to study as a model of PD (Langston et al., 1984b). Interestingly, only a small proportion of users exposed to MPTP developed the syndrome, from which we may speculate that additional genetic susceptibility factors may have been required. It was from studies into the effects of MPTP that mitochondria were ﬁrst implicated in the pathophysiology of PD. The selectivity of MPTP for dopaminergic neurons is a result of speciﬁc metabolic and uptake pathways. MPTP is a protoxin and is metabolized to its active derivative 1-methyl4-phenylpyridinium (MPP+) by monoamine oxidase B (MAO-B) which is found predominantly in glial cells of the CNS. MPP+ is a substrate for the dopamine uptake carrier located on dopaminergic neurons, where it is accumulated by mitochondria in an energy-dependent manner. Singer and colleagues demonstrated that MPP+ was a selective and reversible inhibitor of complex I of the mitochondrial electron transport chain (ETC) (Ramsay et al., 1986; Nicklas et al., 1985) and caused depletion in ATP levels (Singer et al., 1988). The way in which MPP+ inhibits complex I is not known, although it is considered to act at the same site as rotenone (Ramsay et al., 1991). However, one group found that MPP+ decreased the expression of the ND4 subunit of complex I in human neuroblastoma cells (Conn et al., 2001). MPTP has been used to study the consequences of complex I insufﬁciency in animal and cell models. The described effects are protean including increased free radical generation (Cleeter et al., 1992a), ATP depletion, mtDNA depletion (Miyako et al., 1999), nitric oxide (NO) mediated effects (Hantraye et al., 1996a), and cell death (Hartley et al., 1994a). At a time

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when there was much debate about whether PD was caused by genetic or environmental factors, the MPTP phenomenon demonstrated how a single exogenous toxin could cause most of the features of PD and stimulated renewed interest in toxic causes of idiopathic PD.

B. COMPLEX I DEFICIENCY IN IDIOPATHIC PARKINSON’S DISEASE The MPTP model provided a signiﬁcant advance in our understanding of the biochemical mechanisms involved in a parkinsonian syndrome and prompted investigation of whether these changes were present in idiopathic PD. In 1989 a complex I defect of approximately 37% was reported in the substantia nigra of patients with idiopathic PD ( Janetzky, 1994; Schapira et al., 1990a, 1989). Other smaller studies have suggested additional defects in complex II/III (Mizuno et al., 1990) and in the enzyme α-ketoglutarate dehydrogenase (Mizuno et al., 1994). This provided a direct link between the toxin-induced disease and the sporadic form, and further validated the use of the former as a model to study the latter. A number of subsequent observations conﬁrmed that the defect was disease- and regionspeciﬁc. Examination of postmortem brain homogenates revealed no defect in cortex, cerebellum, caudate, putamen, tegmentum, globus pallidus, cingulate cortex, or substantia innominata (Schapira et al., 1990b). The ﬁnding of normal complex I activities in other sites of Lewy body formation in idiopathic PD and dementia with Lewy bodies suggest that there are speciﬁc properties of nigral dopaminergic neurons that confer a predisposition to the development of such a defect. One argument put forward for the complex I dysfunction was that it was merely a reﬂection of neuronal degeneration in this area. In addition, levodopa was shown to cause a reversible inhibition of complex I in rats (Przedborski et al., 1993). Both issues were addressed in studies of brains from patients with multiple systems atrophy (MSA), a related parkinsonian syndrome also characterized by severe dopaminergic nigral neuronal degeneration (Gu et al., 1997; Schapira et al., 1990b). Although less effective in MSA patients, levodopa is also given to most patients in comparable doses to those used in PD. Analysis of substantia nigra and other brain regions in MSA revealed no defect in mitochondrial respiratory chain function. The pattern of respiratory chain dysfunction in PD is also different from those seen in other neurodegenerative diseases, including the complex II/III defect in Huntington’s disease (Gu et al., 1996) and complex IV defect in Alzheimer’s disease (Kish et al., 1992). These ﬁndings would again suggest that the complex I defect is a speciﬁc biochemical ﬁnding rather than a nonspeciﬁc consequence of neuronal degeneration.

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A respiratory chain defect can be distributed either systemically or locally within the tissue of interest. A generalized defect may be due to a systemically distributed toxin or an abnormality of mtDNA or a nuclear gene encoding a mitochondrial protein, whereas a local defect would point toward a local mitochondrial toxin or a particular vulnerability of these cells to a more systemic cause. Therefore examination for the presence of complex I defects in other tissues was a fundamental question in addressing the nature of the cause. Studies in skeletal muscle using magnetic resonance spectroscopy (Penn, 1995; Taylor et al., 1994). Polarography and respiratory chain analysis have yielded conﬂicting results of multiple defects or none at all (Schapira, 1994 for review). The reasons for the differences are not apparent and may reﬂect differences in patient sampling and the methods used. Likewise, in lymphoblasts, a small number of studies were not conclusive (Martin et al., 1996; Barroso et al., 1993; Yoshino et al., 1992). In platelets the results have been more consistent. Respiratory chain analysis in platelet homogenates from PD patients failed to detect a signiﬁcant difference from controls (Mann et al., 1992). However, in studies of puriﬁed platelet mitochondria, the majority of studies have demonstrated a complex I defect albeit generally less severe than that found in PD substantia nigra (Haas et al., 1995; Krige et al., 1992; Parker et al., 1989). Thus it would appear that the complex I defect is not just conﬁned to the substantia nigra. There are several possible explanations for the observed distribution. Firstly, it is conceivable that platelets and dopaminergic neurons have shared properties that determine the site of action of a potential neurotoxin. In this regard it should be noted that platelets, like dopaminergic neurons, possess MAO-B and MPP+ uptake mechanisms. Secondly, an mtDNA abnormality may exhibit different mutant loads in different tissues leading to tissue differences in mitochondrial respiratory chain function and may also account for the conﬂicting results in muscle.

III. Etiology of Parkinson’s Disease

Complex I defects in the substantia nigra in PD stimulated further efforts to discover etiological agents. MPTP illustrated how a mitochondrial toxin could cause a parkinsonian syndrome and thus intensive efforts to ﬁnd endogenous and exogenous neurotoxins were the initial focus. However, interest in the genetic causes of PD yielded more positive ﬁndings with the identiﬁcation of several causative genes and loci. The fact that neither environmental nor genetic causes have been identiﬁed that account for the

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majority of cases is indicative of the fact that PD is likely to be due to a more complex interplay of predisposing genetic and environmental factors.

A. PARKINSON’S DISEASE AND MITOCHONDRIAL TOXINS While MPTP proved to be a valuable model to study the mechanisms underlying the development of parkinsonism, it is the etiological agent in a very small and discrete population. However, this example of toxinmediated parkinsonism stimulated a search for similar MPTP-like compounds in the PD brain. Isoquinolines and β-carbolines were two such compounds that were identiﬁed in parkinsonian brains. 1,2,3,4 tetrahydroisoquinoline (TIQ) alkaloids and their derivatives are produced in a Pictet-Spengler type condensation reaction between endogenous biogenic amines, such as dopamine, adrenaline, and noradrenaline, and their oxidative aldehyde metabolites. TIQ compounds are also found in many common foods, including cheese, banana, egg, milk, ﬂour, and various alcoholic beverages (Niwa et al., 1989; Makino et al., 1988) and readily cross the bloodbrain barrier (Niwa et al., 1988). Thus the presence of TIQs in the brain may be due to exogenously ingested substances or endogenously formed during the metabolism of biogenic amines. A number of TIQ alkaloids have been identiﬁed in brain, cerebrospinal ﬂuid (CSF), and urine of PD patients and controls with some studies suggesting an increase in PD patients over controls (Nagatsu, 1997 for review). The metabolism, biochemical effects, and cytotoxicity of TIQ alkaloids and their derivatives parallel those of MPTP in many ways. For example, TIQ is methylated to N-methyl TIQ and then oxidized by monoamine oxidase to the N-methylisoquinolinium ion, which is a substrate for the dopamine uptake system, mirroring the conversion of MPTP to its toxic derivative (Naoi et al., 1989). A variety of TIQ alkaloids have been shown to be inhibitors of complex I of the mitochondrial respiratory chain (Morikawa et al., 1998; McNaught et al., 1995) and increase production of free radicals (Maruyama et al., 1995a,b) and reduce ATP synthesis (Suzuki et al., 1988). The cytotoxicity of TIQs has been demonstrated in cultured mesencephalic neurons (Nishi et al., 1994) and dopaminergic cell lines (Seaton et al., 1997). The methylated, oxidized derivatives have been reported to demonstrate increased complex I inhibition over the parent molecules (Suzuki et al., 1992). Several animal models have demonstrated that TIQ derivatives are capable of causing a parkinsonian phenotype. Intraventricular administration of 1,2,3,4 tetrahydro-2-methyl-6,7-isoquinolinetriol to rats reduces striatal dopamine (Liptrot et al., 1993), whereas TIQ derivatives cause neurochemical, pathological, and behavioral changes similar to those

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induced by MPTP in rats (Naoi et al., 1996) and monkeys (Yoshida et al., 1990; Nagatsu et al., 1988). In these respects, TIQs represent good candidates for endogenous neurotoxins. There is, however, at present a lack of convincing evidence of their involvement in patients with PD. Epidemiological studies have indicated that pesticide exposure and rural living are associated with a higher risk of developing PD (Gorell et al., 1998; Rajput et al., 1986). Rotenone is a naturally occurring common pesticide derived from the roots of certain plants. Its biochemistry is well characterized as a speciﬁc and highly potent inhibitor of complex I, acting at the same site as MPP+ (Ramsay et al. 1991). Exposure of cells to rotenone causes many of the biochemical events relevant to PD pathogenesis, including production of reactive oxygen species (Saybasili et al., 2001; Barrientos et al., 1999), increased sensitivity to other oxidative stressors (Ishiguro et al., 2001), and aggregation of alpha synuclein (Lee et al., 2002). Unlike MPP+, it is a hydrophobic molecule and thus can penetrate cell membranes without depending on speciﬁc uptake mechanisms. Therefore rotenone would be expected to cause a systemic complex I defect rather than one localized to the substantia nigra. In idiopathic PD the distribution of the defect in brain and platelets may be consistent with the effects of a systemic toxin. Chronic rotenone administration to rats, causing a systemic and partial inhibition of complex I, caused nigrostriatal degeneration, ubiquitin, and alpha synuclein positive cytoplasmic ﬁbrillar inclusions, and a movement disorder characterized by hypokinesia and rigidity (Betarbet et al., 2000). This model thus reproduced many of the features of PD and demonstrated how a systemic complex I defect could cause degeneration of dopaminergic neurons.

B. MITOCHONDRIA AND GENETIC ETIOLOGIES OF PARKINSON’S DISEASE Genetic factors in the etiology of PD may occur in a number of ways. These range from single genes causing PD with high penetrance to genes or haplotypes that render an individual more likely to develop the disease, possibly in conjunction with environmental factors. Such genetic factors may be encoded by nuclear or mitochondrial DNA. Epidemiological studies suggest that ﬁrst-degree relatives of patients with PD are more likely to develop PD than controls (De Michele et al., 1996; Marder et al., 1996; Bonifati et al., 1995; Vieregge et al., 1995; Payami et al., 1994). However, twin studies failed to reveal increased concordance in monozygotic twins compared with dizygotic twins and argued against a signiﬁcant genetic component in the aetiology of PD (Piccini et al., 1999; Vieregge et al., 1992; Marttila et al., 1988a; Bharucha et al., 1986). One of the difﬁculties with twin studies

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involves establishing whether an apparently normal co-twin is affected or not, given the long presymptomatic period of nigral degeneration before clinical symptoms develop. The uptake of 18F-dopa into the striatum as detected by positron emission tomography (PET) was used to circumvent the problems associated with presymptomatic diagnosis in twins. In PD brain striatal uptake is reduced to 50% of control values. Concordance values for nigrostriatal dysfunction using this method were increased in monozygotic twins compared to dizygotic twins thus indicating a genetic involvement (Wooten et al., 1997). Finally families have been described with autosomal dominant, autosomal recessive, and maternal inheritance (Leroy et al., 1998). In a few pedigrees mutations in the alpha synuclein (Polymeropoulos et al., 1997), parkin (Kitada et al., 1998), and ubiquitin carboxy terminal hydrolase L1 genes (Gu et al., 1998) have been identiﬁed. These are rare causes of PD but illustrate one end of the spectrum of the ways in which genetic factors are important. 1. Mitochondrial DNA and Parkinson’s Disease An alternative to the toxic/environmental etiology of mitochondrial dysfunction in PD is that the complex I defect in substantia nigra and platelets is caused by genetic factors. Complex I consists of 41 subunits, 7 of which are encoded by mtDNA and the remainder encoded by nuclear genes. The mitochondrial genome is a circular double stranded molecule of 16.5 kb and encodes 22 transfer RNAs, two ribosomal RNAs, and 13 polypeptides which are all components of the respiratory chain. A number of mutations of mtDNA have been described in association with human disease and the genotype phenotype correlations are complex and poorly understood. Several important features distinguish mtDNA mutations from their nuclear counterparts. One might expect a classical maternal inheritance pattern in PD caused by mtDNA mutations. While pedigrees have been described which observe such an inheritance pattern, the majority of patients with PD have apparently sporadic disease. However, it should be noted that the majority of patients with known mtDNA mutations associated with other neurological diseases, for example, Leber’s hereditary optic neuropathy, have no family history. Secondly, mtDNA mutations usually exhibit heteroplasmy indicating the co-existence of mutant and wild-type molecules in the same tissue and possibly in the same mitochondrion. The threshold of a mutation for biochemical expression may vary between mutations and the dependence of a tissue on oxidative phosphorylation may determine the consequences to a given mutant load. This may explain why carriers of mtDNA mutations may be asymptomatic or oligosymptomatic, thus rendering pedigree patterns complex and resulting in apparently sporadic cases without an obvious maternal inheritance pattern.

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One approach to investigate the role of mtDNA in causing a respiratory chain defect involves the use of cybrid cell lines. The system involves the creation of ρ0 cell lines, which lack mtDNA and thus a functioning respiratory chain, by prolonged culture in ethidium bromide. Platelets, which lack nuclei, from PD patients are thus suitable donors of mtDNA in cell fusion experiments with the ρ0 cells to create PD cybrid cell lines. If a complex I deﬁciency in the original platelets is perpetuated in the cybrids, then it must be caused by a mutation or mutations in mtDNA. Conversely if the complex I defect is lost then the cause is either due to a nuclear defect or a circulating toxin. Platelets from PD patients, speciﬁcally chosen for their low complex I activity, were fused with A549 ρ0 cells and mixed cybrid analysis revealed a 25% deﬁciency of complex I activity (Gu et al., 1998). Clonal cell lines from one patient with such deﬁciency revealed a range of complex I and IV deﬁciencies, COX-1 expression, and mitochondrial membrane potential, as assessed by JC-1 staining, which was consistent with a heteroplasmic mtDNA effect. Swerdlow (1996) reported that in unselected PD cybrids using a neuroblastoma cell line as the host, there was a 20% reduction in complex I activity compared to controls, although how this correlated with enzyme activities in platelets was not stated. The PD cybrids also demonstrated increased production of free radicals and sensitivity to the toxic effects of MPP+. Another study utilizing cybrid fusions with ρ0 HeLa cells, however, showed no difference between PD and elderly controls in restoring respiratory chain function (Aomi et al., 2001). However, it is not clear whether these patients exhibited a complex I defect in their platelets before cybrid fusion. Given the heterogeneity of PD it is reasonable to surmise that patients at the low end of complex I activity are those most likely to be harboring an mtDNA abnormality. Therefore a lack of complex I deﬁciency in unselected PD cybrids does not exclude mtDNA as an important factor in PD. These studies taken together imply that the low complex I activity in PD is due to abnormalities in mtDNA rather than nuclear DNA or a circulating toxin. Numerous studies have looked for mtDNA mutations in PD. MtDNA has a much higher mutation rate than nuclear DNA and a number of reasons have been suggested for this including high levels of oxidative stress in the local environment, the lack of a histone coat, and the DNA polymerase error rate. This, however, makes it more difﬁcult to differentiate between disease causing mutations and polymorphisms. In general a heteroplasmic base change in a conserved region that is absent in controls without disease is more suggestive of a disease-causing mutation. Sporadic case reports have described atypical parkinsonism in association with mtDNA mutations. Thyagarajan et al. (2000) described a heteroplasmic 12S rRNA mutation, in a family with maternally inherited sensorineural deafness, neuropathy, and levodopa responsive parkinsonism,

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that was not present in 270 controls. mtDNA deletions were found in the muscle of two patients with early-onset atypical parkinsonism and features typical of a more systemic mitochondrial disorder, including raised serum creatine kinase and lactate, multiple symmetric lipomatosis, and suggestive muscle biopsy changes (Siciliano et al., 2001). One report described a 4 base pair deletion in the mtDNA encoded cytochrome b gene, abnormalities of which typically cause skeletal muscle weakness, in association with parkinsonism (Rana et al., 2000). The most common primary mutation of Leber’s hereditary optic neuropathy at nucleotide 11778 was found in a family with atypical parkinsonism (Simon et al., 1999). These cases are interesting in illustrating how mitochondrial disorders may manifest as an akinetic rigid syndrome. However, they are rare isolated reports and were generally associated with additional features suggestive of a generalized mitochondrial disorder. Therefore it is unlikely that they are relevant to patients with sporadic PD. The search for mtDNA mutations in PD has generally been inconclusive. Ikebe et al. (1990) described an increase in the 4977 base pair “common deletion” in PD striatum. However, it became apparent that this deletion accumulates in the brain as a consequence of normal aging (Lestienne et al., 1991). Furthermore, RFLP analysis of mtDNA from PD postmortem substantia nigra, putamen, and frontal cortex of patients with complex I defects failed to reveal any mtDNA deletions (Lestienne et al., 1990). However, a more sensitive PCR-based methodology with sequencing of tRNA and complex I subunit genes in substantia nigra of two patients with PD and two controls revealed multiple deletions and mtDNA polymorphisms (Kapsa et al., 1996). This led to suggestions that the accumulation of mtDNA abnormalities in the brain during aging may affect respiratory chain function leading to subtle alterations in the local bioenergetic proﬁle, which in turn may render cells more vulnerable to other toxic and pathophysiological stresses. Several studies have attempted to identify point mutations in PD mtDNA. Studies analyzing tRNA genes (Grasbon- Frodl et al., 1999), complex I genes (Kirchner et al., 2000; Kosel et al., 1998), and complete mitochondrial genomes (Brown et al., 1996; Ikebe et al., 1995) have revealed a multitude of point mutations, although these were not consistent between patients. Other sporadic reports have associated PD with mutations in complex IV (Ozawa et al., 1991), although this was not reproduced on further study (Lucking et al., 1995), and with secondary LHON mutations (Kosel et al., 1998) (which increase the penetrance of a primary LHON mutation). An extensive study for mtDNA variants using restriction endonuclease analysis was undertaken from 73 brains with Alzheimer’s Disease (AD) and PD, 62 AD brains, and 32 blood and 6 muscle samples from PD patients (Shoffner et al., 1993). They identiﬁed an A4336G mutation in the tRNAgln gene in 5.2% of the patients surveyed and 0.7% of age-matched controls. The tRNAgln

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mutation was not reproduced by another study which reported instead mutations in position 15927 and 15928 in the tRNAthr gene (Mayr-Wohlfart et al. 1997). Two further studies identiﬁed a G5460A mutation in the complex I ND2 gene (Kosel et al., 1996; Schnopp et al., 1996). A more recent study, however, reported sequencing data of all complex I and tRNA genes from platelets of 28 PD patients and 8 control subjects, and restriction digest analysis from platelets, blood, or postmortem brain of a further 243 PD patients and 209 controls. They did not ﬁnd any association between PD and the mutations at nucleotides 4336, 5460, or 15927/8 and did not ﬁnd any signiﬁcant differences in the frequencies of any mutations in PD versus control groups (Simon et al., 2000). Vives-Bauza et al. (2002) sequenced the entire mitochondrial genome from eight PD and nine control subjects and found that the two groups did not differ in the total number of either all mutations or missense mutations. The missense mutations found were rare and none differed in frequency between the two groups. The diverse and conﬂicting data that emerge from these studies and the low frequency of the described mutations calls into question their pathogenic signiﬁcance. The concept that PD may be caused by a single mutation may be an oversimpliﬁcation. mtDNA involvement, if any, in PD may involve the subtle alteration of function by complex haplotype effects, vulnerability factors conferred by particular polymorphisms, or the accumulation of deletions or other mutations with age. As yet, data concerning mtDNA in PD have been unrevealing not only in identifying causative mutations, but also in generating rational explanations for how described mutations result in the observed respiratory chain dysfunction. 2. Nuclear Mutations of Mitochondrial Proteins The failure to identify causative mtDNA mutations in PD and the fact that the majority of complex I subunits are encoded by the nuclear genome led to renewed interest in the possibility of nuclear mutations contributing to complex I dysfunction in PD, although this would be in contradiction to the cybrid data. Screening of a 24-kDa subunit of the ﬂavoprotein fraction of complex I, encoded by a single nuclear gene NDUFV2 on chromosome 18, identiﬁed a new polymorphic mutation in the signal peptide (Hattori et al., 1988). It was shown that 23.8% of 123 PD patients were homozygous for this mutation whereas in 113 control subjects this ﬁgure was 11.5%. Other studies have revealed an increase in PD patients of polymorphisms in the E2 enzyme of the α-ketoglutarate dehydrogenase complex and the Mn-SOD gene, both mitochondrial proteins that may be important in the pathogenesis of PD (Kobayashi et al., 1998). α Ketoglutarate dehydrogenase activity is diminished by MPP+ (Mizuno et al., 1987), as well as complex I, and may be reduced in PD brain (Mizuno et al., 1994). Deﬁciency causes reduced production of succinate and hence less electron transfer through

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complex II, causing further respiratory chain insufﬁciency. The described polymorphism, however, does not effect a change in the amino acid sequence of the translated product, which implies that it is not directly important and may reﬂect close proximity to another genetic locus important in PD. Mn-SOD is an important enzyme in regulating the levels of cellular oxidative stress which may play a central role in the pathophysiology of PD. A polymorphism was described in the mitochondrial targeting sequence and the allelic frequency was higher in PD patients (19.2%) than in controls (12.1%) (Shimodo-Matsubayashi et al., 1996). The signiﬁcance of such ﬁndings is not clear, although it is possible that such ﬁndings constitute genetic risk factors. However, current understanding of such concepts is rudimentary and therefore it is not possible to draw deﬁnitive conclusions from such data.

IV. Mitochondrial Dysfunction and the Pathophysiology of Parkinson’s Disease

In addition to complex I deﬁciency, a number of other biochemical and cellular processes, including oxidative stress, iron accumulation, excitotoxicity, inﬂammation, protein aggregation, and apoptosis have been implicated in the pathogenesis of PD. One of the challenges facing PD research is in piecing together these strands, deﬁning their interrelationships, and forming a unifying theory of how these changes lead to neuronal death. Mitochondrial respiratory chain dysfunction may contribute through its effect on ATP production or the generation of free radicals. Furthermore mitochondria may be involved via their roles in calcium buffering and apoptosis. It is not certain whether mitochondrial dysfunction is a primary pathogenic abnormality or a secondary consequence of other pathophysiological events. Nonetheless whichever is the primary mechanism, mitochondrial dysfunction may interact in such a way that triggers a cycle of toxicity leading to cell death.

A. OXIDATIVE STRESS Approximately 98% of molecular oxygen is reduced to water at complex IV of the respiratory chain during oxidative phosphorylation. The remaining molecular oxygen is reduced to H2O2 and superoxide free radicals (Chance et al., 1979). Free radicals have an unpaired electron and are thus a highly reactive species capable of causing oxidative damage to a variety of macromolecules. Cellular antioxidant enzymes operate to prevent the

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deleterious effects of free radicals. Mitochondria are the major site of superoxide generation in the cell and thus occupy a pivotal role in both energy provision and levels of oxidative stress. Oxidative stress reﬂects the balance between the generation of free radicals and cellular antioxidant mechanisms. The basal levels of oxidative stress in the substantia nigra are high, possibly explaining the selective vulnerability of dopaminergic neurons in PD (Olanow, 1990; Halliwell and Gutteridge, 1985). This is due to the metabolism of dopamine, both enzymatically by monoamine oxidase type B, producing hydrogen peroxide and the deaminated metabolites, dihydroxyphenylacetic acid and homovanillic acid, and auto-oxidation which generates neuromelanin, quinone, and semi-quinone species and subsequently other free radicals. A number of observations support the concept that the levels of oxidative stress are increased in PD substantia nigra. Several studies have produced evidence of oxidative damage to proteins, lipids, and DNA in PD substantia nigra. More speciﬁcally, the levels of reactive protein carbonyls (Alam et al., 1997) and lipid hydroperoxides (Dexter et al., 1994a) are markedly increased in the PD substantia nigra. Elevated concentrations of 8-hydroxy2 deoxyguanosine in nuclear and mitochondrial DNA fractions from PD brain suggest increased DNA oxidative damage (Sanchez-Ramos et al., 1994). However, what is apparent from some of the studies is that the increase is not just conﬁned to the substantia nigra and additional factors must explain the selectivity of neuronal degeneration. The possibility that the widespread changes may be a consequence of levodopa treatment is not supported by the observation that marmosets treated with levodopa do not exhibit such changes (Lyras et al., 2002). Iron may play an important role in mediating oxidative stress in PD by acting as an electron donor in a number of redox reactions. In particular, it can interact with hydrogen peroxide (which may be augmented by dopamine metabolism) to generate highly reactive hydroxyl radicals in the Fenton reaction or react with molecular oxygen generating superoxide ions. In control subjects, basal iron levels are high in substantia nigra, globus pallidus, and the striatum. In PD, there is a 35% increase in the iron levels in the substantia nigra (Soﬁc et al., 1991; Dexter et al., 1989). One study using x-ray microanalysis found increased levels of iron in neuromelanin granules ( Jellinger et al., 1992; Hirsch et al., 1991), raising the possibility that neuromelanin may act as a toxic reservoir of iron. It is not known whether the observed increases in iron exist in a free and reactive form or bound to ferritin. One study using antibodies against the H and L forms of ferritin did not identify any difference in ferritin levels between PD patients and controls, suggesting that some of the increase in iron may exist in the reactive state (Mann et al., 1994). The fact that other basal ganglia degenerations,

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such as MSA and progressive supranuclear palsy (Dexter et al., 1992), also have similar increases in iron suggests that increased iron levels are a manifestation of cell loss, rather than a primary mechanism. However, this may not diminish the role iron may play in potentiating the toxic process once underway. Increased oxidative stress can result not only from the increased generation of free radicals but also from the depletion of protective free radical scavengers. Reduced glutathione (GSH) plays an important antioxidant role by reducing hydrogen peroxide, thereby preventing its reaction with iron in the Fenton reaction. Perry et al. (1982) described low levels of reduced GSH in PD substantia nigra pars compacta. Subsequently, other groups conﬁrmed these ﬁndings, reporting a decrease of GSH of approximately 30–40% (Sian et al., 1994; Soﬁc et al., 1992). This was not found in other brain regions and was present in preclinical Lewy body brains (Dexter et al., 1994b). While GSH depletion, using intraventricular buthionine R,S-sulfoximine (BSO) infusion, of 30–70% in rodent models did not induce neuronal degeneration (Toffa et al., 1997; Andersen et al., 1996), GSH depletion conferred increased vulnerability of neurons to MPP+ in rats (Wullner et al., 1996). Cytosolic (copper/zinc) and mitochondrial manganese superoxide dismutase are enzymes important in dismutating superoxide radicals. Both were shown to be increased in PD substantia nigra (Yoritaka et al., 1997; Saggu et al., 1989; Marttila et al., 1988b) while the latter has been reported to be elevated in the CSF of parkinsonian patients (Yoshida et al., 1994), implying that levels of superoxide free radicals may be elevated in PD. The cause of the increased oxidative stress in PD is not known. There is, however, a clear and reciprocal relationship between respiratory chain dysfunction and oxidative stress. Complex I is one of the major sites of superoxide generation in the cells and inhibition of the respiratory chain, particularly at complexes I or III, results in the increased generation of free radicals (Takeshige and Minakami, 1979). Conversely free radicals can themselves damage the respiratory chain. In vitro studies suggest that both complexes I and III are particularly affected (Zhang et al., 1990; Hillered and Ernster, 1983) whereas in vivo studies suggest complex IV and complex I are inhibited (Thomas et al., 1993; Benzi et al., 1991). PD cybrids with low complex I activity demonstrate increased levels of free radicals. Further evidence of the link between mitochondrial dysfunction and free radicals is indicated by a study on the effects of MPP+, which typically causes reversible complex I inhibition, on submitochondrial particles. Prolonged exposure results in a severe and irreversible inhibition of complex I when electron ﬂow through the respiratory chain is prevented by inhibition of complex IV (Cleeter et al., 1992b). This irreversible inhibition is prevented by free radical scavengers indicating that oxidative damage may have occurred as a result

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of respiratory chain inhibition. Therefore free radical mediated damage and mitochondrial dysfunction may form part of a self-amplifying cycle, augmented by elevated cellular iron concentrations.

B. EXCITOTOXICITY AND NO Excitotoxic cell death is a mitochondrially mediated process and has been implicated in a variety of neurological conditions, including other neurodegenerative diseases and cerebral ischaemia. The term refers to the persistent overstimulation of N-methyl-D-aspartate (NMDA) glutamate receptors leading to massive cellular calcium inﬂux and ensuing neuronal death. The NMDA receptor exhibits a voltage-dependent Mg2+ block at normal resting cell membrane potential. Typically the resting potential is approximately –90 mV and is actively maintained by ATP-dependent mechanisms. Therefore a mitochondrial defect, more speciﬁcally in PD the complex I defect observed, may lead to reduced membrane depolarization due to the reduction in ATP production. When the cell membrane potential reaches a threshold of –50 to –60 mV the NMDA receptor block is relieved leading to persistent receptor stimulation. Therefore in PD, complex I deﬁciency may create an environment that favors the development of excitoxicity. This postulated link between ATP production, cell membrane potential, NMDA receptor activation, and glutamate toxicity was demonstrated in cultured chick retinal neurons in which NMDA receptor activation and glutamate toxicity was achieved by either inhibition of oxidative phosphorylation or modulation of the resting membrane potential (Zeevalk et al., 1990, 1991). NMDA overstimulation leads to Ca2+ inﬂux and this event correlates closely with neuronal death. The bulk of the rise in cytosolic calcium levels is buffered by mitochondria resulting in mitochondrial calcium overload. Calcium overload results in the increased production of free radicals, both directly and through the activation of nitric oxide synthase (NOS) (Dykens, 1994). Additionally mitochondrial calcium overload and the increased oxidative stress are both inducers of mitochondrial permeability transition pore opening (see Section IV.C). These various facets of mitochondrial involvement are consistent with the observation that excitoxicity is initiated in areas with high mitochondrial density, resulting in mitochondrial calcium inﬂux in these areas (Bindokas and Miller, 1995) and collapse of the mitochondrial transmembrane potential (White and Reynolds, 1996; Schinder et al., 1996; Ankarcrona et al., 1996; Nieminen et al., 1996). Evidence of a role for excitotoxic cell death in PD has largely been provided by MPTP models in rats, mice, and primates in which NMDA antagonists protected against MPTP and MPP+ induced striatal dopamine depletion (Brouillet

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and Beal, 1993; Storey et al., 1992; Zuddas et al., 1992). A number of studies have demonstrated the neuroprotective effects of the glutamate antagonist, riluzole in MPTP rodent and primate models (Benazzouz et al., 1995; Boireau et al., 1994), although an early clinical trial in PD patients failed to demonstrate a beneﬁt ( Jankovic and Hunter, 2002). Many of the toxic effects of NMDA activation are linked to the generation of NO. In this regard glutamate and NMDA induced toxicity is attenuated in cultured neurons treated with NOS inhibitors and in nNOS deﬁcient mice (Dawson et al., 1991, 1996). NO is present in many tissues and is generated by the conversion of L-arginine to L-citrulline by NO synthase. NO generation can lead to a variety of deleterious effects. Reaction with superoxide ions, levels of which may be elevated by complex I defects, lead to the formation of highly reactive peroxynitrite ions (Beckman and Crow, 1993). This in turn can lead to free radical damage to macromolecules, nitration of tyrosine residues in cellular proteins, oxidation of dopamine to reactive quinone species, and opening of the mitochondrial permeability transition pore (LaVoie and Hastings, 1999; Packer et al., 1997). NO itself can exacerbate mitochondrial dysfunction by binding to and inhibiting complex IV of the respiratory chain and may therefore “prime” the respiratory chain to the effects of complex I inhibition (Cleeter et al., 1994). Thus NO was shown to enhance the inhibitory action of MPP+ on complex I activity in brain submitochondrial particles and from this it was proposed that the reversible inhibition of cytochrome oxidase potentiated the MPP+ induced, irreversible, free radical mediated inhibition of complex I (Cleeter et al., 2001). Finally NO may potentiate oxidative stress by liberating cellular iron making it available in the Fenton reaction. Indirect evidence for excitotoxicity and NO mediated damage in PD has derived from changes detected in PD and from MPTP animal models. Nitrosyl radicals in substantia nigra and elevated CSF nitrite concentrations have been demonstrated in PD patients (Shergill et al., 1996; Quereshi et al., 1995). Lewy bodies are immunoreactive for 3-nitrotyrosine, a marker of peroxynitrite mediated damage (Good et al., 1998), and have also been shown to contain accumulations of nitrated alpha synuclein (Giasson et al., 2000). Three different NOS isoenzymes have been identiﬁed, neuronal NOS (nNOS), endothelial (eNOS), and inducible NOS (iNOS). nNOS is the dominant isoform in the brain while there is little or no expression of iNOS under normal conditions. A number of insults or pathological conditions can, however, induce the expression of iNOS in glial cells and invading macrophages. For example, PD postmortem brain tissue express greatly increased levels of iNOS compared to age-matched controls (Knott et al., 2000). The nNOS inhibitor 7-nitro-indazole protected against MPTP neurotoxicity in mice and baboons (Hantraye et al., 1996b; Schultz et al., 1995)

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and MPP+-induced neuronal degeneration and striatal 3 -nitrotyrosine concentrations are reduced in nNOS deﬁcient mice (Matthews et al., 1997). Similarly, iNOS deﬁcient mice are signiﬁcantly more resistant to MPTP and iNOS expression in mice was found to be upregulated in MPTP treated mice (Liberatore et al., 1999). This animal data indicate a pathophysiological role for both nNOS and iNOS in MPTP toxicity. In summary, there are good conceptual reasons and indirect observations that suggest that excitotoxicity may be important in PD. Mitochondria may be involved in creating conditions in which it is likely to occur, in the intermediate pathways consequent upon calcium inﬂux and also in the effector processes leading to neuronal death.

C. APOPTOSIS AND MITOCHONDRIA It is now clear that mitochondria are involved in several aspects of cell physiology and that the traditionally held view of mitochondria, as organelles whose sole function is cellular energy provision, was too narrow in its scope. In addition to its role in cellular calcium regulation, mitochondria mediate certain apoptotic cell death pathways. Apoptosis refers to the morphology of dying cells described by Kerr et al. (1972) and has become synonymous with the concept of programmed cell death whereby cell death is an active process enacted by intrinsic cellular programs. This form of cell death is thought to play an important role in tissue development and maturation. In contrast to tissue necrosis, apoptosis may take hours to days, involve new gene transcription and protein expression, and is morphologically characterized by condensation of nuclear chromatin into electron dense masses, preservation of plasma and nuclear membranes, appearance of apoptotic bodies, and the lack of inﬂammatory changes. It is known that substantia nigral neurons undergo apoptosis during normal development while apoptosis has been implicated in several other neurodegenerative disorders, including amyotrophic lateral sclerosis, Huntington’s disease and Alzheimer’s disease (Stefanis et al., 1997). Neuronal apoptosis can be induced by a wide range of inducing stimuli, many of which are relevant to PD including glutamate (Mitchell et al., 1994), MPTP and its metabolites (Tatton and Kish, 1997; Dipasquale et al., 1991), 6-hydroxydopamine (6-OHDA) (Walkinshaw et al., 1994), complex I inhibitors (Hartley et al., 1994b), pro-oxidants (Slater et al., 1995), high levels of dopamine (Ziv et al., 1994), and levodopa (Walkinshaw et al., 1995). Therefore studies have attempted to address the question of whether apoptotic changes are present in PD nigral neurons and have yielded conﬂicting results, in part due to problems with the methods used to detect apoptotic changes in

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tissue sections. In situ end labeling (ISEL) techniques have been used widely for this purpose, whereby the 3 ends of cleaved DNA are labeled with a chromagen or ﬂuorophore. However, it has become apparent that ISEL can yield false-positive results in necrotic cell death. Therefore it is considered essential to combine this technique with one that demonstrates characteristic morphological appearances, in order to unequivocally detect apoptosis. One group demonstrated apoptotic changes in PD and diffuse Lewy body disease brains (Mochizuki et al., 1996), although this was not reproduced by another study which failed to show apoptotic changes in PD brain despite positive ISEL detection (Kosel et al., 1997). However, one group, using a double labeling ﬂuorescence technique with ISEL and the cyanine DNA binding dye, YOYO1, detected apoptosis in 1–2% of dopaminergic neurons of the substantia nigra pars compacta (Tompkins et al., 1997). The fact that PD is a chronic disease should also be taken into account when interpreting such studies as they may not truly reﬂect the degree of involvement of apoptosis in postmortem brain. This may explain why in rodent models of PD, involving MPTP and 6-OHDA administration, apoptosis is more readily seen in nigral dopaminergic neurons (He et al., 2000; Tatton and Kish, 1997). The importance of mitochondria in apoptosis was ﬁrst illustrated in cell free systems where it was shown that mitochondria or their products could induce nuclear DNA fragmentation and chromatin condensation (Newmeyer et al., 1994). Their role in apoptosis is mediated by the opening of the mitochondrial permeability transition pore (PTP), a large, nonselective proteinaceous pore complex spanning the inner and outer mitochondrial membranes. During oxidative phosphorylation, energy derived from electron transfer is used by complexes I, III, and IV to actively pump protons out of the mitochondrial matrix across the inner mitochondrial membrane. This creates a pH gradient and potential difference across the inner mitochondrial membrane (ψM ), generating a proton-motive force, which drives the conversion of ADP to ATP by ATP synthase. The greater component of the proton-motive force is the ψM , which therefore approximates to the ATP/ADP ratio. Thus PTP opening results in the collapse of the ψM , uncoupling of oxidative phosphorylation, equilibration of gradients of ions and small solutes across the mitochondrial membrane, and mitochondrial swelling. Dissipation of ψM is one of the earliest changes to occur in apoptotic cell death, before the development of the other ultrastructural features (Wadia et al., 1998). The bioenergetic and biochemical abnormalities that may be present in PD can induce PTP opening, including mitochondrial Ca2+ overload and pro-oxidants (reviewed in Crompton, 1999). Additionally respiratory chain dysfunction results in a reduction in ATP synthesis and mitochondrial membrane potential, both of which can increase the propensity

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of the pore to open. Therefore there is good reason to hypothesize a role for PTP opening in nigral death. Opening of the PTP causes mitochondrial swelling, unfolding of the inner mitochondrial membrane, outer membrane rupture, and the release of pro-apoptogenic factors from the mitochondrial intermembrane space into the cytosol. These include the water soluble electron carrier cytochrome c (Kantrow and Piantadosi, 1997) and a 57-kDa ﬂavoprotein named the apoptosis initiating factor (AIF) (Susin et al., 1999). Both are capable of inducing apoptosis in cells and in cell free systems (Susin et al., 1999; Li et al., 1997; Liu et al., 1996) and these events are thought to occur by the recruitment and activation of caspases. These are a family of cysteine proteases, active at aspartic acid residues, which exist as inactive procaspases and are sequentially activated by cleavage by other caspases or by self-cleavage. This cascade of caspase activation eventually exerts its effects on endonucleases and other effectors of apoptosis. The regulation of PTP-mediated apoptosis may occur as a consequence of functional interactions with pro-apoptogenic or anti-apoptogenic proteins. For example, the bcl-2 family of oncoproteins contains both inhibitors of apoptosis, including bcl-2 and bcl-XL and promoters of apoptosis, including bax and bid. Indirect evidence that the mitochondrial PTP plays a role in PD or models of PD is provided in several studies and largely relies on the properties of cyclosporine, which in addition to its immunosuppressant properties is an inhibitor of PTP opening. MPP+ induces cyclosporine sensitive PTP opening and cytochrome c release in liver and brain mitochondria and also induced apoptosis, in a pheochromocytoma cell line that was inhibited by cyclosporine (Cassarino et al., 1999; Seaton et al., 1998). In rats and mice treated with 6-OHDA, cyclosporine protected against striatal dopamine depletion (Matsuura et al., 1997a,b; Ogawa, 1996). Further evidence is provided in examining proteins involved in the regulation and effector pathways of apoptosis. In MPTP-treated mice increases in the expression of bax (Hassouna et al., 1996) and caspases 3, 8, and 9 (Turmel et al., 2001; Hartmann et al., 2001; Viswanath et al., 2001) have been detected, while bax deﬁcient mice are resistant to MPTP toxicity (Vila et al., 2001). In PD substantia nigra there is an increase in bcl-2 (Hartmann et al., 2000) and activated caspase 3 (Mogi et al., 1996) expression in surviving neurons, the former implying a compensatory upregulation of anti-apoptotic mechanisms.

D. PROTEIN AGGREGATION The description of mutations in the alpha synuclein gene in autosomal dominant PD prompted renewed interest in the role of protein aggregation

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in the pathogenesis of PD. While these mutations are extremely rare, the importance of wild-type alpha synuclein was demonstrated by the fact that Lewy bodies in sporadic PD are strongly immunoreactive for alpha synuclein (Spillantini et al., 1997). Overexpression of mutant and wild-type alpha synuclein in mice leads to the formation of intraneuronal alpha synuclein aggregates (Giasson et al., 2002; Masliah et al., 2000) while, in vitro, mutant alpha synuclein has a greater tendency to aggregate than wild-type (Conway et al., 1998). The description of mutations in parkin and ubiquitin carboxy hydrolase L1, both proteins involved in the ubiquitination of proteins marked for proteasomal degradation, combined with the impairment of proteasomal function noted in the substantia nigra of idiopathic PD (McNaught and Jenner, 2001), further strengthened the hypothesis that protein aggregation may be a common thread in the pathogenesis of PD of different etiologies. It is not yet clear how and where alpha synuclein aggregation relates to the other pathophysiological abnormalities in PD and in particular the mitochondrial dysfunction. However, data is emerging that links complex I inhibition with changes in alpha synuclein. MPTP upregulates expression of the synuclein 1, the rodent homolog of alpha synuclein, and induces its aggregation in baboons (Vila et al., 2000; Kowall et al., 2000). Correspondingly, in cell culture models, MPP+ upregulated alpha synuclein expression in neuroblastoma cells (Gomez-Santos et al., 2002), and complex I inhibition by rotenone induced aggregation of overexpressed alpha synuclein in monkey kidney cells (Lee et al., 2002). From this data one may hypothesize that mitochondrial dysfunction, in those patients for whom it is a significant factor, may alter the levels and propensity to aggregation of alpha synuclein. Theories of how aggregates contribute to the toxic process are currently speculative and may be through the increased generation of free radicals or by direct toxicity of aggregates or their precursor protoﬁbrils.

V. Concluding Remarks

A description of mitochondrial involvement in PD pathogenesis encompasses a number of processes at different points of the pathways leading to cell death. In a complex, etiologically heterogeneous disease, a fundamental and major challenge facing researchers is deﬁning the common pathways in the toxic process. This will generate multiple targets for rational neuroprotective strategies. For example, coenzyme Q10, which may attenuate mitochondrial bioenergetic dysfunction, reduced the loss of striatal dopamine and neuronal loss in MPTP-treated mice and caused a trend toward increased complex I in PD patients (reviewed in Shults et al., 1999).

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A multitude of other agents are currently under scrutiny, including antiexcitotoxic agents, antioxidants, caspase inhibitors, and other anti-apoptotic agents. Consequently there is reason for cautious optimism for the future, in changing the emphasis of PD therapy from improving clinical symptoms, to one of retarding the progressive and inexorable loss of neurons in the disease.

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HUNTINGTON’S DISEASE: THE MYSTERY UNFOLDS?

˚ Peters´en1 and Patrik Brundin Asa Section for Neuronal Survival Wallenberg Neuroscience Center Department of Physiological Sciences Lund University Lund, Sweden

I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII.

Huntington’s Disease Epidemiology and Symptomatology Neuropathology Genetics Other Triplet Repeat Diseases Intracellular Localization of Normal and Mutant Huntingtin Function of Normal and Mutant Huntingtin Cell Death in Huntington’s Disease: Apoptosis and Autophagy Oxidative Stress and Metabolic Dysfunction Dopamine Toxicity Transgenic Mouse Models of Huntington’s Disease Concluding Remarks References

I. Huntington’s Disease

In 1872, George Sumner Huntington presented his classic paper on Huntington’s disease (HD). The early twentieth century saw several publications on the pattern of inheritance and the neuropathology of HD, but it was not until the development of modern molecular genetic techniques in the 1980s that major advances were made. In 1993, the disease-causing HD gene was found by a multicenter effort, organized by the Hereditary Disease Foundation (Huntington’s Disease Collaborative Research Group, 1993). Since then, the development of genetically engineered mice and cells have provided excellent tools not only for the study of pathogenic mechanisms in HD, but also for investigating new clinical interventions. Today research into the pathogenesis of HD has reached a very exciting stage, where the interaction between molecular genetics, mitochondrial biology, 1

To whom correspondence should be addressed.

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and neuroscience could yield a major breakthrough in the understanding of why the HD mutation leads to gradual degeneration of certain groups of neurons.

II. Epidemiology and Symptomatology

The disease has a prevalence of 4–8 per 100,000 in Europe (Harper, 1992). It is characterized by a decline of cognitive, emotional, and motor functions (Quarrel and Harper, 1996). Disturbances in motor function are a classical feature and have led to use of the term Huntington’s chorea (chorea, Greek for dance). Symptoms develop insidiously, and they are initially hard to differentiate from the patient’s normal behavioral repertoire. Typically, the disease presents between 35 and 45 years of age; however, disease onset can vary between childhood and old age, depending on the severity of the genetic mutation. In adult-onset HD, death typically occurs within 15–20 years of onset, usually following infectious complications of immobility. Juvenile cases often progress more rapidly and can lead to death within 7–10 years from disease onset. Interestingly, there is often a marked loss of body weight and a generalized lack of muscle bulk, in spite of increased calorie intake (Sanberg et al., 1981; Kremer and Roos, 1992). It remains unclear whether this is the result of increased energy expenditure due to involuntary movements, or to reduced gastrointestinal absorption, or whether there is a more complex underlying metabolic disturbance involving mitochondria.

III. Neuropathology

The most marked neuropathological features are the dramatic loss of neurons and the development of astrogliosis in the striatum (Vonsattel and DiFiglia, 1998). Combined with some atrophy of the neocortex (20%), the reduction in striatal volume (60%) leads to around 30% loss in total brain weight. In the neostriatum, the GABAergic medium-sized spiny projection neurons, which constitute around 90% of all striatal neurons (Andersson and Reiner, 1991), are the most affected. There is relative sparing of mediumsized aspiny neurons that contain somatostatin, neuropeptide Y, and NADPH diaphorase, as well as of cholinergic interneurons (Vonsattel and DiFiglia, 1998). In the striatum and cortex of HD patients and HD transgenic mice, there are neuronal intranuclear inclusions, and protein aggregates in dystrophic

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neurites (Roizin et al., 1979; Davies et al., 1997; DiFiglia et al., 1997; Becher et al., 1998; Gutekunst et al., 1999). Around 1–5% of striatal neurons in adult HD brains contain nuclear inclusions (DiFiglia et al., 1997; Gutekunst et al., 1999). One spherical inclusion per nucleus is most common, and inclusions are detected in all cortical layers with a higher frequency in juvenileonset forms (38–52% of cortical neurons) compared to adult-onset forms (3–6% of cortical neurons) of HD (DiFiglia et al., 1997). Similar fragments of huntingtin are also found in dystrophic neurites predominantly in cortical layers V and VI. Interneurons that are spared in HD display the highest frequency of neuronal inclusions (Kummerle et al., 1999). The inclusions contain ubiquitinated mutant huntingtin and N-terminal fragments of wildtype huntingtin (Dyer and McMurray, 2001). It remains controversial whether the protein aggregates are protective, toxic, or just an epiphenomenon. The presence of neuronal intranuclear inclusions in symptomatic HD patients, and their absence in presymptomatic patients, suggests that they are closely linked to the onset of the disease (DiFiglia et al., 1997). Transgenic HD mice (R6 lines) also develop neuronal intranuclear inclusions in the cortex and striatum just prior to the onset of symptoms (Davies et al., 1997; Hansson et al., 2001b). The aggregation of mutant huntingtin has also been shown to correlate with toxicity in cultured cells (Cooper et al., 1998; Lunkes and Mandel, 1998; Martindale et al., 1998; Waelter et al., 2001). However, a protective effect of huntingtin aggregation has been proposed in another culture system (Saudou et al., 1998). Huntingtin aggregation and toxicity can also occur in parallel and independently of each other (Kim et al., 1999). In a transgenic mouse with a tetracyclin-regulated expression of exon 1 of the HD gene, inclusions and symptoms appear after transgene expression, and both disappear when the transgene is turned off (Yamamoto et al., 2000). This study shows that the formation of inclusions is a dynamic process, and that neurological symptoms can be reversed, but leaves us without clues as to the relationship between them. If protein aggregates really play an important role in generating symptoms in the patients, these ﬁndings are very important as they suggest that therapy could be applied to HD even after the development of signiﬁcant cell pathology.

IV. Genetics

HD is caused by an expanded, unstable CAG trinucleotide repeat in a gene of unknown function (Huntington’s Disease Collaborative Research Group, 1993). The CAG repeat is located within the coding sequence,

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17 codons downstream of the initiator ATG in exon 1 of the 67 exon gene (Ambrose et al., 1994). It is translated and transcribed with the rest of the gene and constitues part of a protein named huntingtin (Huntington’s Disease Collaborative Research Group, 1993). In normal subjects, the gene contains about 9–34 CAG repeats (Snell et al., 1993), while the presence of 37 or more CAG repeats may give rise to HD (for review, see Gusella and MacDonald, 2000). There is an inverse correlation between the number of CAG repeats and the age of onset (Gusella and MacDonald, 2000). A repeat length of 40–50 is most frequently seen when the onset of the disease occurs in mid life, while longer repeats, usually above 70, cause juvenileonset HD. Genetic polymorphisms adjacent to the CAG repeat have also been suggested to inﬂuence the age of onset in HD patients (Vuillaume, 1998). Huntington’s disease is inherited with genetic anticipation, which is only seen in paternal transmission and is due to repeat instability during spermatogenesis (Ranen et al., 1995). Furthermore, there is a certain degree of tissue mosaicism in terms of CAG repeat numbers in HD patients (Aronin et al., 1995; DeRooij et al., 1995), with the greatest variance of repeat numbers typically found in neurons and sperm (Telenius et al., 1994; Kennedy and Shelbourne, 2000; Ishiguro et al., 2001). In the brain, the regions most affected in HD, cortex and striatum, also display the largest mosaicism, whereas cerebellum, which is relatively spared, has the lowest level of mosaicism (Telenius et al., 1994). Notably, HD transgenic mice exhibit age-dependent repeat expansions in the striatum (up to threefold) (Kennedy and Shelbourne, 2000; Mangiarini et al., 1997).

V. Other Triplet Repeat Diseases

Several other genetic diseases exhibit expanded CAG repeats in coding parts of the genome. They include spinobulbar muscular atrophy (SBMA), dentato-rubral pallido-luysian atrophy (DRPLA), and spinocerebellar ataxias (SCA) 1, 2, 3, 6, and 7. (Gusella and MacDonald, 2000; Zoghbi and Orr, 2000). Also, a CAG repeat expansion in the TATA box binding protein (TBP) has recently been found to cause SCA 17 (Fujigasaki et al., 2001; Margolis et al., 2001; Nakamura et al., 2001). These disorders result in a selective loss of neurons, but with a different anatomical distribution in each disease (Gusella and MacDonald, 2000). It is possible that they are due to a similar polyglutamine-dependent gain of function in the proteins involved, with neuronal intranuclear inclusions as part of the common pathogenic mechanism (Gusella and MacDonald, 2000; Zoghbi and Orr, 2000). Recently, one

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pedigree exhibiting Huntington-like phenotype, but no HD mutation, was found to display a novel expansion of an octapeptide repeat in the prion protein (Moore et al., 2001). Consequently, it appears that a mutation in another protein that has a tendency to misfold can cause a neuropathology analogous to HD. This suggests that advances made in understanding the pathogenesis of any of the slowly progressing neurodegenerative diseases could provide valuable information also about the others.

VI. Intracellular Localization of Normal and Mutant Huntingtin

The messenger RNA (mRNA) of the HD gene is normally expressed in all tissues of the body (Hoogeveen et al., 1993; Strong et al., 1993; Trottier et al., 1994), and the levels of both mRNA (Strong et al., 1993) and protein (Bhide et al., 1996; Gourﬁnkel-An et al., 1997) are not changed in HD. In the brain, huntingtin expression is mainly neuronal, with only low levels in glia (Li et al., 1993; Sharp et al., 1995). Huntingtin is a protein with around 3140 amino acids, depending on the number of CAG repeats, and with a mass of approximately 349 kDa (Huntington’s Disease Collaborative Research Group, 1993). Normal huntingtin is found in the cytoplasm, and mutant huntingtin is localized both to the cytoplasm and to the nucleus (DiFiglia et al., 1995; Sharp et al., 1995; Trottier et al., 1995). In HD patients, however, normal huntingtin can also be found in the nucleus (Hoogeveen et al., 1993). Recently, it was shown that huntingtin present in the nuclear aggregates consists of N-terminal fragments of cleaved wild-type huntingtin that have been recruited by the full-length mutant protein (Dyer and McMurray 2001). Huntingtin is found in vesicle-enriched fractions, and it overlaps with the distribution of synaptosomal membrane proteins (DiFiglia et al., 1995). In some HD cortical and striatal neurons, huntingtin is also associated with cytoplasmic granules and multivesicular bodies, which are involved in retrograde transport and protein degradation (Sapp et al., 1999). Moreover, huntingtin associates with microtubules (Gutekunst et al., 1995; Tukamoto et al., 1997), indicating that it may have a function in vesicle trafﬁcking. Further support for this concept is provided by ﬁndings showing that both mutant and normal huntingtin associate with clathrin-coated vesicles in the Golgi network, the cytoplasm, and the plasma membrane (Velier et al., 1998). Shorter fragments of huntingtin are translocated to the nucleus more efﬁciently, whereas longer fragments are believed to form aggregates in the cytoplasm (Lunkes et al., 1997). It is thought that full-length huntingtin is proteolytically cleaved by proteases such as caspases before translocating

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into the nucleus (Wellington et al., 2000) and that nuclear localization of mutant huntingtin leads to increased toxicity (Saudou et al., 1998; Kim et al., 1999; Peters et al., 1999). Previously it was thought that the cleavage of mutant huntingtin, to generate N-terminal fragments that could enter the nucleus, was a crucial pathogenic event (Huang et al., 1998; Wheeler et al., 2000). However, as mentioned above, recent ﬁndings suggest that the mutation actually prevents the molecule from being cleaved by caspases. Instead, full-length mutant huntingtin enters the nucleus and has been shown to recruit N-terminal fragments of cleaved normal huntingtin into aggregates (Dyer and McMurray, 2001). This opens up the possibility that the disease is due to a “loss of function.” Indeed, Cattaneo and co-workers have proposed that the HD disease mechanism involves a combination of toxicity due to the polyglutamine stretch and a loss of function of wild-type huntingtin transcribed from the normal allele (Cattaneo et al., 2001). Ubiquitin, heat shock proteins and several functional subunits of the proteosome have also been found to co-localize with polyglutamine inclusions (Davies et al., 1997; Cummings et al., 1998; Waelter et al., 2001). In addition, transcription factors are associated with huntingtin in the aggregates

FIG. 1. Intracellular handling of mutant huntingtin and its possible contributions to mechanisms of cell dysfunction and death in HD.

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(Cha, 2000). Moreover, two synaptic proteins, complexin II and α-synuclein are recruited into huntingtin inclusions in HD brains and mouse models of the disease (Mezey et al., 2000; Morton and Edwardsson, 2001). Chaperones are involved in the facilitation of protein folding, especially important under conditions of cellular stress. The two major systems for degradation of cellular contents are autophagy and proteolysis by the ubiquitin/proteasome complex (Klinosky and Emr, 2000). Autophagy involves sequestration and proteolytic degradation of intracellular components in organelles surrounded by double membranes that eventually deliver their contents to lysosomes. Misfolding and altered solubility of proteins are involved in polyglutamine diseases, and the presence of chaperones and ubiquitin in the aggregates indicates that the cells may be trying to degrade mutant huntingtin (Fig. 1). Interestingly, a recent report shows that huntingtin aggregation impairs the function of the ubiquitin–proteasome system, providing further evidence for the role of huntingtin aggregation in the pathogenesis in HD (Bence et al., 2001).

VII. Function of Normal and Mutant Huntingtin

The function of wild-type huntingtin is not known. Targeting disruptions in exon 5 (Nasir et al., 1995), exon 4 (Duyao et al., 1995), and the promoter region (Zeitlin et al., 1995) have shown that the protein is functionally indispensable as nullizygous embryos are developmentally retarded and die in utero. Indeed, recent studies have suggested that loss of the normal function of huntingtin may contribute to cell death in HD. For instance, it has been shown that the absence of wild-type huntingtin decreases the survival of striatal neurons in vitro and in vivo (Dragatsis et al., 2000; Rigamonti et al., 2000), indicating that huntingtin has an anti-apoptotic function. As mutant huntingtin recruits normal huntingtin into aggregates (Huang et al., 1998; Kazantzev et al., 1999; Wheeler et al., 2000; Dyer and McMurray, 2001), it is possible, as mentioned above, that mutant huntingtin kills cells by sequestrating wild-type huntingtin and disrupting cell function. As mentioned previously, huntingtin could normally be involved in intracellular trafﬁcking and play a role in vesicular transport (DiFiglia et al., 1995; Block-Galarza et al., 1997; Velier et al., 1998). A role for wild-type huntingtin in transport and processing of mRNA has also been suggested (Cattaneo et al., 2001). Polyglutamine sequences can link β-strands in proteins into barrels or sheets by hydrogen bonding, forming so-called polar zipper structures (Perutz et al., 1994). β-Sheets formed by the polyglutamine stretch may

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cause huntingtin to aggregate and thereby interfere with vital cellular processes. The presumed normal function of polar zippers is to join speciﬁc transcription factors bound to separate DNA sequences. Transcriptional dysregulation has recently been suggested as an important pathogenetic mechanism in HD (Cha, 2000). Indeed, expanded polyglutamine stretches in transcription factors exhibit reduced ability to mediate transcription (Gerber et al., 1994). Transcription factors that normally contain a polyglutamine stretch include TBP (a transcriptional activator), cyclic AMP response element binding protein (CREB)-binding protein (CBP) (a transcriptional co-activator with histone acetylase function), and nuclear receptor co-activator (ACTR) (a histone acetyltransferase and transcriptional coactivator) (Cha et al., 2000). If huntingtin normally functions as a transcription factor, its activity may be reduced when the number of glutamines exceeds 37. There is normally a balance between the opposing forces of acetyltransferase enzymes that modify proteins to increase genetic activity and histone deacetylase (HDAC) enzymes that reverse these modiﬁcations to reduce gene activity. When mutant huntingtin binds to the acetyltransferase domain of CBP, the function of the transcriptional co-activator is inhibited, which leads to reduced amounts of acetylated histones (Steffan et al., 2001). Administration of HDAC inhibitors restores the reduction in acetylation caused by mutant huntingtin, and blocks polyglutamine-induced neuronal degeneration in Drosophila HD models. This ﬁnding suggests that an altered level of histone acetylation activity is an important, if not crucial, factor in HD pathogenesis (Steffan et al., 2001). Moreover, CBP is recruited into huntingtin aggregates in both transgenic mice and HD patients, leading both to inhibition of CBP-mediated transcription and to toxicity in HD transfected neuroblastoma cells (Nucifora et al., 2001). Moreover, by overexpressing CBP, polyglutamine-induced toxicity is reduced (Nucifora et al., 2001). Interestingly, genes that are regulated by CBP, such as BDNF and enkephalin, are downregulated in HD transgenic mice and patients (Ferrer et al., 2000; Luthi-Carter et al., 2000; Menalled et al., 2000; Zuccato et al., 2001). Several proteins interact with huntingtin (Fig. 1). However, the role of modiﬁed protein–protein interactions in HD pathogenesis is not clear. The proteins huntingtin associated protein 1 (HAP1) (Li et al., 1995), huntingtin interacting protein (HIP1) (Kalchman et al., 1997; Wanker et al., 1997), and SH3-containing Grb2-like protein (SH3GL3) (Sittler et al., 1998) are associated with membrane vesicles, and they interact with the cytoskeleton, indicating a role for huntingtin in vesicle trafﬁcking. The HIP2 is a ubiquitin-conjugating enzyme, linking huntingtin to the ubiquitin system (Kalchman et al., 1996). Calmodulin is a Ca2+-binding regulatory protein that binds more avidly to mutant huntingtin than to wild-type huntingtin

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(Bao et al., 1996). Glyceraldehyde dehydrogenase phosphatase also binds more strongly to mutant huntingtin, and is a protein with multiple functions in glycolysis, transcription and endocytosis (Burke et al., 1996). We hope that modern proteomics will shed more light on the effects of pathological protein–protein interactions in HD.

VIII. Cell Death in Huntington’s Disease: Apoptosis and Autophagy

Several forms of cell death exist including apoptosis, necrosis, autophagy, and intermediate forms, such as parapoptosis (Leist and J¨aa¨ ttel¨a, 2001; Roy and Spolsky, 1999). It is not clear which type of cell death predominates in HD. Parallel death pathways exist, and factors such as cellular energy status and relative activity of speciﬁc proteases (caspases, calpains, cathepsins, etc.) may determine the road to death (see Leist and J¨attel¨a, 2001). Some studies have shown that a subset of neurons and glia in the neostriatum of HD patients appears to undergo apoptosis (Dragunow et al., 1995; Portera-Cailliau et al., 1995; Thomas et al., 1995). However, deﬁnite morphological evidence for an apoptotic process in HD is still missing, and as apoptosis is executed rapidly over hours to days, it is difﬁcult to ﬁnd a sufﬁcient number of cells showing apoptotic features at any single timepoint analyzed in the brain. There are several interesting interactions between huntingtin and proteases involved in apoptosis, i.e., caspases. Caspase activation occurs in HD brains (Ona et al., 1999) and huntingtin is a substrate for caspase-3 (Goldberg et al., 1996). Moreover, in cell culture models, a mutation in the caspase-3 cleavage site in huntingtin reduces toxicity (Wellington et al., 2000) and caspase-8 activation is triggered by polyglutamine aggregation (Sanchez et al., 1999). Wild-type huntingtin can inhibit caspase-9 (Rigamonti et al., 2001). Also, the life span of R6/2 transgenic HD mice is prolonged by intraventricular infusion of a caspase inhibitor or by crossing HD mice with caspase-1 dominant-negative mice (Ona et al., 1999). Interestingly, huntingtin interacts with p53, a regulator of apoptosis (Steffan et al., 2000). Autophagy involves the bulk sequestration and proteolytic degradation of intracellular components. It is induced by pathological conditions such as fasting, metabolic inhibition, hypoxia, and ischemia (Klinosky and Emr, 2000; Stromhaug and Klionsky, 2001) and is activated in sympathetic neurons exposed to apoptotic stimuli (Xue et al., 1999). Brains of HD patients exhibit an increased presence of endosomal/lysosomal organelles and multivesicular bodies (organelles associated with autophagy), and accumulation of lipofuscin (Tellez-Nagel, 1975; Roizin et al., 1979; Sapp et al., 1999). In cell culture models of HD, there

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is an increased number of vacuoles, displaying features of autophagosomes and in some cases containing huntingtin (Kegel et al., 2000; Peters´en et al., 2001b). Both autophagy and apoptosis are induced by the opening of the mitochondrial permeability transition pore (Lemasters et al., 1998), caspase activation, and cytochrome c release (Xue et al., 1999). Possibly, autophagy shares the same inductive phase as apoptosis, but it has a separate execution phase, as it persists even after caspase inhibition (Xue et al., 1999). In conclusion, autophagy may be an important process leading to dendritic pruning and neuronal atrophy in HD.

IX. Oxidative Stress and Metabolic Dysfunction

Oxidative stress has been suggested to occur in brains of HD patients (Coyle and Puttfarcken, 1993; Browne et al., 1999). It can be the consequence of mitochondrial malfunction (Beal et al., 1995) or excitotoxicity (Coyle and Puttfarcken, 1993; Lafon-Cazal et al., 1993). In the HD striatum, increased levels of markers of oxidative stress, e.g., nucleotide 8-hydroxydeoxyguanosine (Browne et al., 1997) and lipofuscin (Browne et al., 1999), have been found. Mitochondrial aconitase activity (the inactivation of which is an indicator of oxidative stress) is greatly reduced in the striatum of HD patients (Tabrizi et al., 1999). However, recently Alam et al. (2000) found no evidence for oxidative damage to lipids, proteins, or DNA in the cortex or striatum of HD patients. A number of studies indicate that the striatum is selectively sensitive to metabolic dysfunction. Alterations in mitochondrial enzymes (Gu et al., 1996; Browne et al., 1997) indicate that defects in energy metabolism could play a role in HD pathogenesis. Further support is provided by reports on damage to basal ganglia following attempted suicide by inhalation of cyanide (Uitti et al., 1985) or carbon monoxide (Klawans and Winer, 1974), both of which inhibit mitochondrial complex IV. Moreover, striatal damage was found in humans that consumed mildewy sugarcane contaminated with 3-nitroproprionic acid (3-NP) (Ludolph et al., 1991). The toxin 3-NP irreversibly inhibits succinate dehydrogenase. Systemic injections of 3-NP into rats and nonhuman primates lead to striatal neurodegeneration (Greene and Greenamyre, 1995; Palﬁ et al., 1996). Intrastriatal administration of 3-NP or malonate (a reversible succinate dehydrogenase inhibitor) causes dose-dependent ATP depletion, increased lactate concentration, and striatal neuronal loss (Beal et al., 1993a,b; Greene and Greenamyre, 1995; Nakao and Brundin, 1997). Energy metabolic changes have been observed in the brains and cerebrospinal ﬂuid of HD patients (Young et al., 1986; Kuwert et al., 1990; Jenkins et al., 1993). A distinct reduction in the activity of

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respiratory chain complexes II and III, and a mild change in the complex IV activity in mitochondria of caudate neurons, but not in platelets, have been observed in HD patients (Gu et al., 1996). A recent study by Guidetti et al. (2001) showed no changes in the activity of mitochondrial complexes I–IV in the brains of presymptomatic and early stage HD patients, suggesting that mitochondrial changes are late events in the disease.

X. Dopamine Toxicity

Changes in the nigrostriatal dopamine (DA) system have been suggested to play an important role in the striatal neurodegeneration observed in HD (for review, see Jakel and Maragos, 2000). Levels of DA D1 and D2 receptors, both of which regulate gene expression, are decreased in early stages of HD in both humans and transgenic mice (Turjanski et al., 1995; Augood et al., 1997; Ginovart et al., 1997; Cha et al., 1998, 1999; Backman and Farde, 2001). Moreover, DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of a molecular weight of 32 kDa), a component of the DA signaling cascade essential for the regulation of striatal physiology (Greengard et al., 1999), is also reduced in HD mice (Bibb et al., 2000; van Dellen et al., 2000). A nigrostriatal pathology has been suggested in HD (Bohnen et al., 2000). Indeed, several studies show that nigrostriatal projections degenerate in HD brains based on TH immunohistochemistry (Ferrante and Kowall, 1987), measurements of vesicular transporters (Bohnen et al., 2000; Suzuki et al., 2001), and loss of DA transporter binding assessed by positron emission tomography (Ginovart et al., 1997). Atrophy of neurons in the substantia nigra has also been reported in brains from HD patients (Oyanagi et al., 1989), and aggregates of mutant huntingtin have been found in the substantia nigra pars compacta (Gutekunst et al., 1999). Importantly, DA is well established to be toxic to striatal neurons both in vivo and in vitro (Filloux and Townsend, 1993; Hastings et al., 1996; Hattori et al., 1998; McLaughlin et al., 1998; Peters´en et al., 2001a,b). Normally, DA is metabolized via monoamino oxidase into 3, 4-dihydrophenylacetic acid (DOPAC) and H2O2. However, DA can autooxidize nonenzymatically to produce H2O2 and DA quinones, or superoxide (O·2− ) and a DA semiquinone. By further reaction, hydroxyl radicals can be formed from H2O2 or superoxide through interaction with transition metal ions (for review, see Sulzer and Zecca, 2000). Quinones can react covalently with cysteinyl residues on proteins, and cause oxidative stress via the above mentioned mechanisms (Sulzer and Zecca, 2000). Dopamine and its metabolites may be well tolerated under normal circumstances in the striatum, but could potentiate cell

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death in HD neurons with impaired energy metabolism. Evidence in support of this theory comes from studies where DA exacerbates, or DA depletion protects from, striatal toxicity induced by mitochondrial toxins (Maragos et al., 1998; McLaughlin et al., 1998; Reynolds et al., 1998). Dopamine has been shown to inactivate mitochondrial complex I of the mitochondrial respiratory chain (Ben-Shachar et al., 1995), although these ﬁndings are controversial (Morikowa et al., 1996).

XI. Transgenic Mouse Models of Huntington’s Disease

The ﬁrst lines of transgenic HD mice (R6 lines) were published in 1996 (Mangiarini et al., 1996). Several other lines of HD mice have been generated since then (Reddy et al., 1998; Schilling et al., 1999; Shelbourne et al., 1999; Hodgson et al., 1999; Wheeler et al., 2000; Yamamoto et al., 2000; Laforet et al., 2001). In all these mice the presence of an expanded CAG repeat causes a progressive neurological phenotype, but the exact nature of the neurologic deﬁcits and the age of onset vary widely. The mice differ with regard to, e.g., the size of huntingtin fragment in the transgene, the number of CAG repeats, the promotor driving the trasngene, and the background strain. The R6 lines overexpress exon 1 of the human HD gene under its endogenous promoter. The transgene contains an expanded CAG repeat of around 117 (R6/1 line) or 150 (R6/2 line) repeats. The R6 lines exhibit decreased striatal and total brain size (Mangiarini et al., 1996), and ubiquitinated nuclear and cytoplasmic inclusion bodies containing both mutant and wild-type huntingtin in striatal cells (Davies et al., 1997; Li et al., 1999). There are changes in neurotransmission, including altered neurotransmitter receptor levels (Cha et al., 1998, 1999), changes in striatal amino acid and DA release (Nicniocaill et al., 2001; and own unpublished observations), decreased expression of striatal signaling genes (Luthi-Carter et al., 2000), deﬁciencies in DA signaling (Bibb et al., 2000), and a progressive loss of the synaptic protein complexin II (Morton and Edwardson, 2001). Reduced levels of DA have been reported in postmortem striatal tissue of 12-week-old R6/2 mice (Reynolds et al., 1999; Fig. 2). Mitochondrial changes and evidence for oxidative stress have been found in the striatum (Tabrizi et al., 2000). The R6 mice exhibit only very limited neuronal death at a very late stage, occurring at around 14–16 weeks of age in R6/2 mice, and it has been described as dark cell degeneration (Iannicola et al., 2000; Turmaine et al., 2000). On the other hand, motor and cognitive dysfunction already start at around 4 weeks of age in R6/2 mice (Carter et al., 1999; Lione et al., 1999; Murphy et al., 2000), indicating that neuronal dysfunction occurs

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FIG. 2. Time schedule depicting sequence of events in the development of the abnormal phenotypes in R6/1 and R6/2 transgenic HD mice. Note that R6/2 mice, which carry more CAG repeats (150) than R6/1 mice (117), also exhibit an earlier onset of the different features of the HD phenotype.

before neuronal loss. This also seems to be the case in HD patients, as motor (Myers et al., 1988) and cognitive deﬁcits (Lawrence et al., 1998) occur prior to any detectable neurodegeneration. Hence, the appearance of neuronal dysfunction seems to precede cell death in HD. In view of the arguments that exicitotoxicity, mitochondrial dysfunction and/or DA toxicity play a role in the pathogenesis of HD ( Jakel and Margos 2000), it is important to consider whether these mechanisms are active in the brains of R6 mice. However, counterintuitively the brains of transgenic HD mice of the R6 lines have so far been found to be resistant to a number of toxic insults, including intrastriatal injections of quinolinic acid (Hansson et al., 1999), DA (Peters´en et al., 2001a), malonate (Hansson et al., 2001a), N-methyl-D-aspartate (NMDA) (Hansson et al., 2001b), and systemic injections of 3-NP (Hickey and Morton, 2000) and kainate (Morton and Laevens, 2000). This resistance to brain insults develops with age and occurs more rapidly in R6/2 than R6/1 mice (Fig. 2). This suggests that the resistance mechanism is related to a prolonged exposure to some form of polyglutamine toxicity. So far, the mechanism is not known, but it may be related to an observed increase in levels of basal cytoplasmic calcium in R6/2 striatal neurons, and an enhanced ability to handle further increases in cytoplasmic calcium (Hansson et al., 2001b). In addition, extracellular levels of striatal DA are reduced by 70% in R6/1 mice. As quinolinate-, DA-, and malonateinduced lesions are partially dependent upon endogenous striatal DA levels

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(Chapman et al., 1989; Filloux and Townsend, 1993; Ferger et al., 1998), this reduction in DA release may partly explain the resistance phenomenon. However, the resistance to intrastriatal injections of quinolinic acid has not been found in other transgenic HD mouse models. Interestingly, a transgenic HD mouse with a yeast artiﬁcal chromosome carrying the full-length HD gene with 72 CAG repeats displays increased sensitivity to quinolinate (Zeron et al., 2002), whereas a transgenic HD mouse with a 3 kilobase portion of the human HD gene exhibits normal sensitivity (Peters´en et al., 2002).

XII. Concluding Remarks

Basic research into HD has stimulated novel clinical drug trials based on the ideas about mechanisms of cell death in the disorder. A recent study investigated the effects of remacemide and coenzyme Q on progression of symptoms in HD (The Huntington’s Disease Study Group, 2001). These drugs are an NMDA antagonist and a mitochondrial electron transfer co-factor, respectively, and were chosen based on the theories that excitotoxicity and energy impairment play roles in the disease. Unfortunately, neither drug was effective, but the trial represents the opening of a new era of trials based on rational theories derived from experimental work in cultures and animals. Despite several important ﬁndings regarding the pathogenesis of HD, several fundamental issues remain to be resolved. For example, what mechanisms underlie the correlation between CAG repeat length and age of onset, and what other factors inﬂuence the appearance of symptoms? How and why is mutant huntingtin toxic, or does the disease actually involve a loss of function of huntingtin? Why are primarily striatal, and to some extent, cortical, neurons selectively affected? Why does it typically take 30–50 years or more for mutant huntingtin to produce symptoms? We hope the coming years will bring answers to these questions and unfold the mystery of HD.

Acknowledgments

We acknowledge the contribution of valuable comments from Zoe Puschban. Our own work described in this review was supported by grants from the Hereditary Disease Foundation, Swedish Medical Research Council, the Kock, Crafoord and S¨oderberg Foundations, the Swedish Association of the Neurologically Disabled, Swedish Society of Medical Research, and Anders Wall Foundation.

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Tabrizi, S. J., Workman, J., Hart, P. E., Mangiarini, L., Mahal, A., Bates, G., Cooper, J. M., and Schapira, A. H. (2000). Mitochondrial dysfunction and free radical damage in the Huntington R6/2 transgenic mouse. Ann. Neurol. 47, 80–86. Telenius, H., Kremer, B., Goldberg, Y. P., Theilmann, J., Andrew, S. E., Zeisler, J., Adam, S., Greenberg, C., Ives, E. J., Clarke, L. A., and Hayden, M. R. (1994). Somatic and gonadal mosaicism of the Huntington’s disease gene CAG repeat in brain and sperm. Nat. Genet. 6, 409–414. Tellez-Nagel, I., Johnson, A. B., and Terry, R. D. (1975). Studies on brain biopsies of patients with Huntington’s chorea. J. Neuropathol. Exp. Neurol. 33, 308–332. Thomas, L. B., Gates, D. J., Ritchﬁeld, E. K., O’Brien, T. F., Schweitzer, J. B., and Steindler, D. A. (1995). DNA end labeling (Tunel) in Huntington’s disease and other neuropathological conditions. Exp. Neurol. 133, 265–272. Trottier, Y., Biancalana, V., and Mandel, J.-L. (1994). Instability of CAG repeats in Huntington’s disease: Relation to parental transmission and age of onset. J. Med. Genet. 31, 377–382. Trottier, Y., Devys, D., Imbert, G., Sandou, F., An, I., Lutz, Y., Weber, C., Agid, Y., Hirsch, E. C., and Mandel, J.-L. (1995). Cellular localization of the Huntington’s disease protein and discrimination of the normal and mutated form. Nat. Genet. 10, 104–110. Tukamoto, T., Nukina, N., Ide, K., and Kanazawa, I. (1997). Huntington’s disease gene product, huntingtin, asssociates with microtubules in vitro. Mol. Brain Res. 51, 8–14. Turjanski, N., Weeks, R., Dolan, R., Harding, A. E., and Brooks, D. J. (1995). Striatal D1 and D2 receptor binding in patients with Huntington’s disease and other choreas. A PET study. Brain 118, 689–696. Turmaine, M., Raza, A., Mahal, A., Mangiarini, L., Bates, G. P., and Davies, S. W. (2000). Nonapoptotic neurodegeneration in a transgenic mouse model of Huntington’s disease. Proc. Natl. Acad. Sci. USA 97, 8093–8097. Uitti, R. J., Rajput, A. H., Ashenhurst, E. M., and Rozdilsky, B. (1985). Cyanide-induced parkinsonism: a clinicopathologic report. Neurology 35, 921–925. van Dellen, A., Welch, J., Dixon, R. M., Cordery, P., York, D., Styles, P., Blakemore, C., and Hannan, A. J. (2000). N-acetylaspartate and DARPP-32 levels decrease in the corpus striatum of Huntington’s disease mice. Neuroreport 11, 3751–3757. Velier, J., Kim, M., Schwarz, C., Kim, T. W., Sapp, E., Chase, K., Aronin, N., and DiFiglia, M. (1998). Wild-type and mutant huntingtins function in vesicle trafﬁcking in the secretory and endocytic pathways. Exp. Neurol. 152, 34–40. Vonsattel, J. P., and DiFiglia, M. (1998). Huntington disease. J. Neuropathol. Exp. Neurol. 57, 369–384. Vuillaume, I., Vermersch, P., Destee, A., Petit, H., and Salbonniere, B. (1998). Genetic polymorphisms adjacent to the CAG repeat inﬂuence clinical features at onset in Huntington’s disease. J. Neurol. Neurosurg. Psychiat. 64, 758–762. Waelter, S., Boeddrich, A., Lurz, R., Scherzinger, E., Lueder, G., Lehrach, H., and Wanker, E. E. (2001). Accumulation of mutant huntingtin fragments in aggresome-like inclusion bodies as a result of insufﬁcient protein degradation. Mol. Biol. Cell. 12, 1393–1407. Wanker, E. E., Rovira, C., Scherzinger, E., Hasenbank, R., W¨alter, S., Tait, D., Colicelli, J., and Lehrach, H. (1997). HIP-1: A huntingtin interacting protein isolated by the yeast two-hybrid system. Hum. Mol. Genet. 6, 487–495. Wellington, C. L., Singaraja, R., Ellerby, L., Savill, J., Roy, S., Leavitt, B., Cattaneo, E., Hackam, A., Sharp, A., Thornberry, N., Nicholson, D. W., Bredesen, D. E., and Hayden, M. R. (2000). Inhibiting caspase cleavage of huntingtin reduces toxicity and aggregate formation in neuronal and nonneuronal cells. J. Biol. Chem. 275, 19831–19838. Wheeler, V. C., White, J. K., Gutekunst, C. A., Vrbanac, V., Weaver, M., Li, X. J., Li, S. H., Yi, H., Vonsattel, J. P., Gusella, J. F., Hersch, S., Auerbach, W., Joyner, A. L., and MacDonald, M. E. (2000). Long glutamine tracts cause nuclear localization of a novel form of huntingtin in

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medium spiny neurons in HdhQ92 and HdhQ111 knock-in mice. Hum. Mol. Genet. 9, 503–513. Xue, L., Fletcher, G. C., and Tolkovsky, A. M. (1999). Autophagy is activated by apoptotic signalling in sympathetic neurons: an alternative mechanism of death execution. Mol. Cell Neurosci. 14, 180–198. Yamamoto, A., Lucas, J. J., and Hen, R. (2000). Reversal of neuropathology and motor dysfunction in a conditional model of Huntington’s disease. Cell 101, 57–66. Young, A. B., Penney, J. B., Starosta-Rubinstein, S., Markel, D. S., Berent, S., Giordani, B., Ehrenkaufer, R., Jewett, D., and Hichwa, R. (1986). PET scan investigations of Huntington’s disease: Cerebral metabolic correlates of neurological features and functional decline. Ann. Neurol. 20, 296–303. Zeitlin, S., Liu, J.-P., Chapman, D. L., Papaioannou, V. E., and Efstratiadis, A. (1995). Increased apoptosis and early embryonic lethality in mice nullizygous for the Huntington’s disease gene homologue. Nat. Genet. 11, 155–163. Zeron, M. M., Hansson, O., Chen, N., Wellington, C. L., Leavitt, B. R., Roy, S., Brundin, P., Hayden, M. R., and Raymond, L. A. (2002). Increased sensitivity to N-methyl-D-aspartate receptor-mediated excitotoxicity in a mouse model of Huntington’s disease. Neuron 33, 849–860. Zuccato, C., Ciammola, A., Rigamonti, D., Leavitt, B. R., Goffredo, D., Conti, L., MacDonald, M. E., Friedlander, R. M., Silani, V., Hayden, M. R., Timmusk, T., Sipione, S., and Cattaneo, E. (2001). Loss of huntingtin-mediated BDNF gene transcription in Huntington’s disease. Science 293, 493–498. Zoghbi, H., and Orr, H. T. (2000). Glutamine repeats and neurodegeneration. Ann. Rev. Neurosci. 23, 217–247.

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MITOCHONDRIA IN ALZHEIMER’S DISEASE

Russell H. Swerdlow Department of Neurology University of Virginia Charlottesville, Virginia 22908

Stephen J. Kish Human Neurochemical Pathology Laboratory Centre for Addiction and Mental Health Toronto, Ontario, Canada

I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV.

Introduction Historical Overview and the Amyloid Cascade Hypothesis Metabolic Dysfunction in Alzheimer’s Disease has been Reported Morphological Studies Demonstrate Mitochondrial Abnormalities in Alzheimer’s Disease PDHC and KGDHC in Alzheimer’s Disease Brain Biochemical Studies of Mitochondrial Enzymes in Alzheimer’s Disease: Is Cytochrome Oxidase Reduction Characteristic of Alzheimer’s Disease? Is a Brain Cytochrome Oxidase Deﬁciency a Robust Feature of Alzheimer’s Disease? Studies of Cytochrome Oxidase in Non-CNS Tissues in Alzheimer’s Disease: Clues to the Origin of the Enzyme Change? Cytochrome Oxidase Dysfunction in Alzheimer’s Disease: Possible Genetic Component? Cytochrome Oxidase Dysfunction in Alzheimer’s Disease: Genetic Studies are Still Inconclusive Cybrid Data Suggest mtDNA Contributes to Alzheimer’s Disease Cytochrome Oxidase Dysfunction Unresolved Issues in Alzheimer’s Disease Cybrid Studies: Where is the mtDNA “Mutation?” Could a Cytochrome Oxidase Defect Cause Alzheimer’s Disease? Concluding Remarks References

The cause of Alzheimer’s disease (AD) is not known but might involve mitochondrial dysfunction. The objective of this review is to establish the strength of the evidence in the scientiﬁc literature that mitochondrial abnormalities are characteristic of AD and might be etiologically involved in the brain degenerative process. This review has been prepared by two INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 53

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Copyright 2002, Elsevier Science (USA). All rights reserved. 0074-7742/02 $35.00

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investigators, both working in the area of mitochondria and AD, who provide differing conclusions regarding the extent of involvement of mitochondrial dysfunction in AD.

I. Introduction

Alzheimer’s disease (AD) is the most common neurodegenerative disorder in the human. It is characterized clinically by a progressive failure of memory and other cognitive systems. Histologically, AD is associated with excessive cerebral cortical accumulation of protein aggregations, senile plaques, and neuroﬁbrillary tangles. It is unknown whether plaques and tangles cause AD neurodegeneration or are a secondary consequence of more primary pathophysiology. Abundant data, from multiple investigators evaluating a myriad of endpoints through a variety of techniques, also report that mitochondria are abnormal, at least in some subjects, in AD. As with plaques and tangles, the location in which mitochondrial dysfunction sits on the causalconsequence continuum is unproven. The objective of this chapter is to establish the strength of the evidence that mitochondrial abnormalities are (1) characteristic of AD; and (2) etiologically involved in the neurodegenerative process. Data detailing AD mitochondrial pathology are described, especially those involving the enzyme cytochrome oxidase, as most mitochondrial studies in AD have involved investigation of this enzyme. This review has been prepared by two investigators considered in the literature to be supportive (RS; Swerdlow et al., 1997; Swerdlow, 2002) and somewhat skeptical (SK; Kish, 1997; Kish et al., 1999) of the hypothesis that mitochondria play an important role in AD degeneration.

II. Historical Overview and the Amyloid Cascade Hypothesis

The patient Auguste D. was institutionalized at a University of Frankfurt asylum in 1902 with symptoms emerging early in her sixth decade, including delusions, paranoia, and memory failure. One of her physicians, Alois Alzheimer, histopathologically surveyed her brain upon her death in 1905. Alzheimer observed extracellular “plaques,” entities ﬁrst described in the late 19th century in persons with epilepsy (for discussion see Berrios, 1990), as well as intracellular “tangles,” which were independently described shortly before the publication of Alzheimer’s report on Auguste D. (see Berrios, 1990).

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Alzheimer was the ﬁrst to make the association between these protein aggregations and presenile dementia. In 1907 he published a report on Auguste D. (Alzheimer, 1907), and over the next several years described several other cases of tangle-and-plaque-associated presenile dementia (reviewed by Graeber et al., 1997). In 1910, his department chairman, Emil Kraepelin, published a medical textbook that referred to this tangle-plaque presenile dementia as “Alzheimer’s disease” (Kraepelin, 1910). The diagnosis of AD remained relatively uncommon until the 1970s. Recognizing that a large number of adults who demented late in life also had plaques and tangles at autopsy, the National Institute of Aging (NIA), founded in 1974, advocated expanding the deﬁnition to include all tangleand-plaque-associated degenerative dementias, regardless of age (reviewed in Katzman, 1986). Since life expectancies have continuously increased in western societies since the time of Alois Alzheimer, application of the expanded deﬁnition has made AD a disorder of aging. By the 1980s, the search for a pathogenic basis was well under way. Infectious and toxic causes were considered, but convincing evidence for these etiologies has not yet emerged. Meanwhile, investigators began to elucidate brain plaque biochemistry, which showed that plaques consisted primarily of an insoluble protein fragment, beta amyloid (Aβ) (Glenner and Wong, 1984). Aβ in turn was derived from the enzymatic degradation of the amyloid precursor protein (APP) (Kang et al., 1987). In 1987 it was shown by several independent groups that the APP gene was localized on chromosome 21 (Goldgaber et al., 1987; Kang et al., 1987; Robakis et al., 1987; Tanzi et al., 1987). This was of substantial interest, since it had long been recognized that trisomy 21 (Down syndrome) subjects invariably develop plaque pathology. In 1991, an APP gene mutation was demonstrated in a family with earlyonset, autosomal dominant, plaque-associated dementia (Goate et al., 1991). Since 1991, APP mutation has been observed in a very limited number of other early-onset (typically presenting before the age of 60), autosomal dominant AD kindreds. As patients with the APP mutations have the clinical and neuropathological characteristics of AD, these genetic ﬁndings have provided compelling support for the etiologic involvement of Aβ in at least one form of AD—i.e., an amyloid precursor protein abnormality is sufﬁcient for the expression of AD. In an attempt to extend the relevance of this ﬁnding to the remaining vast majority of those with late-onset, sporadic AD, Hardy and colleagues proposed the “amyloid cascade hypothesis” (Hardy and Allsop, 1991; Hardy and Higgins, 1992). It was subsequently shown in 1995 that mutation in two other genes is deterministic for an early-onset, autosomal dominant AD phenotype. Indeed, mutation of the presenilin 1 gene on chromosome 14 is responsible for the disease in up to 50% of affected kindreds (Sherrington et al., 1995). Mutation of a homologous gene, presenilin 2 (on chromosome 1), is far less

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common (Levy-Lahad et al., 1995). Data suggest that presenilin proteins might play a role in APP processing, which is taken by some as support for the amyloid cascade hypothesis (Mann et al., 1996; Hardy, 1997). The sum total of cases caused by mutations in each of these deterministic genes appears to account for less than 5% of AD (Lendon et al., 1997). To obtain animal model proof of concept for the amyloid cascade hypothesis, multiple transgenic mouse lines expressing human APP and presenilin mutations were developed. Some of these transgenics developed difﬁculty in a cognitive task (water maze test), which can be ameliorated by immunization against Aβ ( Janus et al., 2000; Morgan et al., 2000). However, some mutant APP transgenics develop plaques but not appropriate clinical phenotype changes (Hsiao et al., 1996). Although mutant APP transgenics develop plaques, only very limited neuronal loss is observed (Takeuchi et al., 2000). Phenotypic changes in transgenic mice may also arise in the absence of plaque pathology (Duff, 2001). Thus, there is proof of concept in humans that APP mutation can cause a rare subtype of early-onset, autosomal dominant AD via a currently unknown mechanism. However, experimental animal proof of concept is as yet lacking (it may never be possible to reproduce all brain characteristics of AD in a rodent). Furthermore, the cause of the common form of late-onset AD is clearly unknown. The extent of involvement of Aβ will be addressed by clinical studies testing the effects of treatments speciﬁcally designed to alter Aβ levels or disposition. The remainder of this chapter addresses the possibility, previously suggested, that mitochondrial dysfunction is a critically important feature of AD which might (or not) incorporate aspects of the amyloid cascade hypothesis in the etiology of this degenerative disorder (Parker et al., 1990; Blass and Gibson, 1991; Beal, 1992; Smith et al., 2002).

III. Metabolic Dysfunction in Alzheimer’s Disease has been Reported

Interestingly, Alois Alzheimer emphasized that he did not believe that plaques or tangles were etiologically important to AD neurodegeneration (cf. Davis and Chisholm, 1999). If this is correct, it becomes essential to deﬁne and describe a more fundamental pathogenesis that leads to neurodegeneration, dementia, and plaque/tangle pathology. One hypothesis that has long intrigued investigators is that AD ultimately arises from a celllevel metabolic failure, in particular, one affecting bioenergetics. Manifestations of this idea date back at least to the 1930s. Citing observations that low oxygen tension at high altitudes renders humans

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encephalopathic, Quastel proposed in 1932 that failure of oxidative metabolism could be the underlying cause of cognitive and psychiatric disorders. In the 1940s, Kety and Schmidt (1948) demonstrated that subjects of advanced age have diminished oxidative metabolism. Friede noted as early as 1965 that alterations of oxidative metabolism occurred in AD patient brains and further proposed that this phenomenon might precede and drive amyloid deposition in the brains of affected subjects. Bowen and colleagues reported in 1979 reduced brain activities for multiple enzymes involved in metabolic and energy metabolism-related processes in AD (Bowen et al., 1979). In the mid-1980s in vitro studies comparing AD and control brain biopsy tissue revealed differences in glucose metabolism (Sims et al., 1983; Sims et al., 1987). The 1980s also provided functional neuroimaging data that corroborated the presence of brain energy dysmetabolism in AD with the demonstration of reduced glucose metabolism on positron emission tomography (PET) (Ferris et al., 1980; Frackowiak et al., 1981; Foster et al., 1983; Friedland, 1983). PET changes in AD appear to precede brain atrophy (Haxby et al., 1986; Hoyer et al., 1988).

IV. Morphological Studies Demonstrate Mitochondrial Abnormalities in Alzheimer’s Disease

Surprisingly, only limited information is available on the actual morphometric analysis of brain mitochondria in AD. However, morphologically abnormal mitochondria have been reported to surround tangles and plaques in brain of patients with AD ( Johnson and Blum, 1970). Wisniewski and colleagues (1970) observed that mitochondrial distortion was an early histopathologic event in degenerating neurites of AD subjects. A recent morphological study of biopsied frontal/temporal cerebral cortex of AD patients reported that neuronal mitochondria were reduced in number on average by approximately 25%, whereas their average size was slightly increased (Hirai et al., 2001).

V. PDHC and KGDHC in Alzheimer’s Disease

Reports of distinct mitochondrial enzyme deﬁciencies date back several decades. In 1980, Perry and colleagues observed decreased activity of pyruvate dehydrogenase complex (PDHC) in postmortem AD brain, a

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ﬁnding also observed by other groups (Sorbi et al., 1983; Sheu et al., 1984; Butterworth and Besnard, 1990; Yates et al., 1990). Deﬁciency of another mitochondrially located, thiamine-dependent enzyme, α-ketoglutarate dehydrogenase complex (KGDHC) has been consistently reported in brain of patients with AD (Gibson et al., 1988; Butterworth and Besnard, 1990; Mastrogiacomo et al., 1993, 1996). However, it must be emphasized that activity of this enzyme is extremely variable in not only postmortem brain of patients with AD but also in control subjects, probably due to the very high susceptibility of this enzyme to inﬂuence by premortem agonal status factors (e.g., prolonged hypoxic death; see Gibson et al., 1988; Mastrogiacomo et al., 1993). Since the majority of AD enzyme values fall within the large range of the control subjects in a study of a large number of subjects (Mastrogiacomo et al., 1993), the results of these investigations are difﬁcult to interpret. In addition, it is now recognized that a brain KGDHC deﬁcit is not speciﬁc to AD, but is also observed in brain of patients with other neurodegenerative conditions (Parkinson disease, Wernicke-Korsakoff syndrome, spinocerebellar ataxia type I, and Friedriech ataxia) (Kish, 1997). Although this indicates that the enzyme reduction in AD may well be a nonspeciﬁc ﬁnding (to date, no defect in genes coding for KGDHC in AD have been conﬁrmed) an abnormality in this energymetabolizing enzyme, if present in living brain, is likely to compromise brain function.

VI. Brain Biochemical Studies of Mitochondrial Enzymes in Alzheimer’s Disease: Is Cytochrome Oxidase Reduction Characteristic of Alzheimer’s Disease?

In the 1980s, Blass and co-workers began formulating and addressing the crucial question of whether systemic metabolic dysfunction occurs outside the brain in AD subjects (Blass and Zemcov, 1984). Relative to control ﬁbroblast cultures, AD ﬁbroblast cultures metabolizing glucose overproduce lactate (Sims et al., 1985). In 1986, Peterson and Goldman reported diminished glucose and glutamine oxidation in AD ﬁbroblasts relative to those from age-matched controls. Cells from AD ﬁbroblast cultures also differed from control ﬁbroblast cells in bioenergetic utilization of glucose, glutamine, and oxygen (Sims et al., 1987), pointing toward a mitochondrial bioenergetic defect in this nonneural tissue. Interestingly, Blass also showed that toxic uncoupling of mitochondrial oxidative phosphorylation induced an exaggerated formation of phosphorylated tau and paired helical ﬁlament antigenic determinants in AD ﬁbroblasts as compared to those in control ﬁbroblasts (Blass et al., 1990).

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Additional information relevant to this issue came from the studies of Parker and colleagues in 1990. Parker had earlier proposed the hypothesis that because mitochondrial DNA (mtDNA) does not adhere to Mendelian genetic principles and only encodes components of the mitochondrial electron transport chain (ETC), non-Mendelian neurodegenerative disorders might exhibit systemic ETC dysfunction (Parker et al., 1989; Parker, 1990). Using this hypothesis as a guide, Parker had previously predicted and demonstrated that activity of the ETC enzyme NADH:ubiquinone oxidoreductase (complex I) was deﬁcient in platelets from patients with Parkinson disease (PD) (for review see Swerdlow, 2000). When Parker and colleagues (1990) evaluated ETC function in AD platelets, this group discovered a circumscribed deﬁciency in the activity of cytochrome oxidase (complex IV). Since this report, most biochemical studies of mitochondial enzymes in AD have focused on cytochrome oxidase. To establish whether the cytochrome oxidase reduction reported by Parker in blood of patients with AD is also present in brain, Kish and coworkers (1992) examined autopsied brain (cerebral cortex, putamen, hippocampus) from 19 patients with AD. Modest changes (–16 to –23%) were observed in the AD group that were statistically signiﬁcant in the frontal and temporal cortices, but with a marked overlap between the individual control and AD values. The general observation that in AD one can identify an abnormality of cytochrome oxidase enzyme activity or protein level both within and outside of brain was subsequently supported and extended (Simonian and Hyman, 1993; Parker et al., 1994a,b; Mutisya et al., 1994; Chagnon et al., 1995; Parker and Parks, 1995; Curti et al., 1997; Gonzalez-Lima et al., 1997; Wong-Riley et al., 1997; Cardoso et al., 1999; Kish et al., 1999; Nagy et al., 1999; Ojaimi et al., 1999; Maurer et al., 2000; Verwer et al., 2000; Cottrell et al., 2001; Hirai et al., 2001; Valla et al., 2001; Bosetti et al., 2002). However, not all groups evaluating cytochrome oxidase in AD detected an abnormality (van Zuylen et al., 1992; Cooper et al., 1993; Reichmann et al., 1993; Cavelier et al., 1995; Schagger and Ohm, 1995; Molina et al., 1997), and among the positive studies there is confusion regarding the brain areas (degenerating versus nondegenerating) which are involved. In principle, these discrepancies could be explained by true variation in brain enzyme differences among patients with AD and/or by differences in methodology for enzyme measurement. In this regard, when discussing the previously reported (and occasionally negatively reported) AD cytochrome oxidase defect, any meta-analysis of data pertaining to the anatomic distribution/defect magnitude debate is complicated by the fact that multiple studies have employed different experimental methods. Methodology has included spectrophotometric determinations of enzyme activity

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in crude homogenates, spectrophotometric determinations of enriched mitochondrial fractions, polarography, histochemical measurements of enzyme activity in tissue slices, and determinations of enzyme protein levels by spectrophotometric quantitation or immunohistochemistry. In spectrophotometric ETC assays, mitochondria are added to a cuvette or other appropriate chamber in the presence of the reduced substrate typically oxidized by the enzyme under study, and if necessary an electron acceptor. Rates are determined by tracking oxidation of the reduced substrate by the enzyme. Most laboratories have performed spectrophotometric cytochrome oxidase determinations in a generally standardized manner. However, there has been no single standardized procedure by which samples are prepared for the assay itself. Samples can vary markedly in mitochondrial concentration and purity, ranging from dilute and impure to concentrated and pure. When assaying ETC enzymes from tissue, the initial step in all sample preparation protocols is to prepare a homogenate. This can be done by manually or mechanistically grinding the tissue so that individual cells are suspended and their plasma membranes disrupted. It is important at this step not to concomitantly destroy mitochondrial membranes. The resulting homogenate at this point can be added to an assay chamber and placed in a spectrophotometer for enzyme rate determinations. Beyond this, different centrifugation strategies can be used to prepare increasingly clean mitochondrial fractions. First, the disrupted cells of the homogenate can be centrifuged at a relatively slow speed to pellet nuclei. The supernatant is harvested and centrifuged at a greater force to pellet remaining large organelles. This pellet is often referred to as a “crude mitochondrial pellet.” To further isolate mitochondria, the contents of the crude mitochondrial pellet are placed on top of a density gradient and ultracentrifuged to separate organelles by density. In brain this allows for isolation of two different “pure” mitochondrial fractions, a less dense synaptosomal mitochondrial fraction, and a more dense nonsynaptosomal fraction (which contains cell body mitochondria). Figure 1 shows a schematic illustration of mitochondrial puriﬁcation strategy. The mitochondrial concentration of the assayed sample determines the scale upon which rates are measured. For example, glial cells contribute largely to brain tissue bulk, and these cells are essentially anaerobic and minimally contribute to brain cytochrome oxidase activity (Wong-Riley, 1989). Therefore, homogenates derived from particular brain regions consist mostly of glial elements and mitochondrial protein comprises only a small amount of total protein, a measure used to standardize rate values between samples. The absolute enzyme rate observed, if expressed as activity/ tissue amount, is consequentially low. Although more labor intensive, the

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Homogenize

mitos

Spin at low g

nuclei

Spin post nuclear supernatant at higher g Tissue

Homogenate membrane mitos and other organelles

Resuspend crude mito pellet

Synaptosomal mitos Cell body mitos

Pure Mitochondrial Fraction

High speed ultracentrifugation through density gradient Crude Mitochondrial Fraction

FIG. 1. Preparation of mitochondrial assay fractions.

advantage of preparing mitochondrial fractions is that the absolute activity per reference denominator will be increased, possibly allowing for a more reliable activity “signal” and a reduction in levels of soluble impurities/factors that might confound enzyme measurement. When enriching mitochondrial fractions, however, it is important to utilize appropriate procedures. Use of an inappropriate (or perhaps any, SK) enrichment technique might also damage mitochondria, and introduce signiﬁcant changes in the environment of cytochrome oxidase which do not occur either in living brain or in crude homogenates. The most common confounding consequence to arise from overly harsh mitochondrial isolation procedures is disruption of marginal mitochondria, which could remove them from the assay fraction, causing an artifactual type II error. Spectrophotometric and polarographic determinations of AD cytochrome oxidase activity are summarized in Table I. To our knowledge, only one investigation has systematically compared cytochrome oxidase activity in a crude brain homogenate to that of an enriched mitochondrial fraction

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TABLE I CYTOCHROME OXIDASE ACTIVITY IN ALZHEIMER DISEASE: SPECTROPHOTOMETRIC AND POLAROGRAPHIC STUDIES Region

COX defect found

COX defect not found

a

Mixed brain regions

Parker et al., 1994

Frontal lobe

Kish et al., 1992 Mutisya et al., 1994a Chagnon et al., 1995 Wong-Riley et al., 1997

Cavelier et al., 1995 Maurer et al., 2000 Bosetti et al., 2002a

Parietal lobe

Mutisya et al., 1994a Chagnon et al., 1995 Wong-Riley et al., 1997

Kish et al., 1992 Reichmann et al., 1993 Mutisya et al., 1994 Cavalier et al., 1995

Hippocampus

Wong-Riley et al., 1997 Maurer et al., 2000 Bosetti et al., 2002a

Reichmann et al., 1993 Kish et al., 1992 Chagnon et al., 1995

Temporal lobe, not speciﬁed as hippocampus

Kish et al., 1992 Mutisya et al., 1994a Mutisya et al., 1994 Wong-Riley et al., 1997 Maurer et al., 2000

Cooper et al., 1993 Reichmann et al., 1993 Chagnon et al., 1995

Occipital lobe

Mutisya et al., 1994a Wong-Riley et al., 1997

Kish et al., 1992 Cavelier et al., 1995

Subcortical

Kish et al., 1992 Chagnon et al., 1995 Wong-Riley et al., 1997

Cerebellum

Wong-Riley et al., 1997

Chagnon et al., 1995 Maurer et al., 2000

Platelets

Parker et al., 1990b Parker et al., 1994b Cardoso et al., 2000a Bosetti et al., 2002a

van Zuylen et al., 1992

Molina et al., 1997a

Lymphocytes Fibroblasts

c

Curti et al., 1997

Unless otherwise indicated, a mitochondrial fraction was not prepared. Unless otherwise indicated, assays were spectrophotometric. a Assay performed on a mitochondrial enriched fraction. b Assay performed on a pure mitochondrial fraction. c Polarographic assay.

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(Mutisya et al., 1994). In the study of Mutisya et al. (1994), who compared cytochrome oxidase activity (adjusted by levels of citrate synthase, a mitochondrial matrix marker) in brain homogenate versus a crude mitochondrial fraction in two brain areas of patients with AD, the percentage enzyme reduction in AD was almost identical in both extracts in the temporal cortex (−30%, homogenate; −27%, mitochondrially enriched) whereas in the parietal cortex a slight activity difference that was not statistically different in the homogenate (−19%) became signiﬁcant in the crude mitochondrial fraction (−30%). These (preliminary) data can be interpreted in one of two ways: (1) the extent of mitochondrial enrichment does not markedly inﬂuence the ability to detect a modest cytochrome oxidase activity change in AD brain (SK), or (2) massive dilution of mitochondria in the homogenate assay fraction makes it difﬁcult to accurately measure mitochondrial enzyme activity rates, causing type II error (RS). Studies have also addressed the issue of brain cytochrome oxidase deﬁciency in AD using histochemical techniques. To measure cytochrome oxidase activity histochemically, ﬁxed brain slices are incubated in the presence of an electron donor, 3,3 -diaminobenzidene (DAB) (Wong-Riley, 1989). The oxidative conversion of DAB to an osmiophilic indamine polymer is accomplished through donation of electrons to an electron acceptor, cytochrome c. For the reaction to proceed over time, cytochrome c that becomes reduced must be cycled back to its oxidized state, which is accomplished by cytochrome oxidase. The amount of DAB reaction product generated can be quantiﬁed by optical densitometry (at the light or electron microscopy level) and serves as a surrogate of cytochrome oxidase activity, and can be used to extrapolate an activity rate for the enzyme. Using these methods, one can determine differences in cytochrome oxidase activity between individual cells and between different subcellular compartments (Kageyama and Wong-Riley, 1982). As shown in Table II, a reduction of brain cytochrome oxidase activity in AD as assessed by histochemistry was observed in all investigations in at least one examined brain area, but with no consistency as to the areas which are spared (e.g., Wong-Riley et al., 1997, enzyme reduction in occipital cortex; Simonian and Hyman, 1993, no enzyme reduction in occipital cortex). In addition to cytochrome oxidase activity measurements, brain levels of the enzyme in AD have also been assessed by measurement of the protein, or protein subunits, by immunohistochemistry in tissue slices or by Western blot analysis in extracts of brain homogenates. As shown in Table III, the results of studies of cytochrome oxidase protein in AD have not been consistent either in terms of the magnitude of the reduction (if any) or in the regional extent of the changes. Kish and colleagues (1999) employed Western blotting to quantify protein levels of two mtDNA-encoded cytochrome oxidase subunits (COI and

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TABLE II BRAIN CYTOCHROME OXIDASE ACTIVITY IN ALZHEIMER DISEASE: HISTOCHEMICAL STUDIES Region

COX defect found

Temporal cortex (not speciﬁed as hippocampus)

Wong-Riley et al., 1997

Hippocampus

Simonian and Hyman, 1993 Wong-Riley et al., 1997 Verwer et al., 2000 Cottrell et al., 2001

Occipital cortex

Wong-Riley et al., 1997

Frontal cortex (posterior cingulate)

Valla et al., 2001

Frontal cortex

Wong-Riley et al., 1997

Parietal cortex

Wong-Riley et al., 1997

Subcortical brain (choroid, lateral geniculate nucleus)

Wong-Riley et al., 1997 Cottrell et al., 2001

Brainstem (inferior colliculus)

Gonzalez-Lima et al., 1997

No COX defect found

Simonian and Hyman, 1993

Valla et al., 2001

TABLE III BRAIN CYTOCHROME OXIDASE PROTEIN LEVELS IN ALZHEIMER DISEASE: IMMUNOHISTOCHEMISTRY, WESTERN BLOT STUDIES Region

COX protein decreased

COX protein not decreased

Temporal cortex (not speciﬁed as hippocampus)

Wong-Riley et al., 1997 Kish et al., 1999 de La Monte et al., 2000

Hirai et al., 2001b

Hippocampus

Wong-Riley et al., 1997 Nagy et al., 1999a Bonilla et al., 1999 de La Monte et al., 2000

Nagy et al., 1999a Verwer et al., 2000 Hirai et al., 2001b

Parietal cortex

Wong-Riley et al., 1997 Kish et al., 1999

Frontal cortex

Wong-Riley et al., 1997

Hirai et al., 2001b

Occipital cortex

Wong-Riley et al., 1997

Kish et al., 1999

Cerebellum

Ojaimi et al., 1999

Hirai et al., 2001

Subcortical brain

Wong-Riley et al., 1997

a

COX subunits decreased in tangle-bearing cells, increased in nontangle-bearing cells. Overall increase inside neurons despite overall decrease in numbers of intact mitochondria. b

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COII) and two nuclear-encoded subunits (COIV and COVIc) in AD temporal, parietal, and occipital cortices. These regions were selected to enable survey of brain regions that show degenerative changes (parietal and temporal cortex), as well as a region that typically shows only limited degeneration (occipital cortex). The brain material used was that in which the authors had previously found diminished cytochrome oxidase activity in temporal but not parietal or occipital cortex homogenates (Kish et al., 1992). Western blot analysis demonstrated lower cytochrome oxidase protein levels (–19 to –47%) of all four enzyme subunits in AD parietal and temporal cortex but not in occipital cortex, in which a slight mean reduction (–12 to –17%) was not statistically signiﬁcant. Although the relationship between the extent of reduction of individual cytochrome oxidase protein subunits and magnitude of loss of enzyme activity in brain homogenates is not known, the implication of this study was that decreased cytochrome oxidase activity in AD brain homogenates might reﬂect in part or perhaps even in toto a loss of absolute cytochrome oxidase protein, with the magnitude of the reduction more marked in brain areas of pathology. These data can be compared with those from the only study to date that evaluated cytochrome oxidase activity in density puriﬁed mitochondrial fractions from AD brains. In that study (Parker et al., 1994a), hemibrains (right hemisphere) from nine AD subjects and eight control subjects were used to prepare pure mitochondrial fractions. Cytochrome oxidase activity was spectrophotometrically determined with a 53% deﬁcit of cytochrome oxidase observed in the AD samples after standardizing activity rates to protein content. As a further “standardization” measure, levels of the complex IV component cytochrome aa3 were determined. The cytochrome aa3 mean in the AD samples was 22% less than that of the control samples, but this was not a statistically signiﬁcant difference. Applying this correction slightly diminished the magnitude of the AD cytochrome oxidase defect, which ranged from 53 to 40% (still statistically signiﬁcant). These data suggest that AD cytochrome oxidase activity reductions arise mostly from a catalytic defect in the enzyme, rather than from a reduction in the amount of enzyme (Parker et al., 1994a). In further support of their study, Parker and Parks subsequently showed an absence of the low K m binding site in AD cytochrome oxidase, indicative of a structural change in the enzyme (Parker and Parks, 1995). Wong-Riley et al. (1997) also measured cytochrome oxidase levels with immunohistochemistry. Using an antibody to brain cytochrome oxidase, AD brains exhibited relative to controls decreased staining of the lateral geniculate nucleus, visual cortex, parietal cortex, hippocampal formation, and entorhinal cortex. The degree to which this generalized diminution in cytochrome oxidase level was contributing to the generalized decrease

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in activity of cytochrome oxidase activity (measured spectrophotometrically and histochemically), also reported in this paper, is unclear. However, in this study immunochemically demonstrated cytochrome oxidase level reductions were small in magnitude, histochemically determined activity decrements slightly more pronounced, and spectrophotometrically demonstrated activity deﬁcits much more profound. As protein levels (as shown by immunohistochemistry) of cytochrome oxidase in AD were diminished by less than 20% from control brain levels, and the magnitude of the biochemical activity reduction (measured in homogenates) typically exceeded 60%, it seems unlikely that referencing homogenate cytochrome oxidase activities to corresponding regional immunohistochemical measurements would eliminate the enzymatic defect. The two most recent studies using immunochemistry to assess cytochrome oxidase protein levels in AD obtained contradictory results. de La Monte et al. (2000) employed a monoclonal antibody to cytochrome oxidase (the subunit was not speciﬁed), and found decreased temporal cortical and hippocampal staining in AD. However, when Hirai et al. (2001) applied a monoclonal antibody speciﬁc for the mtDNA-encoded COI subunit to AD hippocampus, frontal cortex, temporal cortex, and cerebellum, antibody staining was increased in all regions except cerebellum. This was despite the fact that overall there were fewer intact mitochondria per AD neuron than there was per control neuron. The disparate differences in AD cytochrome oxidase immunochemical data are difﬁcult to reconcile. They may indicate brain cytochrome oxidase reductions are not robust in individual AD patients (SK). Alternatively, they may indicate that data from immunochemistry studies are not quantitatively robust enough to yield consistent results when different methods are used from study to study (RS). For example, a structural alteration in the enzyme might increase binding afﬁnity for some of the cytochrome oxidase antibodies used while reducing binding afﬁnity for others. Other data addressing cytochrome oxidase in AD brain have focused on mRNA levels for selected cytochrome oxidase subunits. As shown in Table IV, these studies have generally demonstrated decreased levels of mRNA for one or more subunits in hippocampus and temporal cortex of patients with AD, but not in primary motor cortex, a brain area less likely to degenerate in AD. To address the issue of the relationship between cytochrome oxidase transcript diminution and AD protein aggregation pathology, in situ hybridization was employed to measure COIII mRNA levels in tangle-free and tangle-bearing neurons of AD temporal cortex. Although a more severe reduction in levels of the transcript was observed in tangle-bearing neurons, a statistically signiﬁcant reduction was still observed in neurons that were tangle-free (Hatanpaa et al., 1996).

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TABLE IV BRAIN CYTOCHROME OXIDASE IN ALZHEIMER DISEASE: mRNA STUDIES Region

Decreased mRNA

Hippocampus

Simonian and Hyman, 1994 Chandrasekaran et al., 1998 Aksenov et al., 1999

Temporal cortex (not hippocampus)

Chandrasekaran et al., 1994 Hatanpaa et al., 1996 Chandrasekaran et al., 1997 Chandrasekaran et al., 1998 Hatanpaa et al., 1998

Parietal cortex

Aksenov et al., 1999

Frontal cortex (10 motor)

Cerebellum

Normal mRNA amounts

Chandrasekaran et al., 1994 Hatanpaa et al., 1996 Chandrasekaran et al., 1997 Aksenov et al., 1999

A generic concern of all studies of mRNA in autopsied human brain is the question of the stability of the mRNA obtained postmortem, which is likely to be much less stable than the enzyme protein (Ross et al., 1992). However, the available data do suggest that brain mRNA levels of cytochrome oxidase are, on average, decreased in AD brain, but with the changes more marked in areas of pathology (e.g., temporal versus motor cortex).

VII. Is a Brain Cytochrome Oxidase Deﬁciency a Robust Feature of Alzheimer’s Disease?

In studies of the neurodegenerative disorder PD, our laboratory (SK) has observed that a deﬁciency of the neurotransmitter dopamine in the putamen subdivision of the striatum is an obligatory feature of the disorder and in which no overlap occurs between the range of the individual control and patient dopamine values in our autopsied brain studies. Although this conclusion is made difﬁcult by the absence of individual control and AD enzyme values in most of the published investigations, the available data on the status of brain cytochrome oxidase in AD suggest that a brain cytochrome oxidase (activity/protein) deﬁciency cannot be an essential or robust characteristic (as is the dopamine deﬁciency in PD) of AD, since distinctly normal values are observed in many patients with the disorder (Kish et al., 1992). In this regard, it is not unreasonable to argue that, on logical grounds, a cytochrome oxidase defect cannot cause AD in at least the subgroup of subjects with AD

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who do not have this enzyme reduction. Furthermore, substantial overlap between brain enzyme activity in AD and controls observed in at least one study (e.g., Kish et al., 1992), together with an often reported only modest magnitude of the average activity reduction (e.g., Mutisya et al., 1994) make it much less likely that the enzyme is etiologically involved in the disorder (SK). However, data from multiple laboratories also suggest that examination of autopsied brain of a representative number of patients with AD and control subjects is likely to disclose a mean reduction in enzyme levels, but with the magnitude of the reduction dependent on the extent of pathology in the brain area (SK). Alternatively, it may be argued that the clean separation between PD and control dopamine levels in putamen (as reported by Kish et al., 1988) does not really address whether or not cytochrome oxidase pathology is likely to be important in AD. Some (RS) might argue a more appropriate analogy is to consider the meaning of overlap in complex I activities between PD and control subjects. Because there is overlap, by the reasoning outlined above one would have to conclude that complex I dysfunction is not a “robust” feature of PD, and therefore irrelevant to the disease (RS). [This reasoning is supported by SK if the overlap between control and patient ranges for the outcome measure are substantial and the average magnitude of the reduction only slight.]

VIII. Studies of Cytochrome Oxidase in Non-CNS Tissues in Alzheimer’s Disease: Clues to the Origin of the Enzyme Change?

Clariﬁcation of the regional extent of cytochrome oxidase enzyme reduction within brain and in other organs in AD will help to elucidate its pathogenesis. In this regard, it is essential to establish whether the enzyme defect in some AD patients is restricted or not to pathologic brain areas. For example, if the enzyme reduction is not restricted to degenerating brain areas, then it is unlikely to represent a neurodegeneration artifact. Experimental data indicate that cytochrome oxidase activity could potentially be impaired as a direct consequence of an amyloid-related process or as a consequence of reduced metabolic needs. Under in vitro conditions, Aβ and Aβ derivatives can injure mitochondria (and by implication cytochrome oxidase), and transfected cells expressing Aβ manifest mitochondrial injury as well (Askanas et al., 1996; Mark et al., 1997; Pereira et al., 1998; Caneveri et al., 1999). Evidence also suggests that brain cytochrome oxidase deﬁciency can develop as a secondary, physiologic response to diminished regional metabolic requirements (Wong-Riley, 1989).

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In this scenario, disconnections between cell networks arise, presumably as a consequence of regional degeneration in remote areas. Degeneration of particular neuron groups, presumably occurring as a consequence of a nonmetabolic primary pathology (such as plaque or tangle formation), then deprives related neuron groups of synaptic input. The viable but synaptically deprived neurons respond by downregulating cytochrome oxidase activity, as there is less need to expend energy maintaining ion gradients at synaptic areas. For example, following lesioning of the perforant pathway, within 24 h, cytochrome oxidase activity reductions are observed downstream in the dentate gyrus (Borowsky and Collins, 1989). Wong-Riley (1989) has further described that in brain, oxidative bioenergetic capacity is “coupled” to neuronal activity. Coupling theory sugggests that active neurons have high bioenergetic demands and upregulate their bioenergetic machinery, whereas inactive neurons with lower bioenergetic demands downregulate levels of these components. Cytochrome oxidase probably represents one of the regulatable enzyme components in this process. If one considers only the inconsistent brain homogenate and immunohistochemistry data, it is impossible to conclude whether the enzyme reduction is absolutely restricted or not to areas of neuronal pathogy in which the enzyme defect could simply be explained by a lower demand for energy production in a region of neuronal loss/hypofunction (e.g., activity is decreased primarily in brain areas of pathology; Kish et al., 1992; Kish et al., 1999; activity is markedly decreased in both degenerating and nondegenerating brain areas, Wong-Riley et al., 1997). Therefore, highly relevant to this debate are studies of cytochrome oxidase in non-CNS areas (e.g., platelets, lymphocytes, ﬁbroblasts), in which biochemical ﬁndings are unlikely to represent artifacts of neuronal pathologies or disconnection. What is the evidence for a cytochrome oxidase enzyme reduction in non-CNS tissue in AD? As mentioned above, the ﬁrst study to demonstrate a cytochrome oxidase reduction in blood of AD patients was the investigation of Parker (Parker et al., 1990), who employed plateletpheresis to procure large amounts of assay material from six AD and six control subjects. This group prepared pure mitochondrial fractions from platelets (using a strategy similar to that discussed above for brain) and spectophotometrically assayed ETC activities. Activities of complex I, succinate:cytochrome c oxidoreductase assay (SCR), succinate dehydrogenase (complex II), and cytochrome c reductase (complex III) activities were normal whereas cytochrome oxidase (complex IV) activity was markedly reduced by 50% in the AD group (Parker et al., 1990). In a subsequent report from Parker’s laboratory, in which assays of crude mitochondrial fractions prepared from 120-ml blood samples (19 AD and 17 control subjects) a 17% decrement in cytochrome oxidase activity was reported. In contrast, Complex II and III activities were comparable

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between groups (Parker et al., 1994b). In abstract form, Cardoso and colleagues (1999) subsequently reported diminished (by 17%) cytochrome oxidase activity in crude mitochondrial preparations derived from AD subject venipuncture samples. Yet another group, using crude mitochondrial fractions derived from 40-ml venipuncture samples from 20 AD and 20 control subjects, reported a 30% cytochrome oxidase mean reduction in the AD group (Bosetti et al., 2002). While the four studies listed above each found a readily demonstrable cytochrome oxidase defect in AD platelets, an investigation by van Zuylen and colleagues (1992) did not detect a reduction. In this study, platelets were extracted from 10-ml venipuncture samples and then repeatedly freeze thawed and ﬁnally sonicated to destroy the platelets, with the raw debris then assayed. Surprisingly, the investigators actually found that cytochrome oxidase activity in AD was nearly twice (a statistically signiﬁcant increase) that of age-matched control subjects, a ﬁnding that might derive from their inability to detect signiﬁcant cytochrome oxidase activity in either control or AD samples. For example, the control group cytochrome oxidase signal obtained by van Zuylen et al. (7.6 nmol/min/mg) was 84% less than that of the crude mitochondrial preparation by Bosetti et al. (2002) in the control group (48.5 nmol/min/mg), and 95% less than of the pure mitochondrial preparation (167.1 nmol/min/mg) by Parker et al. (1990). Two other investigations have assessed cytochrome oxidase status in nonCNS AD tissue. In the ﬁrst study Curti and colleagues (1997) reported by polarographic assay that AD ﬁbroblast cytochrome oxidase was reduced, on average, 30% below that of control ﬁbrobasts. This ﬁnding may relate to earlier observations that ﬁbroblasts from both familial and sporadic AD subjects show reduced overall rates of glucose and glutamine oxidation (Sorbi et al., 1995). In the second investigation, Molina and co-workers (1997) failed to demonstrate any AD-related decrement in cytochrome oxidase activity in mitochondrially enriched blood lymphocytes. Interestingly, the methods used in this report did not appear to replicate a complex I PD lymphocyte defect this group had previously shown employing a different technique (Barroso et al., 1993; Martin et al., 1996).

IX. Cytochrome Oxidase Dysfunction in Alzheimer’s Disease: Possible Genetic Component?

Multiple independent studies support the notion that if one appropriately isolates platelet mitochondria from groups of AD and control subjects, and determines cytochrome oxidase activities, the AD group mean rate will be reduced. The presence of a cytochrome oxidase defect outside of brain

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suggests that it is necessary to consider a possible underlying genetic pathogenesis for the enzyme activity reduction. In order to discuss potential underlying gene lesions, it is necessary to review ﬁrst mitochondrial genetics in general and cytochrome oxidase genetics in particular. Cytochrome oxidase consists of 13 protein subunits that derive from both nuclear and mitochondrial genomes. Ten subunits are nuclear-encoded, and three are encoded on mitochondrial genes. AD epidemiologic studies suggest mutation of mtDNA might be more relevant to cytochrome oxidase deﬁciency than nuclear DNA mutation since a nuclear DNA mutation would be expected to manifest with an autosomal dominant inheritance pattern, which appears not to be the case for the vast majority of those with AD (Lautenschlager et al., 1996). Still, having an affected ﬁrst degree relative does increase somewhat the lifetime risk of developing the disorder. For this reason, it is probably more appropriate to consider the common sporadic form of AD as being “pseudosporadic.” If risk increase is then due to inheritance, the fact that Mendelian inheritance patterns are not observed raises the possibility that a non-Mendelian genetic factor may be involved. In humans, the one recognized subset of genes that does not play by Mendelian rules are those located on mtDNA. Mitochondrial genetics differs from nuclear genetics in several fundamental ways. One major difference is heteroplasmy. Heteroplasmy is the mtDNA correlate of nuclear heterozygosity. Heteroplasmy occurs when not all mtDNA copies within a given cell are identical. In fact, various ratios of wild-type (wt):non-wt mtDNA can co-exist inside a cell and probably even within a single mitochondrion. Heteroplasmic ratios can also vary over time, particularly in postmitotic cells such as neurons. Whether or not non-wt mtDNA present within a heteroplasmic cell carries a phenotypic consequence depends on the burden of the non-wt species. If the burden is high enough to surpass a deﬁned threshold, then functional consequences result. If not, an associated phenotypic change may not manifest. Mitotic (replicative) segregation, which can give rise to mitochondrial mosaicism, further complicates the impact of a given heteroplasmy. Because mitochondria are dispersed from parent to daughter cells through cytoplasmic partition, cells in different tissues or organs and even different cells within a given tissue or organ can display heteroplasmic variation. Heteroplasmic thresholds may also vary between cell types, with the most aerobically active cells being the most likely to express an altered phenotype. Lastly, as an individual’s mitochondrial issue derives from the oocyte, mtDNA is essentially maternally inherited. It is important to point out, however, that although mtDNA is maternally inherited, for the reasons described above mtDNA diseases are more likely to present sporadically or

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pseudosporadically than within the framework of a recognizable matrilineal pedigree ( Johns et al., 1991; Newman, 1993). In this regard, a maternal inheritance bias has been reported in some (Duara et al., 1993; Edland et al., 1996) but not all (Lautenschlager et al., 1996; Ehrenkrantz et al., 1999) investigations in AD. Regarding mtDNA sequence variation, substitutions that occur in one of the 13 mtDNA structural genes (all of which encode subunits of the ETC) can occasionally lead to an amino acid change in a protein, and these sequence deviations are “nonsynonymous.” “Synonymous” substitutions are more common, and do not alter amino acids. Sequence deviation also occurs within the 22 tRNA or 2 rRNA mitochondrial genes (the so-called “synthetic” genes), which are neither synonymous nor nonsynonymous since they are not translated into protein. Whether a sequence deviation represents a mutation is to some degree speculative. By deﬁnition, any sequence variant that is present in more than 1% of a tested population is considered a “polymorphism” rather than a “mutation.” However, a sequence substitution that is common in one population and considered to represent a polymorphic variant may appear exceedingly rare in another population and qualify as a mutation. Particular criteria are therefore often applied when considering the pathogenic potential of an mtDNA sequence substitution. Nonsynonymous substitutions are generally held to represent better etiologic candidates than synonymous substitutions. Substitutions occurring in phylogenetically conserved sites are more suspect than those not occurring at conserved sites. Substitutions that lead to radical amino acid changes are considered more likely disease candidates. Finally, segregation of a particular mtDNA genotype with a disease phenotype helps determine the signiﬁcance of a given sequence deviation. Even when these guidelines are applied, however, it is not always evident which mtDNA sequence substitutions are disease-relevant and which are not. Two fundamentally different models address how mtDNA abnormalities might play a role in AD disease pathophysiology. One proposes that mtDNA acquires somatic mutation over the lifetime of an individual, most likely from oxidative stress (Linnane et al., 1989; Wallace, 1992). The sum of these acquired mutations, which may include deletions or point mutations, diminishes the pool of normal ETC enzymes, which are perhaps replaced by miscoded protein subunits. Oxidative phosphorylation impairment occurs, with adverse consequences on mitochondrial physiology and cell viability. Under this hypothesis, mtDNA derangement represents secondary, albeit degeneration-relevant, pathology. The other hypothesis pertinent to AD contends that inherited mtDNA mutations are primarily responsible for altered mitochondrial functioning in persons with non-Mendelian diseases that feature systemic mitochondrial

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defects. This model, ﬁrst proposed by Davis Parker in the late 1980s, was developed in an attempt to explain the apparent pseudosporadic inheritance of non-Mendelian disorders (Parker et al., 1989; Parker, 1990, 1991). The most applicable group of disorders in general is the late-onset, sporadic neurodegenerative diseases. Parker’s hypothesis assumes that clinically deﬁned entities such as AD, PD, and amyotrophic lateral sclerosis (ALS) each encompass a set of genetically heterogeneous, phenotypically similar disorders. For example, AD can arise from mutation of a number of different nuclear genes. Such cases are typically recognizable because they are usually early-onset (presenting before the age of 60) and tend to present within an obvious autosomal dominant framework. Nevertheless, those without an autosomal dominant family history but who have an affected ﬁrst degree relative do see an increase in their lifetime AD risk (Lautenschlager et al., 1996), suggesting the presence of a gene contribution. Under Parker’s hypothesis, when a disorder shows both pseudosporadic epidemiology (raising the possibility of a nonMendelian genetic component) and also mitochondrial dysfunction (again raising the possibility of an mtDNA component), it is worthwhile to consider the presence of contributing inheritable mtDNA mutation or variation.

X. Cytochrome Oxidase Dysfunction in Alzheimer’s Disease: Genetic Studies are Still Inconclusive

Although the human genome project now reveals the existence of nuclear ETC gene variability, there are no data to date implicating mutation or variation of nuclear cytochrome oxidase subunit genes in AD. However, the literature does contain a number of reports in which investigators have attempted to address the possibility of somatic and inherited mtDNA mutation in AD. Relevant to the former possibility is the fact that mtDNA mutation rates are high, far exceeding those for nuclear DNA mutation (Wallace et al., 1987; reviewed in Linnane et al., 1989). There are also data showing excessive mtDNA levels of oxidative adducts in AD brain (Mecocci et al., 1994; de La Monte et al., 2000; Hirai et al., 2001). Consequences of oxidative mtDNA damage may also include generation of an ∼5-kb deletion, the so-called “common deletion” (mtDNA4977 ) (Beckman and Ames, 1998). The COIII gene resides within this deleted stretch. mtDNA4977 has been quantiﬁed in AD brain. Although several groups reported no change in mtDNA4977 levels (Blanchard et al., 1993; Sheu et al., 1994; Cavelier et al., 1995; Chandrasekaran, 1996; Chang et al., 2000), others

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have demonstrated increased levels of mtDNA4977 (Corral-Debrinski et al., 1994; Hamblet and Castora, 1997; Hirai et al., 2001). In the positive studies, despite an absolute increase in the mtDNA4977 , the burden in neurons remained low (<1%), and therefore, the relevance of the increase may be more meaningful as a marker of pathology than as a cause. Documenting somatic point mutations in mtDNA is complicated by the fact that, relative to wild-type sequence, the burden of an individual acquired point mutation is proportionally quite low and may not be detected under normal surveillance paradigms. Chang and colleagues (2000) therefore employed an experimental strategy that would permit detection and quantiﬁcation of very low levels of speciﬁc mtDNA mutations. The mutations screened for were any that would eliminate an AvaII restriction site typically present at nt16390 of the mtDNA (AvaII cuts GGACC sequences). Detection sensitivity was achieved through an initial incubation of genomic DNA (extracted from parietal lobe tissue, hippocampus, cerebellum, and lymphocytes) in AvaII. The restricted DNA was subsequently added to a PCR reaction with primers ﬂanking the nt16390 AvaII restriction site. Thus, the only subject-derived mtDNA template available for ampliﬁcation was that which did not originally contain the AvaII site. The principal sequence change found was a C→T transition at the ﬁfth nucleotide of the restriction target sequence, which could potentially arise from oxidative stress-induced misreplication. Employing this strategy Chang found that compared to controls, levels of this C→T D-loop transition were ∼3-fold elevated in AD parietal, hippocampal, cerebellar, but not lymphocyte samples, suggesting that somatic mtDNA mutation is excessive in AD brain. Lin and co-workers (2002) employed a strategy in which individual COI mtDNAs were isolated and expanded prior to sequencing. Mitochondrial DNA was prepared from both autopsied brain association and primary visual cortices of AD patients, with the mtDNA PCR ampliﬁed using primers for the COI gene. The aim of this strategy was to detect low abundance heteroplasmies, presumably those arising from somatic mutation. Compared to that of samples from young subjects, the frequency of low abundance heteroplasmic mutation was elevated in elderly controls and in AD subjects. These data support the concept that mtDNA mutation acquisition occurs with aging. Over the assessed COI stretch, the sum of the assorted low-abundance heteroplasmies between different mtDNA molecules from a given aged individual was cumulatively high, and there was a negative correlation between overall mutational burden and cytochrome oxidase activity. However, there was no statistical difference between the elderly control and AD groups in the number of total mutations detected. In addition to epidemiologic surveys, polymorphism association studies have evaluated whether particular mtDNA variants contribute to AD

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risk. The most extensively investigated polymorphism has been the tRNAGln A → G transition at nt4336, in which a positive association with AD has been reported in some studies (Shoffner et al., 1993; Hutchin and Cortopassi, 1995; Egensperger et al., 1997) but not in others (Wragg et al., 1995; Hutchin et al., 1997; Tanno et al., 1998; Zsurka et al., 1998; Chagnon et al., 1999; Chinnery et al., 2000; Rodriguez-Santiago et al., 2001; Edland et al., 2002). Similarly, ﬁndings of increased nt5460 G → A/T polymorphism (in the ND2 gene) in AD (Lin et al., 1992) have not been conﬁrmed (Petruzzella et al., 1992; Kosel et al., 1994; Janetzky et al., 1996; Hutchin et al., 1997; Chagnon et al., 1999; Rodriguez-Santiago et al., 2001; Edland et al., 2002). Several studies have employed direct PCR-based sequencing methods to survey substantial portions of AD subject mitochondrial genomes. Brown and colleagues (1996) sequenced the entire mtDNA from autopsied brain of three patients with the disease who demonstrated neuropathological features of both AD and PD. Multiple deviations from the reported Cambridge sequence (Anderson et al., 1981) were revealed in all three patients. Some of these deviations were polymorphisms previously postulated by some to associate with disease. For example, one patient had the tRNAGln A4336G transition, and one carried a T4216C transition considered to increase the penetrance of some Leber’s disease mtDNA mutations. Although other novel substitutions were uncovered, none of the deviations were common to all three patients. This study clearly points out one of the difﬁculties of surveying mtDNA for sequence deviations, which are frequent. It must be emphasized that any given individual will differ from the Cambridge sequence at multiple recognized positions, and it is also not uncommon to detect novel substitutions. Thus, when considering mtDNA substitutions in individual patients with sporadic disease, a difﬁculty arises in proving causality. In disorders in which large, recognizable maternal kindreds are available (as is occasionally the case with Leber’s disease), it is relatively easy to determine whether a given substitution segregates with the phenotype. However, when no kindred with multiple, unequivocally affected and unaffected members is available (which is by deﬁnition the case with sporadic disease), interpretation of the results of such analysis is problematic. Examples of other AD mtDNA sequence studies detecting substitutions of uncertain signiﬁcance include: two heteroplasmic, amino acid altering, novel mtDNA substitutions in the COII gene of a single 82-year-old AD subject (Qiu et al., 2001), 12S rRNA mtDNA mutations in AD subjects (one transition and one insertion) not present in 59 controls (Tanno et al., 1998), and multiple candidate mutations described by Edland in AD subjects undergoing mtDNA analysis (Edland et al., 1999). Only one study has addressed the possibility that patterns of mtDNA sequence deviation might contribute to AD risk (Chagnon et al., 1999).

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Chagnon et al. (1999) sequenced the CO, tRNA, and rRNA mtDNA genes from patients with AD and from controls. With the exception of two substitutions that were actually less common in AD subjects (and therefore postulated to perhaps be protective), no single detected substitution was found to occur at a different frequency between groups. However, a combination “haplotype” that included Cambridge sequence deviations at nt5633, nt7476, and nt15812 was present in 4/85 AD subjects screened but 0/83 controls. This preliminary ﬁnding raises the possibility that the risk in AD of certain individual mtDNA substitutions is minimal but additive, or that it is mostly relevant combinations of mtDNA substitution that confer risk. The Parker hypothesis of causative, inherited mtDNA mutation in pseudosporadic, non-Mendelian, late-onset neurodegenerative diseases permits (if not predicts) an important role for low-abundance heteroplasmic mutations. This is because disease-associated, low-level heteroplasmy would favor sporadic epidemiologic patterns and might even account for phenotypic divergence between identical twins. Testing this hypothesis has proven far more complex than might be expected, primarily because standard PCRbased sequence strategies are only adequate to detect a priori unknown mutations that are present in greater than ∼30–40% abundance. Thus, with nuclear DNA sequencing, when heterozygosity at a particular nucleotide exists, overlapping peaks are apparent on chromatogram inspection since each different DNA species is present at the 50% level. However, under conditions of low-abundance heteroplasmy, at the relevant position the chromatogram reveals a major nucleotide peak that can overlie a less obvious minor peak, corresponding to the less abundant mtDNA species, which becomes impossible to discern from background noise and goes undetected. To date, there are only two published studies that attempted to detect low-abundance heteroplasmies over extended regions of AD mtDNA. One was the study of Lin et al. (2002) limited to part of the COI gene, which was discussed earlier in this section. These investigators reported that lowabundance heteroplasmy was common in AD brain (although it did not appear to occur more frequently than it did in age-matched controls). The second investigation was that of Davis and colleagues (1997) in which genomic DNA was extracted from control and AD subject blood samples via a boiling protocol. In this procedure, separate primer sets annealing upstream and downstream to the three mtDNA CO genes were used to generate amplicons that were ligated into plasmids. These constructs were used to transfect E. coli, and individual expanded colonies were selected for plasmid harvesting. Each retrieved plasmid was then used for clonal sequencing. By this approach, ﬁve “linked” Cambridge sequence deviations were found distributed through portions of the amplicons corresponding to the mtDNA COI and COII genes. Compared to wild-type sequence, these deviations represented a minority species and were believed to reﬂect a low-abundance

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heteroplasmic haplotype. The deviant sequence was ampliﬁed from virtually all genomic DNA samples prepared, including both control and AD subjects. However, the proportion of deviant to wild-type sequence was higher overall in those with AD. The investigators interpreted this as evidence of an inheritable, low-abundance mtDNA heteroplasmy that was increased in AD and of potential pathogenic signiﬁcance and diagnostic utility. Subsequently, however, two other laboratories and one of the investigators associated with the original paper showed that the ﬁve linked mutations were actually not from mtDNA, but rather arose through co-ampliﬁcation of a nuclear mtDNA pseudogene (Hirano et al., 1997; Wallace et al., 1997; Davis and Parker, 1998). This led to retraction of the Davis report (Davis et al., 1997, 1998). Still, the reason why pseudogene ampliﬁcation in AD subjects exceeded that of controls remains unclear. Likely explanations include the possibility that there was less extractable mtDNA in the AD samples, that there was simply less mtDNA in the AD samples, or that mtDNA extraction or ampliﬁcation efﬁciency was reduced in the AD samples. At least for brain, data exist to support these scenarios. Reduction of mtDNA in AD has been reported (Lezza et al., 1999; de La Monte et al., 2000). Hirai et al. (2001) observed that although total mtDNA was increased in several neuronal populations in AD, much of this mtDNA was present in digestive organelles and therefore potentially unsuitable for extraction or ampliﬁcation. In this study reduced numbers of intact mitochondria were observed, and thus the amount of ampliﬁable mtDNA was likely reduced as well. Therefore, although the conclusions of the Davis publication were incorrect, these data do provide some evidence that mtDNA is altered in AD (see also Brown et al., 2001). Partly due to the complexities of mitochondrial genetics and associated methodological approaches, and also because it is difﬁcult to demonstrate genetic causality in a potentially heterogeneous sporadic disorder, it has not been possible to prove or disprove whether mtDNA abnormality does play a pathogenic role in AD. Available data suggest that there is unlikely to be a single common homoplasmic mtDNA mutation that is singularly deterministic for the disorder. Parker’s hypothesis that non-Mendelian (i.e., mtDNA) gene inheritance may contribute to non-Mendelian, pseudosporadic disease development also predicts an important role for low-abundance heteroplasmic mutation (Parker, 1990, 1991), a possibility that still needs to be systematically addressed. Finally, inadvertent and intended competitive PCR of genomic DNA from both blood and brain of AD subjects suggests that mtDNA might be abnormal in AD (Davis et al., 1997; Brown et al., 2001). This abnormality could represent sequence or nonsequence-related pathology. In further consideration of the possibility that mutation-free mtDNA may still contribute to AD pathogenesis, it is conceivable that ETC components encoded from certain “normal” mtDNA variants may simply not “work well”

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with particular ETC protein components produced from certain polymorphic nuclear ETC genes. Failure of particular mtDNA and nuclear DNAencoded ETC component combinations to work efﬁciently together could be relevant to AD and even aging, as such could account for ETC defects. The extremely high prevalence of AD in the most elderly of the population in some ways makes this an attractive concept. After all, if as many as half of those over the age of 85 qualify for a diagnosis of AD (Evans et al., 1989), can a causative genetic factor really be considered a mutation?

XI. Cybrid Data Suggest mtDNA Contributes to Alzheimer’s Disease Cytochrome Oxidase Dysfunction

Techniques for accurate and comprehensive mtDNA sequencing are still evolving. Meanwhile, other strategies have attempted to address the question of whether mtDNA aberration could play a role in late-onset, sporadic AD. Supportive data have derived from studies of cytoplasmic hybrids (cybrids). King and Attardi (1989) developed cybrid methodology in the late 1980s, following reports from other laboratories showing that it was possible to deplete endogenous mtDNA from cell culture lines and still expand the cells (Desjardins et al., 1986). Cells rendered completely free of endogenous mtDNA were termed ρ0 cells, since prior to its localization in mitochondria, cytoplasmic nucleic acid was designated as nucleic acid (Ephrussi et al., 1949). To cause depletion of mtDNA the cells were maintained in medium containing ethidium bromide, a DNA mutagen that carries a net positive charge and concentrates within negatively charged mitochondria. Mitochondrial concentrations high enough to disrupt mtDNA replication but not chromosome replication can therefore be achieved. Through extended ethidium bromide exposure it is possible to remove all detectable mtDNA. However, with appropriate metabolic support ρ0 cells will grow. Exposure of the cells to high glucose medium supplemented with pyruvate (to help cycle glycolytic pathway components and byproducts) and uridine (to bypass a block that arises in the dihydroorotate dehydrogenase catalyzed step of pyrimidine synthesis) are usually sufﬁcient for viability (King and Attardi, 1996). After generation of an osteosarcoma ρ0 cell line, King and Attardi (1989) succeeded in repopulating mtDNA-depleted cells with exogenous mtDNA. In this process ﬁbroblasts were enucleated and, with fusion of the resultant cytoplasts with the ρ0 cells or by cytoplast microinjection into ρ0 cells, the mitochondria and hence mtDNA of the donor cytoplast propagated and restored aerobic competency. King and Attardi described the resultant

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cytoplasmic hybrids as “cybrids,” a term previously used by other investigators evaluating the effects of combining cytoplasmic contents from different cells (Bunn et al., 1974). This procedure was later simpliﬁed by production of cybrid cell lines by fusing platelets with ρ0 cells (Chomyn et al., 1994). Compared to ﬁbroblasts, platelets are easier to employ as mtDNA donors since these nonnucleated blood cells are readily acquired and do not require propagation in culture or enucleation prior to mitochondrial transfer. In the early 1990s, cybrids were employed to study the physiologic effects of known mtDNA mutations (King et al., 1992; Masucci et al., 1995). Speciﬁcally, cybrid cell lines with different heteroplasmic ratios were evaluated biochemically to determine thresholds at which phenotypic consequences occurred. By the mid 1990s, other groups began adapting cybrid methodology to screen for mtDNA mutation in persons with candidate diseases, in particular, the late-onset, sporadic neurodegenerative diseases. Cybrids appeared to represent an ideal system for such a study, since in cybrid cell lines produced from a common ρ0 parent line, nuclear DNA is clonal and should not account for differences between lines. Since the cells are expanded in culture, the investigator determines the environment. Thus, unsuspected toxins are no longer a concern. An illustration of the cybrid technique is provided in Fig. 2. During expansion of cybrid lines, cells undergo a sufﬁcent number of divisions so that any transferred components that do not propagate or replicate degrade over time and dilute well over a millionfold. Furthermore, mtDNA only encodes parts of the ETC. Thus, if one wishes to demonstrate

Platelet from subject with disease

Cybrid expresses disease subject s mito genes

Expand Line

Compare phenotypes of cells with same nuclear but different mtDNA backgrounds

ρ 0 cell

Platelet from control subject

Cybrid expresses control subject s mito genes

Expand Line

FIG. 2. The cybrid technique.

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that mitochondrial genomes are different between two individual mtDNA donors, surveying ETC function in the two resulting cybrid lines can provide relevant information. If ETC enzyme activity is different between the cybrid lines, the most parsimonious explanation is that the difference arises from differences in mtDNA. Davis and colleagues published the ﬁrst AD cybrid series in 1997. In this study, 45 cybrid cell lines expressing platelet-derived mitochondrial genes from 45 different AD subjects and 20 control cybrid cell lines from 20 different age-matched control subjects were generated. The ρ0 cells used were derived from the SH-SY5Y human neuroblastoma line (Miller et al., 1996). Complex I activity means were equivalent between AD and control cybrid groups. The mean cytochrome oxidase activity for the AD cybrid group was ∼80% that of the control group. This suggested CO gene(s) between AD and control subjects were not equivalent, and that this could account for the lower cytochrome oxidase rates in the AD cybrids. This cybrid series was also evaluated for oxidative stress using the dye 2 ,7 -dichlorodihydroﬂourescein diacetate (DCF-DA), which upon reaction with reactive oxygen species (ROS) converts to a ﬂuorescent derivative that can be quantiﬁed with a ﬂuorocytometer. DCF-DA ﬂuorescence in the AD lines was increased by approximately 25% over that in the control cybrid lines, suggesting mtDNA-derived differences in cytochrome oxidase between the groups was responsible for a secondary ROS elevation in the AD cybrids. A second AD cybrid series was published later that year (Swerdlow et al., 1997). In this study, a human NT2 teratocarcinoma ρ0 cell line was used to generate 15 AD and 9 age-matched control cybrid cell lines. Platelets were again the source of mtDNA donation. Cytochrome oxidase activities were spectrophotometrically determined by following the conversion of reduced cytochrome c to oxidized cytochrome c in a cuvette. Relative to that of the control lines, AD cytochrome oxidase mean activity was depressed by 16%, again suggesting that at least part of the AD cytochrome oxidase defect ultimately arises from mtDNA. DCF-DA studies were also performed, and again increased ﬂuorescence was observed in AD cybrid lines. To corroborate that this increase in DCF-DA ﬂuorescence was indeed due to ROS overproduction (and not speciﬁcally due to ROS underscavenging), activities of several antioxidant enzymes were determined. Glutathione reductase, glutathione oxidase, Cu/Zn superoxide dismutase, and Mn superoxide dismutase activies were all elevated. These data suggest that an mtDNA-determined cytochrome oxidase defect causes secondary oxidative stress in AD. In addition to cytochrome oxidase deﬁciency and ROS overproduction, additional studies (mostly performed using SH-SY5Y AD cybrids) reveal additional interesting pathology in AD cybrids. Sheehan et al. (1997) evaluated

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mitochondrial calcium homeostasis in AD cybrids in which cytoplasmic calcium measurements were determined by ﬂuorescence microscopy using a ﬂuorescent dye, fura-2 AM. Under basal conditions, cytoplasmic calcium was higher in AD cybrid cells than in control cybrid cells. Cells were then exposed to a protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), which uncouples electron transport by rendering the inner mitochondrial membrane permeable to H+ and eliminates the mitochondrial membrane potential , with resultant matrix depolarization. When matrix depolarization occurs, electrochemically mediated compartmentalization of cations within mitochondria is no longer possible, resulting in efﬂux of mitochondrial calcium from the matrix. This increases cytoplasmic concentration of the ion. In AD cybrids, the magnitude of the CCCP-induced cytoplasmic calcium increase was less than that of control cybrids, suggesting that mitochondrial calcium sequestration in AD cybrid mitochondria is reduced as a consequence of mtDNA-derived differences between AD and control cybrid cytochrome oxidase. In related experiments, AD and control cybrid lines were exposed to carbachol, a cholinergic agonist that triggers release of calcium from certain cell compartments (especially endoplasmic reticulum) through its activation of phosphoinositide-mediated second messenger systems. To reverse this calcium transient, the ion is moved into other areas that can store the ion, including mitochondria. In AD cybrids, recovery from carbachol-induced cytoplasmic calcium transients was delayed, again suggesting impaired calcium sequestration by AD cybrid mitochondria (Sheehan et al., 1997). Perhaps relevant to the calcium data are experiments addressing status in AD cybrid mitochondria. Tetraphenylphosphonium ion (TPP+) accumulation in AD cybrid whole cells is reduced relative to that of control cybrid whole cells (Cassarino et al., 1998). Incubating cell lines in 5 μM cyclosporin increased TPP+ accumulation in both groups, and following cyclosporin treatment there were no longer differences in total TPP+ accumulation. Although absolute measurements are not possible from such experiments on nonisolated mitochondria, these data suggest that compared to control cybrids, AD cybrid mitochondria are relatively depolarized. Relevant to this possbility is another study that evaluated in AD cybrids with the cationic potentiometric dye JC-1 (Khan et al., 2000). JC-1 can exist in two different forms, including a monomeric form that emits in the green spectrum and an aggregate form that emits in the red spectrum. Using confocal microscopy, red and green ﬂuorescence can be quantiﬁed. Khan et al. (2000) calculated aggregate:monomer ratios for cybrid mitochondria, and found that relative to control cybrid mitochondria, in AD cybrids aggregate:monomer ratios overall were lower. The simplest interpretation of these experiments is that is relatively decreased in AD cybrid mitochondria.

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AD cybrid cells also differ from control cybrid cells in aspects potentially relevant to apoptotic cell death pathways. One consequence of mitochondrial depolarization is the release of cytochrome c from the organelle to the cytoplasm. Cytoplasmic cytochrome c levels are increased in AD cybrids (Khan et al., 2000). In cytoplasm, cytochrome c forms part of an apoptosome that leads to activation of the caspase 3 enzyme. Relative to control cybrids, AD cybrids manifest increased caspase 3 activity (Khan et al., 2000). Mitochondrial ultrastructure is altered in AD cybrids. Excessive bizarreshaped and enlarged mitochondria are observed, often with disrupted cristae. Increased amounts of degenerating mitochondria and mitochondria undergoing lysosomal digestion are apparent (Trimmer et al., 2000). Various cellular signaling mechanisms are also altered in AD cybrids. De Sarno et al. (2000) reported that phosphoinositide-mediated signaling systems are more active in AD than control cybrids and that AP-1 DNA binding activity is reduced. In a separate study, Bijur et al. (1999) found that heat shock factor-1 (HSF-1) DNA binding activity was lower than normal in AD cybrids, possibly a result of the cytochrome oxidase-determined state of chronic oxidative stress exhibited by AD cybrid cell lines. Alteration of heat shock protein (HSP) systems could have protein aggregation-related implications, since HSPs serve as antiaggregants. Perhaps relevant to this ﬁnding are data describing overproduction in AD cybrids of Aβ1-40 and Aβ1-42 proteins, which further aggregate in culture (Khan et al., 2000). To date, at least 60 AD cybrid cell lines have been concomitantly generated and assayed against at least 29 control cybrid cell lines. Comparisons of these AD cybrid cell lines versus controls have shown cytochrome oxidase activity deﬁciency, oxidative stress, altered calcium homeostasis, altered mitochondrial morphology, reduced , activation of programmed cell death pathways, perturbed amyloid precursor protein processing with Aβ aggregation, and altered cellular signaling mechanisms. These results are described in eight different reports from three groups, several studies of which were performed in blinded fashion. In these reports, two different neuronal-like ρ0 cell lines were utilized to generate cybrids, the human neuroblastoma SH-SY5Y line and the human teratocarcinoma NT2 cell line. One AD cybrid study, however, did not ﬁnd reduced cytochrome oxidase activity in cell lines containing AD subject mtDNA (Ito et al., 1999). The explanation for this discrepancy is unknown, but probably relates to the multiple methodologic differences between the positive studies and the Ito study. In this regard, Ito examined only three AD and three control cell lines, employed a human cervical carcinoma cell line (HeLa cell) nuclear background, apparently used frozen instead of fresh platelets, and only assayed particular manually selected clones from each overall cybrid culture.

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XII. Unresolved Issues in Alzheimer’s Disease Cybrid Studies: Where is the mtDNA “Mutation?”

AD cybrid studies were designed and carried out to evaluate possible differences between AD and control individual mtDNA, and most of these studies support this possibility. However, it must be emphasized that the cybrid studies do not disclose how AD and non-AD mtDNA are actually different. Such information must derive from actual direct analyses of the mtDNA itself. In the absence of correlative sequence data, some hesitate to accept inferences supported only by biochemical-physiologic cybrid data in the absence of direct mtDNA data (SK; Schon et al., 1998). Consistent with the views of cybrid detractors, available sequence data appear to rule out the possibility that a single homoplasmic or high percentage heteroplasmic mutation causes AD in most subjects. Further sequence efforts thus need to evaluate for low abundance heteroplasmies, compound microheteroplasmies, or unique Cambridge sequence deviations (either alone or in combination) occurring in individual patients. Certainly, in Mendelian genetics the presence or absence of frank mutation often deﬁnes whether or not a particular gene is relevant to a disease. This concept may or may not generalize to mitochondrial genetics. Mitochondrial genes are highly polymorphic, and mtDNA gene products function in the context of proteins encoded by nuclear genes that are themselves polymorphic. It is possible distinct combinations of nonmutated ETC components do not function identically. In this scenario, normal mtDNA variation could contribute to AD risk, while at the same time explaining why mtDNA polymorphism-AD associations are difﬁcult to establish. It could potentially account for observed overlap between individual data points from disease and control cybrid cell lines. This genetic model is also compatible with the extremely high prevalence of AD in late life, in that it does not require the majority of affected persons to harbor an actual pathogenic mutation. It also can be argued that the modest magnitude of the AD cybrid cytochrome oxidase defect underestimates the extent of the enzyme reduction in brain, as postmitotic brain cells, unlike platelets, may experience certain events that further insult mtDNA integrity, such as long-term oxidative stress. Furthermore, factors relating to mtDNA transfer during cybrid cell line generation may select against more dysfunctional mitochondria harboring a greater mtDNA mutational burden. Following fusion, cells with more dysfunctional mitochondria may undergo further negative selection, again minimizing defect magnitude.

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XIII. Could a Cytochrome Oxidase Defect Cause Alzheimer’s Disease?

The phenotypic consequences of cytochrome oxidase deﬁciency have been evaluated in rats exposed to discrete amounts of the inhibitor sodium azide via a subcutaneous osmotic minipump in which a 35–39% inhibition of enzyme activity is typically induced (Bennett et al., 1996). Rats exposed to the enzyme inhibitor demonstrate defective spatial and nonspatial learning, abnormalities of hippocampal long-term potentiation (Bennett et al., 1992), and poor learning and memory performance on the radial arm maze and Morris water maze (Bennett and Rose, 1992). Gabuzda and co-workers (1994) found both cytochrome oxidase inhibition by sodium azide and ETC uncoupling by CCCP facilitate production of an 11.5-kDa, carboxy-terminal-containing APP derivative. This derivative appears to escape the α secretase-mediated cleavage of APP into soluble components. By sparing the 39–43 amino acid Aβ stretch from α secretase-mediated cleavage, this 11.5-kDa derivative could ultimately facilitate production of the APP 4 kD Aβ metabolite. Consistent with this possibility, levels of soluble, α secretase-generated APP metabolites decline following bioenergetic impairment in PC12 cells (Webster et al., 1998). Extending these observations, Khan et al. (2000) observed both intracellular and extracellular Aβ1-40 and Aβ1-42 protein elevations in cultures of cybrid cell lines expressing platelet-derived mitochondrial genes from sporadic AD subjects. In addition, when AD cell ﬂasks were maintained at high conﬂuency for several days, immunochemistry revealed large extracellular accumulations of Aβ1-40. These extracellular Aβ1-40 accumulations were morphologically similar to plaques seen in AD brain (Fig. 3). Such

FIG. 3. “Plaquette” in an AD cybrid culture. This picture reveals an extracellular aggregation surrounded by AD SH-SY5Y cybrid cells. The aggregation has been stained with a primary antibody to Aβ1-40. (Courtesy of Drs. James P. Bennett and Shaharyar Khan.)

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Aβ aggregations were not observed in control cybrid cultures maintained under identical conditions. AD ﬁbroblasts develop hyperphosphorylated tau antigenic determinants when exposed to CCCP (Blass et al., 1990). These antigenic determinants can be compared to those observed in paired helical ﬁlament tau. Although this does not prove that mitochondrial dysfunction can induce paired helical ﬁlament tau formation and aggregation, this ﬁnding complements data showing that mitochondrial dysfunction can activate kinase systems that phosphorylate tau, a prerequisite aggregation event (Bush et al., 1995; Luo et al., 1997).

XIV. Concluding Remarks

In closing, the authors of this review offer the following comments. SK: 1. At present, a much more compelling case can be made for the amyloid cascade hypothesis of AD than for the mitochondrial hypothesis of AD, as proof of concept in the human has been described in which a hereditary amyloid (APP) abnormality causes (albeit by unknown mechanism) a form of AD. Conversely, no causal association between any mitochondrial abnormality and AD has ever been reported. 2. In future studies, examination of large numbers of patients with AD will probably reveal a modest overall reduction of brain cytochrome oxidase activity. However, high scatter between the range of AD and control values will indicate that the change is not a robust deﬁning feature of the disorder and therefore unlikely to be etiologically important for at least many patients with AD. 3. The available data are too discrepant, even in studies of mitochondrially enriched fractions (see especially Bosetti et al., 2002) to establish whether a cytochrome oxidase reduction occurs in nondegenerating brain areas in AD, as would be expected if the enzyme defect were “systemic.” 4. In future studies, much more attention should be focused on investigations of the behavior of cytochrome oxidase in non-CNS (e.g., platelets, ﬁbroblasts, cybrid cells) versus CNS cells in AD as peripheral cell studies, which provide the strongest support for the mitochondrial hypothesis of AD, are much less affected by confounds related to degenerative processes. The ﬁnding of decreased cytochrome oxidase activity in non-CNS tissue of some patients with AD is exciting but, after more than a decade of intense investigation, still requires explanation.

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5. A mitochondrial etiological hypothesis of AD, although quite plausible, will not be accepted until an actual mtDNA “mutation” (the smoking gun) is discovered. Investigators studying AD cybrid and blood cells must devote much more effort to characterization in the cells of the actual mtDNA abnormality (if present) assumed to explain the enzyme defect. RS: 1. Confusion regarding the consistency and/or distribution of the AD cytochrome oxidase defect arises primarily from discrepancies between homogenate-based studies. Enriched mitochondrial fraction data, meanwhile, suggest reduced cytochrome oxidase activity in degenerating AD brain regions, nondegenerating AD brain regions, and AD platelets. The ability of enriched mitochondrial fraction studies to detect defects where homogenates do not suggests that either purifying mitochondria creates a speciﬁc cytochrome oxidase defect artifact, or homogenate-based assays are less sensitive. The latter explanation seems more likely. In any case, it should be possible to experimentally resolve this issue. 2. It is possible to relate cytochrome oxidase dysfunction and mitochondrial abnormalities to multiple AD phenomena. Under experimental conditions, both seem able to drive AD-related pathology. This suggests mitochondria in general and cytochrome oxidase function speciﬁcally are relevant to AD pathophysiology. Arbitrarily declaring observed AD mitochondrial dysfunction simply is not “robust” enough to be pathophysiologically relevant does not detract from data indicating it is relevant. 3. Observations of cytochrome oxidase dysfunction in both cerebral and noncerebral AD subject tissues are consistent with a potential underlying genetic basis. Cybrid studies support this possibility, but the hypothesis remains unproven (or disproven) at a nucleotide sequence level. Future research on low abundance heteroplasmies, compound microheteroplasmies, and polymorphism combinations is necessary. A better understanding of how particular cytochrome oxidase mitochondrial genenuclear gene polymorphism combinations inﬂuence enzyme physiology is required. 4. The strength of a particular AD paradigm should be determined by its ability to explain any and all aspects of the disease. Comprehensive hypotheses of AD pathogenesis must take into account cerebral and extracerebral mitochondrial dysfunction. At this point, it is not possible to exclude a role for cytochrome oxidase dysfunction in either AD pathophysiology or pathogenesis.

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Acknowledgments

Supported by grants from U.S. National Institute of Aging (RHS) and NIH NINDS (SJK).

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Sheu, K. F., Lai, J. C., and Blass, J. P. (1983). Pyruvate dehydrogenase phosphate (PDHb) phosphatase in brain: Activity, properties, and subcellular localization. J. Neurochem. 40, 1366–1372. Sheu, K. F., Chen, X., and Schon, E. A. (1994). Are there deletions of mtochondrial DNA in Alzheimer’s disease? Neurology 44, A390. Shoffner, J. M., Brown, M. D., Torroni, A., Lott, M. T., Cabell, M. F., Mirra, S. S., Beal, M. F., Yang, C. C., Gearing, M., and Salvo, R., et al. (1993). Mitochondrial DNA variants observed in Alzheimer disease and Parkinson disease patients. Genomics 17, 171–184. Simonian, N. A., and Hyman, B. T. (1993). Functional alterations in Alzheimer’s disease: Diminution of cytochrome oxidase in the hippocampal formation. J. Neuropathol. Exp. Neurol. 52, 580–585. Simonian, N. A., and Hyman, B. T. (1994). Functional alterations in Alzheimer’s disease: Selective loss of mitochondrial-encoded cytochrome oxidase mRNA in the hippocampal formation. J. Neuropathol. Exp. Neurol. 53, 508–512. Sims, N. R., Bowen, D. M., Neary, D., and Davison, A. N. (1983). Metabolic processes in Alzheimer’s disease: Adenine nucleotide content and production of 14 CO2 from [U-14 C]glucose in vitro in human neocortex. J. Neurochem. 41, 1329–1334. Sims, N. R., Finegan, J. M., and Blass, J. P. (1985). Altered glucose metabolism in ﬁbroblasts from patients with Alzheimer’s disease. N. Engl. J. Med. 313, 638–639. Sims, N. R., Finegan, J. M., Blass, J. P., Bowen, D. M., and Neary, D. (1987). Mitochondrial function in brain tissue in primary degenerative dementia. Brain Res 436, 30–38. Sims, N. R., Finegan, J. M., and Blass, J. P. (1987). Altered metabolic properties of cultured skin ﬁbroblasts in Alzheimer’s disease. Ann. Neurol. 21, 451–457. Smith, M. A., Drew, K. L., Nunomura, A., Takeda, A., Hirai, K., Zhu, X., Atwood, C. S., Raina, A. K., Rottkamp, C. A., Sayre, L. M., Friedland, R. P., and Perry, G. (2002). Amyloid-beta, tau alterations and mitochondrial dysfunction in Alzheimer disease: The chickens or the eggs? Neurochem. Int. 40, 527–531. Sorbi, S., Bird, E. D., and Blass, J. P. (1983). Decreased pyruvate dehydrogenase complex activity in Huntington and Alzheimer brain. Ann. Neurol. 13, 72–78. Sorbi, S., Piacentini, S., Latorraca, S., Piersanti, P., and Amaducci, L. (1995). Alterations in metabolic properties in ﬁbroblasts in Alzheimer disease. Alzheimer Dis. Assoc. Disord. 9, 73–77. Swerdlow, R. H., Parks, J. K., Cassarino, D. S., Maguire, D. J., Maguire, R. S., Bennett, J. P., Jr., Davis, R. E., and Parker, W. D., Jr. (1997). Cybrids in Alzheimer’s disease: A cellular model of the disease? Neurology 49, 918–925. Swerdlow, R. H. (2000). Role of Mitochondria in Parkinson’s Disease. In “Molecular Mechanisms of Neurodegenerative Diseases”(M. F. Chesselet, ed.), pp. 233–270. Humana Press Inc., New Jersey. Swerdlow, R. H. (2002). Mitochondrial DNA–related mitochondrial dysfunction in neurodegenerative diseases. Arch. Pathol. Lab. Med. 126, 271–280. Takeuchi, A., Irizarry, M. C., Duff, K., Saido, T. C., Hsiao Ashe, K., Hasegawa, M., Mann, D. M., Hyman, B. T., and Iwatsubo, T. (2000). Age-related amyloid beta deposition in transgenic mice overexpressing both Alzheimer mutant presenilin 1 and amyloid beta precursor protein Swedish mutant is not associated with global neuronal loss. Am. J. Pathol. 157, 331–339. Tanaka, M., Kovalenko, S. A., Gong, J. S., Borgeld, H. J., Katsumata, K., Hayakawa, M., Yoneda, M., and Ozawa, T. (1996). Accumulation of deletions and point mutations in mitochondrial genome in degenerative diseases. Ann. N.Y. Acad. Sci. 786, 102–111. Tanno, Y., Okuizumi, K., and Tsuji, S. (1998). mtDNA polymorphisms in Japanese sporadic Alzheimer’s disease. Neurobiol. Aging 19, S47–S51.

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Tanzi, R. E., Gusella, J. F., Watkins, P. C., Bruns, G. A., St. George-Hyslop, P., Van Keuren, M. L., Patterson, D., Pagan, S., Kurnit, D. M., and Neve, R. L. (1987). Amyloid beta protein gene: cDNA, mRNA distribution, and genetic linkage near the Alzheimer locus. Science 235, 880–884. Trimmer, P. A., Swerdlow, R. H., Parks, J. K., Keeney, P., Bennett, J. P., Jr., Miller, S. W., Davis, R. E., and Parker, W. D., Jr. (2000). Abnormal mitochondrial morphology in sporadic Parkinson’s and Alzheimer’s disease cybrid cell lines. Exp. Neurol. 162, 37–50. Valla, J., Berndt, J. D., and Gonzalez-Lima, F. (2001). Energy hypometabolism in posterior cingulate cortex of Alzheimer’s patients: Superﬁcial laminar cytochrome oxidase associated with disease duration. J. Neurosci. 21, 4923–4930. Van Zuylen, A. J., Bosman, G. J., Ruitenbeek, W., Van Kalmthout, P. J., and De Grip, W. J. (1992). No evidence for reduced thrombocyte cytochrome oxidase activity in Alzheimer’s disease. Neurology 42, 1246–1247. Verwer, R. W., Jansen, K. A., Sluiter, A. A., Pool, C. W., Kamphorst, W., and Swaab, D. F. (2000). Decreased hippocampal metabolic activity in Alzheimer patients is not reﬂected in the immunoreactivity of cytochrome oxidase subunits. Exp. Neurol. 163, 440–451. Wallace, D. C., Ye, J. H., Neckelmann, S. N., Singh, G., Webster, K. A., and Greenberg, B. D. (1987). Sequence analysis of cDNAs for the human and bovine ATP synthase beta subunit: Mitochondrial DNA genes sustain seventeen times more mutations. Curr. Genet. 12, 81–90. Wallace, D. C. (1992). Mitochondrial genetics: A paradigm for aging and degenerative diseases? Science 256, 628–632. Wallace, D. C. (1994). Mitochondrial DNA sequence variation in human evolution and disease. Proc. Natl. Acad. Sci. USA 91, 8739–8746. Wallace, D. C., Stugard, C., Murdock, D., Schurr, T., and Brown, M. D. (1997). Ancient mtDNA sequences in the human nuclear genome: A potential source of errors in identifying pathogenic mutations. Proc. Natl. Acad. Sci. USA 94, 14900–14905. Webster, M. T., Pearce, B. R., Bowen, D. M., and Francis, P. T. (1998). The effects of perturbed energy metabolism on the processing of amyloid precursor protein in PC12 cells. J. Neural. Transm. 105, 839–853. Wisniewski, H., Terry, R. D., and Hirano, A. (1970). Neuroﬁbrillary pathology. J. Neuropathol. Exp. Neurol. 29, 163–176. Wong-Riley, M. T. (1989). Cytochrome oxidase: An endogenous metabolic marker for neuronal activity. Trends Neurosci. 12, 94–101. Wong-Riley, M., Antuono, P., Ho, K. C., Egan, R., Hevner, R., Liebl, W., Huang, Z., Rachel, R., and Jones, J. (1997). Cytochrome oxidase in Alzheimer’s disease: Biochemical, histochemical, and immunohistochemical analyses of the visual and other systems. Vision Res 37, 3593–3608. Wragg, M. A., Talbot, C. J., Morris, J. C., Lendon, C. L., and Goate, A. M. (1995). No association found between Alzheimer’s disease and a mitochondrial tRNA glutamine gene variant. Neurosci. Lett. 201, 107–110. Yates, C. M., Butterworth, J., Tennant, M. C., and Gordon, A. (1990). Enzyme activities in relation to pH and lactate in postmortem brain in Alzheimer-type and other dementias. J. Neurochem. 55, 1624–1630. Zsurka, G., Kalman, J., Csaszar, A., Rasko, I., Janka, Z., and Venetianer, P. (1998). No mitochondrial haplotype was found to increase risk for Alzheimer’s disease. Biol. Psychiatry 44, 371–373.

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CONTRIBUTIONS OF MITOCHONDRIAL ALTERATIONS, RESULTING FROM BAD GENES AND A HOSTILE ENVIRONMENT, TO THE PATHOGENESIS OF ALZHEIMER’S DISEASE

Mark P. Mattson1 Laboratory of Neurosciences National Institute on Aging Gerontology Research Center Baltimore, Maryland 21224-6825, and Department of Neuroscience Johns Hopkins University School of Medicine Baltimore, Maryland, 21205

I. Overview of Neurodegenerative Cascades in Alzheimer’s Disease II. Mitochondrial Alterations in Alzheimer’s Disease Patients and Experimental Models A. Perturbed Energy Metabolism B. Oxyradical Production C. Dysregulation of Calcium Homeostasis D. Apoptotic Cascades III. Genetic Factors and Mitochondrial Alterations in Alzheimer’s Disease A. APP Mutations B. Presenilin Mutations C. Apolipoprotein E IV. Environmental Factors and Mitochondrial Alterations in Alzheimer’s Disease A. Calorie Intake B. Intellectual and Physical Activities C. Folic Acid V. Conclusions References

I. Overview of Neurodegenerative Cascades in Alzheimer’s Disease

Alzheimer’s disease (AD) involves dysfunction and degeneration of neurons in brain regions involved in learning and memory, emotional behaviors, and stress responses. Histological examination of brain tissue from AD patients reveals two prominent abnormalities, extracellular accumulation 1 To whom correspondence should be addressed at Laboratory of Neurosciences, National Institute on Aging Gerontology Research Center 4F01, 5600 Nathan Shock Drive, Baltimore, MD 21224. [email protected]/gov

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of insoluble aggregates of amyloid β-peptide (Aβ) in the form of plaques, and intraneuronal formation of ﬁlamentous aggregates of the microtubuleassociated protein τ , which are called neuroﬁbrillary tangles (see Yankner, 1996, for review). Abnormal proteolytic processing of the amyloid precursor protein (APP), as the result of genetic and environmental factors that promote AD, results in increased production of a long form of Aβ (Aβ1–42) that readily forms amyloid ﬁbrils. The Aβ is thought to play a major role in promoting dysfunction of synapses and neuronal degeneration and death by rendering neurons vulnerable to age-related increases in levels of oxidative stress and impairments in cellular energy metabolism (see Mattson, 1997, for review). Contributing to the neurodegenerative action of Aβ is disruption of cellular calcium homeostasis (Mattson et al., 1993a; Mark et al., 1995). When in an aggregating form, Aβ induces membrane lipid peroxidation by a mechanism requiring Cu+ and/or Fe2+ (Butterﬁeld et al., 1994; Hensley et al., 1994; Lynch et al., 2000). Membrane lipid peroxidation causes impairment of the function of membrane ion-motive ATPases (Na+/K+- and Ca2+-ATPases), and glucose transporters and glutamate transporters (Mark et al., 1995, 1997a,b; Blanc et al., 1998), resulting in membrane depolarization and reduced energy availability to cells. Accordingly, neurons exposed to aggregating Aβ become hypersensitive to excitotoxicity and apoptosis (Fig. 1). Studies of APP mutant transgenic mice provide further evidence

FIG. 1. Proteolytic processing of APP, and mechanisms for modulation of mitochondrial function by amyloid β-peptide and secreted forms of APP. See text and references (Mattson, 1997; Mattson and Camandola, 2001) for discussion. Aβ, amyloid β-peptide; APP, amyloid precursor protein; GDPK, cyclic GMP-dependent proein kinase; HNE, 4-hydroxy-2,3-nonenal; ROS, reactive oxygen species.

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supporting the hypothesis that Aβ induces cellular oxidative stress and renders neurons vulnerable to excitotoxic and ischemic insults (Zhang et al., 1997; Pappolla et al., 1998). Considerable evidence suggests that many neurons that die in AD do so by a form of programmed cell death called apoptosis (see Mattson, 2000, for review). The increased oxidative stress and elevated intracellular calcium levels induced by Aβ can activate an apoptotic cascade involving prostate apoptosis response-4 (Par-4), and mitochondrial membrane alterations that lead to release of cytochrome c and activation of caspases. Analyses of brain tissue from AD patients reveals alterations in expression of apoptosis-related genes including Bcl-2 family members, Par-4, and DNA damage response genes (Su et al., 1994; Masliah et al., 1998; Guo et al., 1999a). In addition, expression proﬁle analysis of thousands of genes in brain tissue samples from AD patients and age-matched control patients revealed a marked decrease in expression of anti-apoptotic gene called NCKAP1 (Suzuki et al., 2000). Moreover, overactivation of glutamate receptors can elicit changes in the cytoskeleton of neurons similar to those seen in neuroﬁbrillary tangles in AD (Mattson, 1990; Stein-Behrens et al., 1994), and can induce apoptosis (Glazner et al., 2000). The ability of secreted forms of APP, and neurotrophic factors and cytokines to protect neurons against Aβ-induced death (Mattson et al., 1993b; Goodman and Mattson, 1994; Barger et al., 1995; Mark et al., 1997c), provides further evidence for the involvement of apoptosis in AD. In addition, studies of the pathogenic actions of mutations in APP and presenilins-1 and 2 have provided quite convincing evidence that neurons die by apoptosis in AD (see Section III.B).

II. Mitochondrial Alterations in Alzheimer’s Disease Patients and Experimental Models

A. PERTURBED ENERGY METABOLISM Functional brain imaging studies in AD patients have demonstrated consistently a deﬁcit in cellular glucose utilization in brain regions affected by the disease, and this energy deﬁcit appears to precede and accompany the neurodegenerative process (Munch et al., 1998). In addition, Blass and coworkers have reported that enzymatic activity of α-ketoglutarate dehydrogenase is markedly reduced in brain tissue from AD patients (Blass, 2001). Cytochrome oxidase activity was reported to be decreased signiﬁcantly in brain tissue from AD patients compared to age-matched control patients (Kish et al., 1992; Mutisya et al., 1994). The defect in cytochrome c oxidase appears to be widespread, and may precede the neurodegenerative process because activity levels of the enzyme are also decreased in platelets from AD

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patients (Parker et al., 1994). Additional studies of postmortem brain tissue, and of ﬁbroblasts and lymphocytes from living patients, have provided evidence for a widespread abnormality in mitochondrial energy metabolism in AD (Blass, 1993). Experimental ﬁndings further support a role for impaired mitochondrial ATP production in the pathogenesis of AD. Exposure of cultured hippocampal neurons to Aβ results in ATP depletion (Mark et al., 1995), and cellular glucose uptake is decreased in the brains of mice expressing an AD-linked mutant form of APP (Dodhart et al., 1999). Interestingly, peripheral glucose metabolism is also altered in APP mutant mice, such that they become hypoglycemic when subjected to food deprivation or restraint stress (Pedersen et al., 1999). Genetic analyses have provided evidence for associations between mutations in genes encoding mitochondrial proteins including cytochrome c oxidase and AD (Davis et al., 1997). Studies in which “cybrid” cells are produced by fusing platelets from AD patients or control patients with neuroblastoma cells have shown that mitochondria from AD patients exhibit aberrant calcium homeostasis and increased production of oxyradicals (Sheehan et al., 1997).

B. OXYRADICAL PRODUCTION It is very clear that levels of cellular oxidative stress are increased in neurons involved in the neurodegenerative process in AD. Increased levels of protein oxidation, membrane lipid peroxidation, and oxidative damage to DNA are observed in association with neuritic plaques and neuroﬁbrillary tangles (Smith et al., 1991; Moccoci et al., 1994; Good et al., 1996; Smith et al., 1997; Fu et al., 1998). Lipid peroxidation products, including 4-hydroxynonenal, are increased in the cerebrospinal ﬂuid of AD patients releative to age-matched control patients (Lovell et al., 1997) and in degenerating neurons (Montine et al., 1997), suggesting a widespread increase in cellular oxidative stress in the brain. Cellular responses to the increased oxidative stress are suggested by increased expression of antioxidant enzymes such as catalase, Mn-SOD and Cu/Zn-SOD (superoxide dismutase)(Bruce et al., 1997). Lipid peroxidation induced by Aβ may contribute to mitochondrial dysfunction and neuronal death in AD as indicated by the ability of antioxidants that suppress membrane lipid peroxidation (e.g., vitamin E, propyl gallate and uric acid) or detoxify 4-hydroxynonenal (e.g., glutathione) to stabilize mitochondrial function and promote the survival of neurons exposed to Aβ (Goodman and Mattson, 1994; Mark et al., 1995, 1997a; Keller et al., 1998a). Epidemiological and clinical data support the oxidative stress hypothesis of mitochondrial dysfunction and neuronal degeneration in AD. Increased

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dietary supplementation with vitamins E and C is associated with reduced risk for AD (Morris et al., 1998), and estrogen replacement therapy in postmenopausal women may decrease their risk for AD (Tang et al., 1996). Estrogen exhibits antioxidant activity, and suppression of membrane lipid peroxidation may account for its ability to protect neurons against Aβ and Fe2+ (Goodman et al., 1996). A clinical trial of vitamin E in patients with mild AD resulted in a signiﬁcant slowing of the progression of the disease (Sano et al., 1997). Moreover, the beneﬁt of anti-inﬂammatory drugs in AD patients may result from their well-established antioxidant activity. Finally, dietary restriction, a manipulation that extends life span and reduces incidence of age-related diseases (Weindruch and Sohal, 1997), can reduce levels of oxidative stress and protect hippocampal and cortical neurons against death in animal models relevant to the pathogenesis of AD (Bruce-Keller et al., 1999; Zhu et al., 1999).

C. DYSREGULATION OF CALCIUM HOMEOSTASIS Neurons degenerating in the brains of AD patients exhibit increased levels of intracellular calcium, as suggested by increased activation of calciumdependent proteases (Nixon et al., 1994) and increased overall concentrations of calcium (protein-bound and free) in neuroﬁbrillary tangle-bearing neurons (Murray et al., 1992). Levels of calcium/calmodulin-dependent protein kinase II are increased in neurons prone to degeneration (McKee et al., 1990), and this kinase may play a role in the formation of neuroﬁbrillary tangles as it can associate directly with paired helical ﬁlaments (Xiao et al., 1996). In addition, transglutaminase, a calcium-activated enzyme, is increased in AD, and has been shown to induce crosslinking of τ protein, the major component of neuroﬁbrillary tangles (Miller and Johnson, 1995). Experimental studies in cell culture and animal models relevant to AD provide support for a cause–effect relationship between altered calcium homeostasis and neuronal dysfunction and death. For example, overactivation of glutamate receptors, and other conditions that result in a sustained elevation of intracellular Ca2+ levels, can induce alterations in the neuronal cytoskeleton similar to those seen in neuroﬁbrillary tangles (Mattson, 1990; Stein-Behrens et al., 1994). What factors result in disruption of neuronal calcium homeostasis in AD, and what are the links between altered calcium homeostasis and mitochondrial dysfunction? It is very clear that increased levels of oxidative stress and impaired energy metabolism, as occur in AD, can compromise the ability of neurons to properly regulate intracellular calcium levels (Mattson, 1997). It has been directly demonstrated that Aβ can impair the ability of neurons to regulate intracellular calcium levels, and that this dysregulation

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of calcium homeostasis contributes to the synaptic dysfunction and cell death caused by Aβ (Mattson et al., 1993a; Mark et al., 1995; Keller et al., 1997). On the other hand, secreted form of amyloid precursor protein (sAPPα) signaling can stabilize calcium homeostasis by activating potassium channels (Furukawa et al., 1996) and the transcription factor NF-κB (Barger and Mattson, 1996). Moreover, mutations in presenilins that cause AD may promote neuronal degeneration by a rather direct effect on neuronal calcium homeostasis (Guo et al., 1996, 1997, 1999a). It has been shown that sustained elevations of the cytoplasmic calcium concentration can promote mitochondrial oxyradical (superoxide) production, membrane depolarization, and ATP depletion (Guo et al., 1998b, 1999b), suggesting a contribution of altered calcium homeostasis to mitochondrial dysfunction in AD. Conversely, impairment of mitochondrial function can result in increased cytoplasmic calcium levels, and studies of experimental models of AD suggest that mitochondrial impairment may indeed promote dysregulation of neuronal calcium homeostasis (Keller et al., 1998; Kruman et al., 1998; Kruman and Mattson, 1999).

D. APOPTOTIC CASCADES Mitochondria play pivotal roles in apoptosis in many different cell types including neurons in both physiological settings and disease states (D’Mello, 1998; Mattson, 2000). A major role for apoptosis in the pathogenesis of AD is suggested by studies documenting increased DNA damage and caspase activity, alterations in expression of apoptosis-related genes such as Bcl-2 family members, and Par-4 and DNA damage response genes in the brains of patients (Su et al., 1994; Guo et al., 1998a; Masliah et al., 1998; Chan et al., 1999). Amyloid β-peptide can induce apoptosis in cultured neurons directly (Loo et al., 1993; Mark et al., 1995) and can greatly increase their vulnerability to death induced by conditions, such as increased oxidative stress and reduced energy availability, that are known to occur in the brain during aging (Mattson et al., 1993a; Guo et al., 1996, 1997). Amyloid β-peptide promotes apoptosis by inducing membrane lipid peroxidation, which impairs the function of membrane ion-motive ATPases and glucose and glutamate transporters, resulting in membrane depolarization, ATP depletion, excessive Ca2+ inﬂux and mitochondrial dysfunction (Mark et al., 1995, 1997a). Accordingly, antioxidants that suppress lipid peroxidation and drugs that stabilize cellular Ca2+ homeostasis can protect neurons against Aβ-induced apoptosis. Additional evidence supporting a role for apoptosis in AD comes from studies showing that neurotrophic factors and cytokines known to prevent neuronal apoptosis can protect neurons against Aβ-induced death

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(Barger et al., 1995; Mark et al., 1997c). Moreover, APP is a substrate for caspase-3 (Weidemann et al., 1999), and caspase-mediated cleavage of APP can release a C-terminal peptide called C31 that is a potent inducer of apoptosis (Lu et al., 2000). Interestingly, the process of apoptosis may begin in synaptic terminals. Thus, Aβ, which is deposited prominently in synaptic regions, can induce Par-4 production, caspase activation, and mitochondrial dysfunction in cortical synaptosomes and dendrites (Mattson et al., 1998; Duan et al., 1999). The latter studies showed that Par-4 can induce mitochondrial dysfunction (membrane depolarization and oxyradical production) in synapses and dendrites. Interestingly, mutations in presenilin-1 (PS1) that cause early-onset inherited forms of AD have been shown to have adverse effects on synaptic function, and to promote synaptic degeneration and neuronal apoptosis (Guo et al., 1997, 1999; Begley et al., 1999; Parent et al., 1999). Mutant PS1 facilitates apoptosis at an early step prior to Par-4 production, mitochondrial dysfunction, and caspase activation (Fig. 2). As described below, mutant PS1 may promote apoptosis by perturbing calcium regulation in the endoplasmic reticulum in a manner that increases calcium release from ryanodinesensitive stores when neurons are exposed to potentially damaging oxidative and metabolic insults (Guo et al., 1997; Chan et al., 2000). These disturbances

FIG. 2. Mechanisms whereby genetic aberrancies, aging, and environmental factors promote neuronal degeneration in AD. See text for discussion. EAA, excitatory amino acid; Par-4, prostate apoptosis response-4.

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in calcium homeostasis have been shown to be present in synaptosomes isolated from PS1 mutant transgenic mice (Begley et al., 1999), suggesting that this mechanism might contribute to the synaptic pathology in AD patients. III. Genetic Factors and Mitochondrial Alterations in Alzheimer’s Disease

It has been proposed that mutations in mitochondrial DNA (Chang et al., 2000; Chinnery et al., 2001; Qui et al., 2001) and in nuclear genes encoding mitochondrial proteins (Sheu et al., 1999) underlie some cases of AD. A. APP MUTATIONS Although most cases of AD occur very late in life and in a manner suggesting no major genetic basis, some cases of AD present with an early onset (30–50 years old) and are inherited in an autosomal dominant manner (Hardy, 1997). Familial forms of AD can be caused by mutations in genesencoding APP (located on chromosome 21), presenilin-1 (chromosome 14), and presenilin-2 (chromosome 1). Amyloid precursor protein is a large integral membrane protein with a single membrane-spanning domain and a large extracellular region that possesses several biologically active domains (Fig. 1). An enzyme activity called α-secretase cleaves APP in the middle of the Aβ sequence at the cell surface resulting in the release of an extracellular portion of APP (sAPPα) that has been shown to play important roles in modulating synaptic plasticity and neuron survival (Mattson et al., 1993b; Mattson, 1994; Furukawa et al., 1996; Ishida et al., 1997). Alternative cleavage of APP at the N-terminus of Aβ by an enzyme called β-secretase (or BACE) generates a membrane-associated C-terminal fragment that contains intact Aβ (Vassar et al., 1999). The BACE-derived APP product is then further cleaved by an enzyme activity called γ -secretase that liberates fulllength Aβ (Aβ1–40 or Aβ1–42) from cells. The APP mutations that cause AD are located at either end, or in the middle, of the Aβ sequence, and alter the proteolytic processing of APP in a manner that increases production of Aβ and decreases production of sAPPα. As now described, Aβ promotes mitochondrial dysfunction, whereas sAPPα stabilizes mitochondrial function. Exposure of cultured neurons to Aβ results in oxidative stress and elevations of intracellular calcium levels, which promotes mitochondrial oxyradical production and membrane depolarization (Keller et al., 1997; Mattson et al., 1997). Overexpression of mitochondrial Mn-SOD in cultured cells protects them from being killed by Aβ (Keller et al., 1998a), demonstrating

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a pivotal role for mitochondrial superoxide production in the neurotoxic action of Aβ. Mitochondrial membrane permeability transition is central to the cell death process induced by Aβ because agents that inhibit the permeability transition, such as cyclosporine A, protect cells against Aβ-induced death (Keller et al., 1998a). Moreover, Aβ induces uptake of calcium into mitochondria, which appears to contribute to impairment of mitochondrial function and depletion of ATP in neurons (Mark et al., 1995). While much attention has focused on the neurotoxic action of Aβ, the decreased production of sAPPα that results from APP mutations may also play a major role in mitochondrial dysfunction and cell death. Exposure of neurons to sAPPα results in membrane hyperpolarization and increased resistance of neurons to glutamate-induced cell death due to reduced calcium inﬂux through N-methyl-D-aspartate (NMDA) receptors and voltagedependent channels(Furukawa et al., 1996; Furukawa and Mattson, 1998). The sAPPα activates a membrane receptor linked to cyclic GMP production resulting in activation of cyclic GMP-dependent protein kinase (GDPK). GDPK, in turn activates a phosphatase that dephosphorylates, and thereby activates, a high-conductance potassium channel. In addition, GDPK activates the transcription factor NF-κB, which can stabilize mitochondrial function by inducing the expression of Mn-SOD, Bcl-2, and the calciumbinding protein calbindin (Barger and Mattson, 1996; Mattson et al., 1997; Camandola et al., 2000).

B. PRESENILIN MUTATIONS Presenilin-1 (PS1) and presenilin-2 (PS2) are homologous integral membrane proteins that contain eight transmembrane domains with both the amino and carboxy termini, and a large hydrophilic loop domain, located on the cytoplasmic side of the membrane (Hardy, 1997). In addition to the 46 kDa PS1 holoprotein, many cells contain 26 kDa N-terminal and 17 kDa C-terminal PS1 cleavage products. Presenilins are expressed in various cell types where they are localized primarily in the endoplasmic reticulum (ER). In the brain, PS1 is expressed at high levels in neurons and at lower levels in glial cells. More than 70 different missense mutations in PS1 have been linked to familial early-onset AD. The sites of the mutations are scattered throughout the PS1 protein, but “hot spots” are located adjacent to transmembrane domain 2 and in the hydrophilic loop region. In addition to the missense mutations, one familial Alzheimer’s disease (FAD) kindred has been shown to harbor a mutant form of PS1 in which exon 9 is deleted. Mutations in PS2 are a more rare cause of FAD, with only a few mutations having been identiﬁed to date.

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FIG. 3. Possible mechanisms whereby presenilin mutations promote mitochondrial dysfunction in Alzheimer’s disease. See text and references (Guo et al., 1998a,b, 1999a; Mattson et al., 2000b) for discussion. AMPA, 2-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; GRP78, glucose regulated protein-78; IP3R, inositol trisphosphate receptor; NMDA, N-methylD-aspartate receptor; PTP, permeability transition pore; ROS, reactive oxygen species; RyR, ryanodine receptor; VDCC, voltage-dependent calcium channel.

A primary defect in neurons expressing PS1 mutations is an alteration in calcium signaling in the ER such that there is a greater pool of calcium available for release (Guo et al., 1996, 1997; Chan et al., 2000). Calcium release through ryanodine- and inositol triphosphate (IP3)-sensitive channels is increased when neurons are stimulated with glutamate or agonists that activate receptors linked to IP3 production (Fig. 3). PS1 may interact directly or indirectly with ryanodine receptors (Chan et al., 2000), but the speciﬁc molecular alterations that lead to perturbed cellular calcium homeostasis are unknown. Nevertheless, studies of transfected pheochromocytoma 12 (PC12) cells and PS1 mutant knockin mice have shown that the enhanced ER calcium release caused by PS1 mutations leads, secondarily, to impaired mitochondrial function (Guo et al., 1997, 1999a; Keller et al., 1998b). Speciﬁcally, mitochondrial membrane depolarization, calcium uptake, and oxyradical production are increased in neurons expressing mutant PS1 when the cells are subjected to oxidative, metabolic, and excitotoxic insults. Accordingly, overexpression of Mn-SOD or the calcium-binding protein calbindin, and treatment with cyclosporine A can counteract the cell death-promoting action of PS1 mutations (Guo et al., 1998b, 1999b). Hippocampal and cortical neurons in PS1 mutant knockin mice exhibit increased vulnerability to excitoxicity and ischemic injury (Guo et al., 1999a; Mattson et al., 2000a). The PS1 mutations may exert their effects in synapses as suggested by studies

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demonstrating abnormal synaptic transmission in hippocampal slices from PS1 mutant transgenic mice (Parent et al., 1999). Mitochondrial dysfunction, associated with abnormal calcium homeostasis, was documented in studies of cerebral cortical synaptosomes from PS1 mutant transgenic mice (Begley et al., 1999). C. APOLIPOPROTEIN E The apolipoprotein E alleles one inherits can affect their risk of AD, such that individuals with an E4 allele are at increased risk (Strittmatter et al., 1993). Cerebrovascular effects of ApoE4 might contribute to its ADpromoting action. On the other hand, ﬁndings have provided evidence that apoE possesses isoform-speciﬁc antioxidant activity (Miyata and Smith, 1996). Interestingly, ApoE isoforms differ in that E2 contains two cysteine residues, E3 contains one cysteine residue, and E4 contains none. Studies of interactions of pure apoE proteins with the neurotoxic aldehyde 4-hydroxynonenal showed that the isoforms differ in the amount of the aldehyde they can bind, with the order E2 > E3 > E4 (Pedersen et al., 1999). The ApoE 2 and ApoE3 were able to protect cultured hippocampal neurons against Aβ-induced death, whereas ApoE4 was ineffective. The latter ﬁndings may explain the relationship between apoE genotype and the susceptibility to AD. With regard to mitochondrial alterations, ApoE4 may promote mitochondrial dysfunction by exacerbating 4-hydroxynonenal modiﬁcation of mitochondrial regulatory proteins (Kristal et al., 1996). Indeed, it was reported that 4-hydroxynonenal inhibits mitochondrial respiration at several sites in the electron transport chain (Picklo et al., 1999). IV. Environmental Factors and Mitochondrial Alterations in Alzheimer’s Disease

A. CALORIE INTAKE Emerging ﬁndings suggest that individuals with a high calorie intake are at increased risk of age-related neurodegenerative disorders including AD. Epidemiological data supporting a link between calories and neurodegenerative disorders include studies of a cohort of people in New York City in which those with the lowest calorie intakes were at the lowest risk of Parkinson’s disease (Logroscino et al., 1996) and AD (Mayeux et al., 1999). Moreover, it was recently reported that genetically similar populations living in different environments have markedly different risks of AD, and that a major difference in lifestyles is that those in the high-risk environment have a markedly

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greater calorie intake (Hendrie et al., 2001). Experimental data supporting an inﬂuence of calorie intake on risk include the well-established observation that dietary restriction increases life span and suppresses age-related molecular alterations in the brain, and that dietary restriction increases resistance of neurons in the brain to oxidative and excitotoxic insults relevant to the pathogenesis of AD (Bruce-Keller et al., 1999; Zhu et al., 1999). Interestingly, dietary restriction can also enhance neurogenesis (Lee et al., 2000), which may provide a larger reserve of neural cells for maintenance of cognitive function during aging. A hormesis-like mechanism may explain how dietary restriction beneﬁts the brain. Studies of rats and mice have shown that dietary restriction induces the expression of heat-shock protein-70 and glucose-regulated protein-78 in neurons in the hippocampus, cerebral cortex, and striatum (Lee et al., 1999a; Duan and Mattson, 1999; Yu and Mattson, 1999). These two stress-responsive protein chaperones have been shown to protect neurons against excitotoxic, metabolic, and oxidative insults relevant to AD (Lowenstein et al., 1991; Yu and Mattson, 1999; Yu et al., 1999). That a cellular stress response is involved in the neuroprotective effect of dietary restriction is further supported by studies showing that rats and mice given 2-deoxy-D-glucose, a nonmetabolizable glucose analogue known to induce an energetic stress, exhibit increased resistance of neurons in their brain to excitotoxic, ischemic, and oxidative insults in experimental models relevant to AD, stroke, and Parkinson’s disease (Duan and Mattson, 1999; Lee et al., 1999a; Yu and Mattson, 1999). Interestingly, dietary restriction can induce the expression of several neurotrophic factors in the brain, prominent among which is brain-derived neurotrophic factor (BDNF) (Lee et al., 2000; Duan et al., 2001). The BDNF can suppress oxyradical production and stabilize mitochondrial function, and can protect neurons against excitotoxic and metabolic insults (Cheng and Mattson, 1994; Mattson et al., 1995). The BDNF appears to make a major contribution to the neuroprotective effect of dietary restriction because the effect is signiﬁcantly attenuated when a BDNF blocking antibody is infused into the lateral ventricles (Duan et al., 2001). Dietary restriction and 2-deoxy-D-glucose administration can stabilize mitochondrial function in synapses. Thus, mitochondrial membrane potential is maintained and oxyradical production decreased after exposure to Aβ, iron, and mitochondrial toxins in cortical synaptosomes from rats maintained on dietary restriction (Guo et al., 2000). The latter study further showed that glucose transport is enhanced in synaptosomes from rats maintained on dietary restriction compared to rats fed ad libitum. Levels of heatshock protein-70 and glucose-regulated protein-78 were increased in synaptosomes from rats maintained on dietary restriction. A similar stabilization

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of mitochondrial function was observed in synaptosomes from rats given 2-deoxy-D-glucose. Thus, dietary restriction can protect synapses and neurons against dysfunction and death induced by Aβ and other insults relevant to AD, suggesting that dietary restriction in adult life may decrease risk of AD in humans.

B. INTELLECTUAL AND PHYSICAL ACTIVITIES There is an inverse relationship between educational attainment and risk for AD such that more educated persons are at reduced risk (Evans et al., 1997). For example, a recent study of a population of nuns demonstrated that those with the best linguistic abilities were at reduced risk for AD (Snowdon et al., 1996). These kinds of data suggesting that intellectual activity is neuroprotective are supported by data from studies of animals maintained in “enriched environments. For example, Greenough and co-workers showed that rats raised in enriched environments exhibit increased complexity of dendritic arbors and synapses in the hippocampus and cerebellum, suggesting an increased functional reserve (Black et al., 1989; Kleim et al., 1997). During aging synaptic density decreases in hippocampus in rats, and maintenance of rats in an enriched environment can prevent such age-related synaptic loss (Saito et al., 1994). Environmental enrichment also stimulates neurogenesis and improves learning and memory ability (Kemperman et al., 1997; Nilsson et al., 1999). Environmental enrichment may also increase resistance of neurons to injury and promote recovery after injury, as demonstrated by the amelioration of functional deﬁcits caused by bilateral lesions of the frontal cortex in rats (Kolb and Gibb, 1991). Environmental enrichment can also improve outcome after a stroke in rats ( Johansson, 1996), suggesting an attenuation of the delayed neurodegenerative process or enhanced recovery of function of surviving neurons. Indeed, postinjury environmental enrichment improves learning and memory performance in a water maze test after lesion of cholinergic basal forebrain neurons in rats (van Rijzingen et al., 1997). Being a couch potato is not a good thing, for neither your body nor your brain. Experimental ﬁndings suggest that physical activity may also counteract the adverse effects of aging and disease on the brain. For example, mice allowed access to a running wheel exhibit increased neurogenesis and improved learning and memory compared to sedentary mice (van Praag et al., 1999). In addition, epidemiological studies in humans show that regular vigorous physical activity can reduce risk for ischemic stroke (Lee et al., 1999b); although it has not been established that prior physical activity can improve outcome after a stroke, this would seem plausible. Physical activity

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can also beneﬁt the brain after injury. Exercise after brain injury improved functional outcome in rats, and the improved outcome was associated with enhanced structural plasticity in the motor cortex ( Jones et al., 1999).

C. FOLIC ACID Folic acid deﬁciency may increase risk of several different age-related diseases including coronary artery disease (Swain and St Clair, 1997), stroke (Elkind and Sacco, 1998), and several types of cancer (Choi and Mason, 2000). By causing a methyl donor deﬁciency state, a low intake of folic acid may promote accumulation of DNA damage as the result of uracil misincorporation, hypomethylation, and reduced ability to repair the damaged DNA. Recent ﬁndings suggest that increased levels of homocysteine is a consequence of folic acid deﬁciency that plays an important role in the pathogenesis of cardiovascular disease and stroke (Refsum et al., 1998), and possibly AD (Clarke et al., 1998; Snowdon et al., 2000) and Parkinson’s disease (Kuhn et al., 1998). Homocysteine is produced from methionine, an amino acid that plays a key role in the generation of methyl groups required for numerous biochemical reactions. Homocysteine can be remethylated to methionine by enzymes that require folic acid and cobalamin (vitamin B12), or it can be catabolized by cystathionine β-synthase, a pyridoxine (vitamin B6)dependent enzyme, to form cysteine (Scott and Weir, 1998). One-carbon metabolism is crucial for normal DNA synthesis and repair, because one carbon groups are essential for the de novo synthesis of both purines and pyrmidine thymidylate (Choi and Mason, 2000). It was recently reported that homocysteine can induce apoptosis of cultured rat hippocampal neurons (Kruman et al., 2000), suggesting that homocysteine can directly damage neurons. Exposure of cultured hippocampal neurons to homocysteine can induce apoptosis and increase their vulnerability to excitotoxicity (Kruman et al., 2000). Homocysteine induces apoptosis by a mechanism involving increased DNA damage, activation of poly(ADP-ribose) polymerase and p53. When hippocampal neurons are incubated in medium deﬁcient in folic acid and methionine, or with elevated homocysteine levels, they become extremely vulnerable to death induced by Aβ (Kruman et al., 2001). Folic acid deﬁciency or Aβ alone cause DNA damage that can be repaired, whereas exposure to Aβ under conditions of methyl donor deﬁciency results in increased DNA damage as the result of impaired DNA repair. When maintained on a folic acid deﬁcient diet, amyloid precursor protein mutant transgenic mice, but not wild-type mice, exhibit increased cellular DNA damage and hippocampal neurodegeneration. When taken together with

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the epidemiological data (Clarke et al., 1998; Snowdon et al., 2000), the data obtained in cell culture and animal models of AD suggest that DNA damage induced by folic acid deﬁciency and homocysteine results from uracil misincorporation that may sensitize neurons to DNA damage and death by creating an imbalance in deoxynucleotide precursors required for DNA repair.

V. Conclusions

Several different genetic and environmental factors have been identiﬁed that either cause, or increase the risk of, AD. Each genetic causal or predisposition factor promotes increased oxidative stress and disrupts cellular calcium homeostasis in neurons. Mitochondrial function is compromised by oxyradicals and calcium overload resulting in further oxyradical production, impaired calcium homeostasis, and activation of apoptotic caspases. Age-related DNA damage (nuclear and mitochondrial) may endanger neurons and render them vulnerable to excitotoxicity and apoptosis. Environmental factors that may predispose to AD include excessive caloric intake, folic acid insufﬁciency, and disuse of neural circuits involved in learning and memory. Based upon the mechanisms described above, novel preventative approaches for AD include dietary restriction, mental aerobics, and dietary supplementation with folic acid and certain anti-oxidants. Therapeutic strategies may include antioxidants, anti-inﬂammatory agents, and drugs that stabilize mitochondrial function.

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MITOCHONDRIA AND AMYOTROPHIC LATERAL SCLEROSIS

Richard W. Orrell∗ ,1 and Anthony H. V. Schapira∗ ,† ∗ University Department of Clinical Neurosciences Royal Free and University College Medical School, and † Institute of Neurology University College London London NW3 2PF, United Kingdom

I. Introduction II. Clinical Features III. Pathogenic Hypotheses A. Evidence for Mitochondrial Abnormalities in ALS B. Lymphocytes C. Platelets D. Skin E. Brain F. Spinal Cord G. Muscle H. Liver IV. Transgenic Mouse Models of ALS A. Cell Lines B. Organotypic Spinal Cord Cultures V. Conclusion References

I. Introduction

Amyotrophic lateral sclerosis (ALS) is a disease of motor neurons, and is sometimes referred to as “motor neuron disease” (MND). There are a number of other rarer diseases of motor neurons, including genetically identiﬁable conditions—for example, spinal muscular atrophy (Survival Motor Neuron gene) (Lefebvre et al., 1995), and bulbospinal muscular atrophy or Kennedy’s disease (a trinucleotide CAG repeat in the androgen receptor gene) (Merry et al., 1998). The distinguishing pathological feature of ALS is the presence of both upper motor neuron (corticospinal tract) and lower motor neuron (anterior horn cell) involvement. More speciﬁc pathological features include a range 1

To whom correspondence should be addressed.

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of nuclear inclusions, especially ubiquitinated protein aggregates (Ince et al., 1998). ALS affects individuals of all ages, the incidence increasing with age, especially over age 50 years. Rare juvenile forms are also recognized. Amyotrophic lateral sclerosis is up to two times more frequent in males than females. The mean age at onset is 61 years and mean duration of disease 3.6 years (Eisen and Krieger, 1998). There is, however, a small proportion of patients with more prolonged survival, sometimes over a period of 20 years (Orrell et al., 1995). Amyotrophic lateral sclerosis is found worldwide, with a variable incidence of around 2:100,000 per year, and prevalence of 6:100,000 population (Eisen and Krieger, 1998). The cause of the disease is unclear. Approximately 5% of patients have a family history of another affected individual with ALS (Orrell and Figlewicz, 2001). Some of these families show typical autosomal dominant inheritance. Around 20% of all families have mutations in the gene for copper/zinc superoxide dismutase (SOD1) (Rosen et al., 1993). These are largely point mutations, but with occasional nonsense, insertion, and deletion mutations (Orrell, 2000). A small proportion of apparently sporadic patients with ALS also have SOD1 mutations. For one particular mutation of SOD1, D90A, inheritance is also autosomal recessive, with only the homozygote individuals manifesting the disease. These patients are found particularly in northern Sweden and Finland (Andersen et al., 1996). Altogether, SOD1 mutations account for only 1–2% of patients with ALS. Mutations of a gene encoding alsin, a protein with three guanine–nucleotide exchange factor domains, a putative GTPase regulator, has been identiﬁed in a rare autosomal recessive form of ALS (ALS2) (Hadano et al., 2001; Yang et al., 2001). Loci for three other forms of autosomal-dominant forms of ALS are recognized, but the genes not yet identiﬁed. Epidemiological studies have suggested toxic exposure to heavy metals, and electrical or physical trauma, as possible precipitants, although with no further proof of causation. A particular cluster of patients in the western Paciﬁc, including Guam and the Kii peninsula in Japan, was recognized to have a combination of ALS and Parkinson’s disease. The incidence peaked at 179:100,000 for males in 1960 (Zhang et al., 1996). This has since fallen considerably toward levels found elsewhere in the world. The cause of this epidemic was not identiﬁed, but hypotheses included the toxic ingestion of the cycad plant (O’Gara et al., 1964), or other environmental factors. The pathological features in Guamian ALS are distinct from those in ALS found elsewhere, with neuroﬁbrillary tangles similar to those found in Alzheimer’s disease present in regions including hippocampus, substantia nigra, locus coeruleus, and anterior horn cells (Hirano et al., 1966).

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II. Clinical Features

The initial clinical presentation of ALS is with features of either lower motor neuron disorder, upper motor neuron disorder, or a combination of both. Typical features include weakness of a hand or foot, with additional muscle wasting and twitching of the muscle (fasciculations) as a result of the lower motor neuron denervation. Reﬂexes in the limb are diminished. Upper motor neuron involvement presents with stiffness or spasticity of the limb, and muscle spasm may be experienced. The reﬂexes are brisk. The combination of both upper and lower motor neuron involvement leads to a variable manifestation of a combination of these features, which may vary through the course of the disease (Fig. 1; see color insert). The cranial nerves may also be involved. In a bulbar palsy (lower motor neuron) there may be weakness of eye closure or mouth movement, and weakness, wasting, and fasciculation of the tongue. In a pseudobulbar palsy (upper motor neuron) there is stiffness or spasticity of face and tongue movement, and disturbance of swallowing reﬂexes. This leads to difﬁculties with speech and swallowing. Unfortunately, the condition progresses relentlessly, leading to increasing immobility, and to loss of speech and swallowing. Cognition is generally unimpaired, although some mild abnormalities may be found on psychological testing. There may be emotional lability, especially related to pseudobulbar palsy. The most common ﬁnal cause of death is respiratory insufﬁciency as a result of muscle weakness of the chest and accessory respiratory muscles, or from aspiration pneumonia as a result of the impaired swallowing reﬂexes. There is no cure for ALS. Clinical trials have demonstrated that riluzole has some effect in slowing the course of the disease, although the effects of this are modest (Lacomblez et al., 1996; Miller et al., 2000). The precise mode of action of riluzole is uncertain, but it was initiated because of its inhibition of glutamate release—glutamate excitotoxicity being implicated in the pathogenesis of ALS. Riluzole is taken as a tablet twice daily, and its relative lack of serious side effects (occasional disturbance of liver function and fatigue are the most common problems) has encouraged its use in slowing disease progression of patients with ALS. Other strategies for management of disability include physiotherapy, occupational therapy, aids, and appliances. Assistance with speech (including electronic aids), assistance with swallowing (including percutaneous endoscopic gastrostomy—a small tube implanted through the skin into the stomach to enable feeding), and respiratory support (including noninvasive ventilation, which may be performed intermittently using a mask, avoiding invasive tracheostomy) may be required as the disease progresses (Eisen and Krieger, 1998).

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III. Pathogenic Hypotheses

The pathogenesis of ALS remains uncertain. The ﬁnding of SOD1 mutations (Rosen et al., 1993), with reduced SOD1 enzyme activity in erythrocytes, lymphocytes, and brain tissue (Bowling et al., 1993; Deng et al., 1993; Robberecht et al., 1994), initially suggested the possibility of increased free radical mediated damage. More extensive studies, in particular the use of transgenic mouse models, have suggested a gain of function factor, more consistent with the autosomal-dominant inheritance. In particular, in transgenic mice expressing SOD1 mutations found in humans with ALS, mice overexpressing the mutant SOD1, in the presence of normal SOD1 enzyme activity, developed weakness and wasting of the muscles, and features thought similar to human ALS (Gurney et al., 1994). Increased expression of wild-type SOD1 does not cause disease. A range of other hypotheses remain for the causation of ALS. These include excitotoxicity, including a disturbance of glutamate neurotransmission. A wide range of growth factors and their receptors have been studied in experimental situations. These have been developed in clinical therapeutic studies, using brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial-derived neurotrophic factor (GDNF), and insulin-like growth factor (IFG). No clear effect on the disease course of ALS has been demonstrated. Involvement of other neurotransmitters and cellular receptors has also been proposed. Abnormalities of voltage-dependent Ca2+ channels have been investigated, and a possible immunological basis explored. A further consideration is neuroﬁlament pathology. This has been demonstrated in both human and animal models, together with an occasional patient with a mutation in the neuroﬁlament gene. These may be primary or secondary events. The sequence of events that leads to motor neuron death, and the disease ALS, remains unclear. The SOD1 gene mutations are one key, but the initiators may be multifactorial, presumably with common converging pathways. The ﬁnal mechanism of motor neuron death is also uncertain. It is probable that this is by apoptosis, although necrosis may also occur.

A. EVIDENCE FOR MITOCHONDRIAL ABNORMALITIES IN ALS Mitochondrial pathology and function has been studied in a number of tissues, in humans with ALS, and in animal models of ALS. A range of mitochondrial abnormalities have been reported, as summarized in the following paragraphs. The evidence is supportive of mitochondrial defects in

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patients with ALS, but it is not clear whether these are primary or secondary events in the pathogenesis of ALS.

B. LYMPHOCYTES Curti et al. (1996) studied oxidative metabolism and calcium homeostasis in peripheral blood lymphocytes of 21 patients with ALS and in 21 controls. There was no difference in basal oxygen consumption rate, cytochrome c oxidase activity, catalase activity, and lactate production. The authors commented that SOD1 and catalase showed high variability among subjects, and a larger sample size would be preferred. Measurements were not possible in all samples. The increase in basal oxygen consumption induced by an uncoupler of oxidative phosphorylation, 1 μM carbonyl cyanide-p -triﬂuoromethoxyphenylhydrazone (FCCP), was depressed in ALS patients. The resting level of free cytosolic calcium was higher in lymphocytes of ALS patients. Increase in free Ca2+ when challenged by a K+ channel blocker or an uncoupler of oxidative phosphorylation (FCCP) was similar in ALS and control. The results suggested subtle changes in mitochondrial function, not present at rest, but exposed by stressful conditions. The calcium changes may be secondary to mitochondrial dysfunction, or to other factors such as expression of cytosolic calcium binding protein. Increased intracellular Ca2+ may cause cell death, and increase mitochondrial production of reactive oxygen species.

C. PLATELETS Swerdlow et al. (1998) had previously demonstrated mitochondrial dysfunction in Alzheimer’s disease and Parkinson’s disease, using a cybrid system. This involves transfer of mitochondrial DNA (mtDNA) into mtDNAdepleted immortalized cells (ρ 0 cells). Isolated platelets were fused with human SHSY5Y neuroblastoma cells, containing no mtDNA. In these cell lines, any difference between control and patient cell lines should be attributable to differences in donor mtDNA. A maximum of 11 patients with ALS and 12 controls were studied. The cybrid system was used to study electron chain activities of complex I, III, and IV. A 19% defect of mean complex I ( p < 0.001) was observed. Mean complex III activity was 21% lower in the ALS cybrid line, and mean complex IV activity 16% lower, but these differences were not statistically signiﬁcant.

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Cytosolic calcium was measured before and after exposure to 10 μM carbonyl cyanide m -chlorophenylhydrazone (CCCP) in 7 ALS and 4 control cybrid cell lines. Carbonyl cyanide m -chlorophenylhydrazone is an electron transport uncoupler that triggers mitochondrial calcium efﬂux. Before CCCP, mean cytosolic Ca2+ was 170 nM in ALS cybrids, and lower in control cybrids at 143 nM ( p < 0.01). Following CCCP, mean cytosolic calcium was 270 nM in ALS, but higher at 361 nM in controls ( p < 0.0001). Control cybrid cytosolic calcium concentration increased by 154% after CCCP, whereas ALS cybrid cytosolic calcium increased by only 58%. Free radical scavenging enzyme activities were studied in 6 ALS and 8 controls. Glutathione reductase, glutathione peroxidase, total SOD, MnSOD, and catalase were all signiﬁcantly reduced, suggesting an increase in ALS cybrid reactive oxygen species production. Cu/Zn SOD was unchanged. When cybrids were exposed to the complex I inhibitor, MPP+ (N-methyl4-phenylpyridinium ion), there was no signiﬁcant difference in percentage of cell death between ALS and control. Electron microscopy demonstrated a proportion of abnormally swollen mitochondria, with surviving cristae peripherally distributed, in ALS cybrids, but these changes were seen only rarely in control cybrids. Intermediate ﬁlament aggregates were also observed more frequently in ALS cybrids. These differences between ALS and control cybrids should be attributable to differences in ALS donor mtDNA. These could be either somatic or inherited. The impaired mitochondrial calcium sequestration may be a trigger for apoptosis. The increased free radical scavenging enzymes suggest increased production of reactive oxygen species. The ultrastructural changes, with neuroﬁlament aggregation and mitochondrial abnormalities, have similarities to ﬁndings in patients and other models of ALS.

D. SKIN McEachern et al. (2000) studied skin ﬁbroblast cultures from 10 patients with ALS, 1 patient with familial ALS and the G82R SOD1 mutation, and 10 controls. Mitochondria were isolated, and superoxide production assayed. The MnSOD, glutathione peroxidase, NFκB (nuclear factor κB), Bcl-2 (B-cell leukemia/lymphoma 2), and Bax (Bcl-2 associated X protein) were measured by immunoblot. The MnSOD(SOD2)was markedly elevated in the mitochondria of three patients with ALS, and moderately increased in one patient with ALS. There was no elevation of MnSOD in the patient with an SOD1 mutation. In the mitochondria prepared from skin ﬁbroblasts of patients with elevated MnSOD, superoxide production was half

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that in the control cell lines; in the other patients superoxide production was similar to controls. Glutathione peroxidase, NFκB, and Bax were similar in the mitochondrial fraction. The Bcl-2, an antiapoptotic protein, was decreased in the mitochondrial fraction of three of the four ALS patients with increased MnSOD. There was no mutation of the MnSOD cDNA sequence. The authors suggest that it is unlikely that ALS patients have mitochondrial respiratory chain defects, but the increase in MnSOD and reduced Bcl-2 may be a result of an upstream signaling abnormality. The overactivity of MnSOD may lead to overproduction of hydroxyl radicals, leading to neuronal damage or death.

E. BRAIN Samples from Brodmann area 6 of 11 sporadic ALS (SALS) brains, 3 familial ALS with A4V SOD1 mutations (FALS1), and 1 familial ALS with no SOD1 mutation (FALS0) were studied to quantify oxidative damage (Bowling, 1993). Superoxide dismutase activity was measured in cytosolic (primarily SOD1) and mitochondrial fractions (primarily SOD2). Oxidative damage to cytosolic proteins (protein carbonyl content), and activities of the mitochondrial electron transport chain complexes were assessed. Cytosolic SOD activity (SOD1) was reduced by 39% in the FALS1 patients compared to control, with no other changes in SOD1 or SOD2. Protein carbonyl content was increased by 85% in the SALS patients. Citrate synthase-corrected complex I activity was increased by 55% in the FALS1 patients compared to controls. Complex I activity was not signiﬁcantly different in SALS patients. There were no changes in complexes II–IV. The authors concluded that the increased oxidative damage (protein carbonyl content) in the SALS group may indicate a common ﬁnal pathway for neuronal death in SALS and FALS. However, how the increase in complex I activity in the FALS1 group could contribute to pathogenesis remains uncertain. Mitochondrial involvement in ALS was explored further by Dhaliwal and Grewal (2000). The common mitochondrial DNA deletion mutation (mtDNA4977 ) was measured in brain tissue from six ALS patients and four controls. The motor cortex (Brodmann area 4) was compared to the temporal cortex (Brodmann area 17). The level of mtDNA4977 was more than 30 times higher in Brodmann area 4 (range 15–250 times) in the ALS patients, but only 3 times higher in the controls. Increases of mtDNA4977 have also been demonstrated in brain, skeletal muscle, and heart of healthy older individuals, and the brains of patients with Alzheimer’s disease and Huntington’s disease, as well as ischemic heart disease.

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F. SPINAL CORD Borthwick et al. (1999) considered that mtDNA may be preferentially susceptible to oxidative damage in patients with ALS. Mitochondrial DNA is lacking in protective histones, and has less effective repair mechanisms (Richter, 1988). Histochemical techniques were used to study mitochondrial enzyme activity in individual motor neurons from spinal cord of six ALS patients, six controls, and one patient with multiple mitochondrial deletions. Sequential measurements of cytochrome c oxidase (COX) and SDH activity were made. A reduction of COX activity was demonstrated in the spinal cord motor neurons of ALS patients, suggesting mitochondrial DNA abnormalities.

G. MUSCLE An out of frame mutation of mtDNA has been identiﬁed in a single patient with ALS (Comi et al., 1998). The man developed progressive spastic paraplegia at 29 years age, with additional lower motor neuron features. This progressed to include bulbar features, with death at age 34 years. There was no family history of similar disease. The mutation was a heteroplasmic 5 base pair deletion in the 5 end of the mitochondrial DNA COI gene, which encodes subunit I of COX-I. This leads to premature termination of the protein, reduced from 513 to 42 amino acids. The proportion of mutant vs wild-type mtDNA in two muscle biopsies was 47 and 69%. Thirteen percent of ﬁbers appeared as ragged-red ﬁbers. An isolated COX deﬁciency (43% of normal) was detected by biochemical analysis. Mitochondrial DNA abnormalities have been investigated in skeletal muscle in a series of studies using techniques that include functional imaging of saponin-permeabilized muscle ﬁbers (Wiedemann et al., 1998; Vielhaber et al., 1999, 2000). If muscle ﬁbers are treated with a low concentration of the saponin glycoside, a selective perforation of the sarcolemma is achieved, leaving the mitochondria and sarcoplasmic reticulum intact. Fourteen patients with ALS, 28 patients with mild myopathic EMG abnormalities, and 3 patients with spinal muscular atrophy (SMA) were used as controls (Wiedemann et al., 1998). Enzyme activities of lactate dehydrogenase, adenylate kinase, creatine kinase, aspartate aminotransferase, citrate synthase, cytochrome c oxidase, succinate:cytochrome c reductase, and NADH:cytochrome c reductase were measured in muscle homogenates. The only signiﬁcant changes were reduced NADH:cytochrome c reductase in ALS and increased aspartate aminotransferase in SMA.

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In saponin-permeabilized skeletal muscle ﬁbers, signiﬁcantly lower maximal rates of respiration were demonstrated with the NAD-dependent substrates, glutamate with malate, and pyruvate with malate ( p < 0.01). In ALS patients, the average ﬂux control coefﬁcient of NADH:CoQ oxidoreductase, 0.32, was signiﬁcantly higher than controls, 0.2 ( p < 0.05). Further studies utilized autoﬂuorescence images of mitochondrial NAD(P)H or ﬂuorescent ﬂavoproteins, acquired in different functional states of the permeabilized ﬁbers. NAD(P)H is ﬂuorescent in the reduced state, and ﬂavoprotein is ﬂuorescent in the oxidized state. The ratio of these autoﬂuroescence signals is used as an indicator of the redox state of the mitochondrial NAD system. There was decreased maximal respiratory activity with NAD-dependent substrates and higher sensitivity of respiratory activity to amytal (which speciﬁcally inhibits NADH:CoQ oxidoreductase). This was concluded to be compatible with the diminished activity of NADH:CoQ reductase in the muscle of patients with ALS. Elevated redox states of the mitochondrial NAD system suggested a respiratory chain defect. The changes were not observed in SMA, suggesting this is not a secondary effect of denervation. In a similar study of muscle biopsies from 26 patients with ALS, 28 controls, 6 SMA, and 2 hexosaminidase A (Tay-Sachs disease) (Vielhaber et al., 1999), NADH:cytochrome c reductase was again lower in ALS, 2.1 U/g wwt (wet weight), than controls, 3.5 U/g wwt ( p < 0.01). Mitochondrial DNA was examined using Southern blot techniques. Multiple deletions were demonstrated in one ALS patient, and low mtDNA/18S ribosomal DNA (rDNA) ratios in 14 patients. Using similar autoﬂuorescence imaging of saponin-permeabilized muscle ﬁbers, the distribution of defective mitochondria was examined by confocal microscopy. This showed a heterogeneous distribution within the ALS muscle, both within and between ﬁbers. Mitochondrial function was also studied in skin ﬁbroblasts. Cytochrome c oxidase was reported to be reduced in ALS, but only three patients were studied. The study was further extended (Vielhaber et al., 2000), including ﬁve from the previous study (Wiedemann et al., 1998). Similar ﬁndings to the previous study were made, with additional reduced cytochrome c oxidase activity in the ALS group. Multiple deletions of mtDNA were demonstrated in one patient, and depletion of intact mtDNA in other patients. Cytochrome c oxidase negative ﬁbers (1–2%) and COX-negative core ﬁbers (1–10%) were observed. Membrane-associated MnSOD (usually increased in mitochondrial myopathy) was signiﬁcantly lower in ALS than control patients. Raggedred ﬁbers were rare. The low membrane-associated MnSOD was proposed as an explanation for the alteration of mtDNA, through increased oxygen radicals.

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H. LIVER Ultrastructural mitochondrial abnormalities have been reported in the liver of patients with ALS. Masui et al. (1985) examined liver biopsies from 10 ALS patients. They found a higher incidence of intramitochondrial inclusions and reduced density of mitochondria, which were enlarged. Nakano et al. (1987) studied liver biopsies from 21 ALS patients. Mitochondrial abnormalities were found in all patients, with features of bizarre giant mitochondria, intramitochondrial paracrystalline inclusions, disorganization of the lamellar structure of rough endoplasmic reticulum, increased number of smooth endoplasmic reticulum, and parasinusoidal ﬁbrosis. Paracrystalline inclusions were highly speciﬁc for ALS.

IV. Transgenic Mouse Models of ALS

A number of mutations of SOD1 associated in humans with ALS have been used to create transgenic mice. These mice are generally heterozygous, with high SOD enzyme activity. They develop a neurodegenerative disease, with many similarities to human ALS. The development of disease, in the presence of normal or higher than normal SOD enzyme activity, suggests a gain of toxic function, dominant, effect. Pathological features similar to those found in human ALS include axonal spheroids, Lewy body-like inclusions, increased ubiquitin, fragmentation of Golgi apparatus, and selective loss of motor neurons. Membranebound vacuoles, derived from dilated mitochondria, in G93A mice (Dal Canto and Gurney, 1994) and G37R mice (Wong et al., 1995), have been observed. Closer analysis of pathological changes related to functional disease stage, rather than animal age, in G93A mice has demonstrated that mitochondrial abnormalities are most prominent in the early stages (Kong and Xu, 1998). At these early time points there is a decline in neuronal function, but neuronal death is a mild feature. At the “pre muscle weakness stage” there were abundant abnormal mitochondria in dendrites and axons, but few in cell bodies. Mitochondrial abnormalities included dilated and disorganized cristae, leakage of the outer membrane, broken outer membrane, and vacuoles carrying remains of mitochondria. The onset of muscle weakness, or rapid declining stage, was associated with massive mitochondrial vacuolation, and degeneration of motor neurons. It is suggested that the toxic effect of mutant SOD1 damages mitochondria, triggering reduction in motor neuron function and onset of clinical disease (Kong and Xu, 1998). Vacuolation decreases as the disease progresses further. Vacuoles are not a

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a

FIG. 2a. Effects of 1 and 2% creatine supplementation on survival in G93A transgenic mice. (a) Cumulative probability for survival. (b) Mean survival. 䊉 and , control (unsupplemented diet); 䊏, 1% creatine diet; 䉱 and , 2% creatine diet. Survival was signiﬁcantly increased in mice fed creatine. Asterisk: p < 0.05; number symbol: p < 0.001. (Reproduced with the kind permission of the Nature Publishing Group from Klivenyi et al., 1999.)

prominent feature in human ALS. However, this may be a result of human postmortem examination usually being performed at a late stage of the disease. The presence of mitochondrial abnormalities, but preserved motor neurons, in the early stages suggests that early intervention at this stage may rescue motor neurons from death. Oral administration of creatine (1 or 2%) to G93A SOD1 transgenic mice produced a dose-dependent improvement in motor performance, reduced loss of motor neurons, and prolongation of survival by 9% with 1% creatine, and 18% with 2% creatine (Klivenyi et al., 1999) (Fig. 2). Increases in biochemical indices of oxidative damage (3-nitrotyrosine and free radical generation using microdialysis), normally observed in G93A SOD-1 transgenic mice, were not observed in mice fed 1% creatine. Genetic inactivation of mitochondrial SOD (SOD2) in mice generates a phenotype of dilated cardiomyopathy, hepatic lipid accumulation, and early neonatal death (Li et al., 1995). Treatment with the SOD mimetic,

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b

FIG. 2b. (continued )

manganese 5, 10, 15, 20 –tetraKis (4-benzoic acid) porphyrin, rescues the mice from this, and prolongs survival. The animals develop a movement disorder by three weeks of age (Melov et al., 1998). Another model of motor neuron disease is the progressive motor neuronopathy (pmn) mouse. Interruption of the mitochondrial membrane is observed preferentially in motor neurons. When treated with an orally active antiapoptotic molecule (CGP 3466B) that binds to glyceraldehyde-3phosphate dehydrogenase, there is a 57% prolongation of life span, and preservation of body weight and motor performance. The mitochondrial changes were markedly reduced, and close to those of controls. The selective involvement of mitochondria in the motor neurons supports a pathogenic association (Sagot et al., 2000).

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A. CELL LINES G93A human mutant SOD1 mice have altered electron transport enzymes. Expression of G93A in neuroblastoma SH-SY5Y cells results in loss of mitochondrial membrane potential and elevated cytosolic calcium concentration (Carri et al., 1997). Buffering intracellular energy levels may exert neuroprotective effects. Creatine stabilizes mitochondrial creatine kinase and inhibits opening of the mitochondrial transition pore (O’Gorman et al., 1996).

B. ORGANOTYPIC SPINAL CORD CULTURES A model of organotypic rat spinal cord cultures demonstrated that motoneurons were more vulnerable than dorsal horn neurons to mitochondrial inhibition with malonate (Kaal et al., 2000). A free radical scavenger α-phenyl-N-tert-butylnitone, an AMPA (DL-α-Amino-3-hydroxy-5-methyl-4isoxazole propionic acid)/kainate receptor blocker 6-cyano-7-nitroquinoxaline-2,3-dione, and riluzole (licensed for the treatment of human ALS) were protective against malonate-induced motoneuron death. This suggested that chronic mitochondrial inhibition leads to motor neuron death. Protection by the caspase inhibitor N-benzyloxycarbonyl–Val–Ala– Asp–ﬂuoromethyl ketone and z–Asp–Glu–Val–Asp ﬂuromethyl ketone, suggested that neuronal death was apoptotic.

V. Conclusion

It is probable that a number of pathogenic pathways contribute to neuronal death in amyotrophic lateral sclerosis. The considerations may be similar to those in other neurodegenerative diseases—for example, Parkinson’s disease. The speciﬁcity of ALS for upper and lower motor neurons remains unexplained at present. Disruption of normal physiological mechanisms, including glutamate neurotransmission, free radical and calcium homeostasis, and neurotrophic factors, may all include abnormalities of the respiratory chain complex and mitochondrial function. The ﬁnal pathway of neuronal death, by apoptosis or necrosis, may also be inﬂuenced by mitochondrial function. It remains to be clariﬁed whether the observations of mitochondrial changes and abnormalities in tissue from ALS patients, and animal models, are primary or secondary phenomena in the pathogenesis of ALS. If mitochondria are found to have a critical function in the

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pathogenesis of ALS, this will be important in the understanding of the disease, and in the development and application of therapies that may be appropriate across a range of neurodegenerative diseases or aging processes.

References

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Klivenyi, P., Ferrante, R. J., Matthews, R. T., et al. (1999). Neuroprotective effects of creatine in a transgenic animal model of amyotrophic lateral sclerosis. Nat. Med. 5, 347–350. Kong, J., and Xu, Z. (1998). Massive mitochondrial degeneration in motor neurons triggers the onset of amyotrophic lateral sclerosis in mice expressing a mutant SOD1. J. Neurosci. 18, 3241–3250. Lacomblez, L., Besimon, G., Leigh, P. N., et al. (1996). Dose-ranging study of riluzole in amyotrophic lateral sclerosis. Lancet 347, 1425–1431. Lefebvre, S., Burglen, L., Reboullet, S., et al. (1995). Identiﬁcation and characterization of a spinal muscular atrophy-determining gene. Cell 80, 155–165. Li, Y., Huang, T. T., Carlson, E. J., et al. (1995). Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutase. Nat. Genet. 11, 376–381. Masui, Y., Mozai, T., and Kakehi, K. (1985). Functional and morphometric study of the liver in motor neuron disease. J. Neurol. 232, 15–19. McEachern, G., Kassovska-Bratinova, S., Raha, S., et al. (2000). Manganese superoxide dismutase levels are elevated in a proportion of amyotrophic lateral sclerosis patient cell lines. Biochem. Biophys. Res. Commun. 273, 359–363. Melov, S., Schneider, J. A., Day, B. J., et al. (1998). A novel neurological phenotype in mice lacking mitochondrial manganese superoxide dismutase. Nat. Genet. 18, 159–163. Merry, D. E., Kobayashi, Y., Bailey, C. K., et al. (1998). Cleavage, aggregation and toxicity of the expanded androgen receptor in spinal and bulbar muscular atrophy. Hum. Mol. Genet. 7, 693–701. Miller, R. G., Mitchell, J. D., and Moore, D. H. (2000). Riluzole for amyotrophic lateral sclerosis (ALS)/motor neuron disease (MND) (Cochrane review). In “The Cochrane Library,” issue 2. Oxford: Update Software. Nakano, Y., Hirayama, K., and Terao, K. (1987). Hepatic ultrastructural changes and liver dysfunction in amyotrophic lateral sclerosis. Arch. Neurol. 44, 103–106. O’Gara, R. W., Brown, J. M., and Whiting, M. G. (1964). Induction of hepatic and renal tumors by topical application of acqueous extract of the cycad nut to artiﬁcial skin ulcers in mice. Third Conference on Toxicity of Cycads. Fed. Proc. 23, 1383. O’Gorman, E., Beutner, G., Wallimann, T., and Brdiczka, D. (1996). Differential effects of creatine depletion on the regulation of enzyme activities and on creatine-stimulated mitochondrial respiration in skeletal muscle, heart, and brain. Biochim. Biophys. Acta 1276, 161–170. Orrell, R. W. (2000). Amyotrophic lateral sclerosis: Copper/zinc superoxide dismutase (SOD1) gene mutations. Neuromusc. Disord. 10, 63–68. Orrell, R. W., King, A. W., Hilton, D. A., Campbell, M. J., Lane, R. J. M., and deBelleroche, J. (1995). Familial amyotrophic lateral sclerosis with a mutation of SOD1: Intrafamilial heterogeneity of disease duration associated with neuroﬁbrillary tangles. J. Neurol. Neurosurg. Psychiat. 59, 266–270. Orrell, R. W., and Figlewicz, D. A. (2001). Clinical implications of the genetics of ALS and other motor neuron diseases. Neurology 57, 9–17. Richter, C., Park, J. W., and Ames, B. (1988). Normal oxidative damage to mitochondrial and nuclear DNA is extensive. Proc. Natl. Acad. Sci. USA 85, 6465–6467. Robberecht, W., Sapp, P., Kristina, M., et al. (1993). Cu/Zn superoxide dismutase activity in familial and sporadic amyotrophic lateral sclerosis. J. Neurochem. 62, 384–387. Rosen, D. R., Siddique, T., Patterson, D., et al. (1993). Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 362, 59–62. Sagot, Y., Toni, N., Perrelet, D., et al. (2000). An orally active anti-apoptotic molecule (CGP 3466B) preserves mitochondria and enhances survival in an animal model of motoneuron disease. Br. J. Pharmacol. 131, 721–728.

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SECTION VI MODELS OF MITOCHONDRIAL DISEASE

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MODELS OF MITOCHONDRIAL DISEASE

Danae Liolitsa∗ and Michael G. Hanna∗ ,† ∗ Centre

for Neuromuscular Disease and † Department of Molecular Pathogenesis Institute of Neurology Queen Square London, WC1N 3BG, United Kingdom

I. II. III. IV.

V.

VI.

VII. VIII.

IX. X.

1

Introduction Classiﬁcation of mtDNA Defects Causing Respiratory Chain Disease Cell Models Employed to Study mtDNA Defects Cell Models of Respiratory Chain Disease Associated with Speciﬁc mtDNA Defects A. Cell Models of Respiratory Chain Disease Associated with mtDNA Rearrangements B. Cell Models of Respiratory Chain Disease Associated with Point Mutations in the Mitochondrial tRNA Gene for Leucine (UUR) C. Cell Models of Respiratory Chain Disease Associated with Point Mutations in the Mitochondrial tRNA Gene for Lysine D. Cell Models of Respiratory Chain Disease Associated with Point Mutations in Protein-Coding Genes Classiﬁcation of Nuclear DNA Defects Causing Respiratory Chain Disease A. Defects in Nuclear Genes Encoding Subunits of the Respiratory Chain Complexes B. Defects in Nuclear Genes Encoding Proteins Involved in the Assembly of the Respiratory Chain Complexes C. Defects in Nuclear Genes Encoding Factors for Intergenomic Communication D. Defects in Nuclear Genes Encoding Factors Indirectly Related to Respiratory Chain Cell Models of Respiratory Chain Disease Associated with Nuclear DNA Defects A. Application of Cell Models for the Identification of Nuclear Gene Defects Associated with Respiratory Chain Diseases B. Cell Models of Respiratory Chain Disease Associated with Nuclear DNA Defects Causing the mtDNA Depletion Syndrome Application of Cell Models for the Development of Therapeutic Strategies in mtDNA Disease Animal Models of Respiratory Chain Disease A. Animal Models of Respiratory Chain Disease Associated with mtDNA Defects B. Animal Models of Respiratory Chain Disease Associated with Nuclear DNA Defects Other Models of Respiratory Chain Disease Conclusions References

To whom correspondence should be addressed.

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Copyright 2002, Elsevier Science (USA). All rights reserved. 0074-7742/02 $35.00

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I. Introduction

All eukaryotic cells produce energy in the form of adenosine triphosphate (ATP) by the process of oxidative phosphorylation (OXPHOS) in the mitochondria. Mitochondrial ATP production is dependent upon nuclear and mitochondrial gene-encoded proteins. Failure in mitochondrial ATP production due to molecular alterations in nuclear and/or mitochondrial genes leads to cellular dysfunction, and it is associated with many respiratory chain diseases—the mitochondrial encephalomyopathies (Wallace, 1992; DiMauro and Moraes, 1993). These diseases are not uncommon, and they cause signiﬁcant morbidity and mortality (Chinnery et al., 2000; Pulkes and Hanna, 2001). The ﬁrst mutations in mitochondrial DNA (mtDNA) associated with respiratory chain disorders were discovered in 1988 (Holt et al., 1988; Wallace et al., 1988a,b). More than a hundred additional pathogenic mtDNA mutations have been identiﬁed (Servidei, 2000). Nuclear gene defects have started to be described. However, the identiﬁcation of nuclear genetic factors is constrained due to the large number of candidate genes involved and due to the rarity of homogenous populations to carry out linkage analyses. While there is good evidence that these mutations in both mtDNA and nuclear DNA are the cause of respiratory chain mitochondrial encephalomyopathies, relatively limited information is available regarding the precise mechanisms of their pathogenicity. Furthermore, there is no effective treatment for these patients. In an effort to elucidate the molecular mechanisms of disease pathogenesis, cell and animal models have been developed. The aim of this chapter is to describe recent research to develop such models of respiratory chain disease. The models that have been developed can be used to address a number of different issues. Some of these are listed below: 1. To demonstrate whether the cause of a given respiratory chain disease is due to a nuclear or a mitochondrial DNA defect. 2. To identify nuclear genes. 3. To study molecular mechanisms of nuclear gene defects. 4. To study molecular mechanisms of identiﬁed mtDNA defects. These may include studies of a. Mitochondrial transcription and translation. b. Threshold effect. c. Role of mtDNA polymorphisms. d. The effect of nuclear background. 5. To understand the mechanisms of segregation and the factors that control the tissue-speciﬁc distribution of heteroplasmic mtDNA mutations.

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6. To develop therapies for the treatment of the respiratory chain diseases.

II. Classiﬁcation of mtDNA Defects Causing Respiratory Chain Disease

There are two main groups of mtDNA defects: large-scale rearrangements and point mutations. Rearrangements include duplications and deletions, and are usually sporadic, arising from the ampliﬁcation of a single mutational event during mtDNA replication. Major rearrangements are commonly found in patients with the Kearns-Sayre syndrome (KSS), in some patients with progressive external opthalmoplegia (PEO) (Zeviani et al., 1998), and in patients with Pearson’s syndrome (Rotig et al., 1995). Muscle biopsy of these patients reveals ragged-red ﬁbers (RRFs), the histochemical hallmark reﬂecting massive proliferation of mitochondria. Such RRFs usually stain weakly or not at all with cytochrome oxidase (COX) due to the overall impairment of mitochondrial protein synthesis. In contrast, point mutations are usually present in the maternal germ line and may be ampliﬁed during oogenesis, sometimes resulting in a large shift in mutant load between mother and offspring. Pathogenic point mutations have been observed in protein coding genes, and two common disorders of this type are Leber’s hereditary optic neuropathy (LHON) and neuropathy ataxia retinitis pigmentosa (NARP). In general, muscle biopsy in such cases does not reveal ragged-red or COX-negative ﬁbers. Point mutations are also found in ribosomal RNA (rRNA) and transfer RNA (tRNA) genes, and are associated with a wide range of neurological disorders. rRNA gene mutations are most commonly found in association with nonsyndromic deafness. The commonest diseases associated with tRNA gene mutations are myoclonic epilepsy with ragged-red ﬁbers (MERRF) (Hammans et al., 1993), and mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS) (Ciafaloni et al., 1992). The mechanisms by which point mutations and rearrangements in the mtDNA differentially affect tissues and hence result in variable disease phenotypes are poorly understood. Not only is it the case that the same mtDNA mutation may be associated with a wide range of clinical phenotypes, but also that the same clinical phenotype may result from a variety of different mitochondrial defects at different positions in mtDNA (Morgan-Hughes and Hanna, 1999). The pathogenetic mechanisms of speciﬁc mtDNA mutations have been examined in various cell culture systems and animal models; the relationships between genotype and biochemical defect have been analyzed.

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III. Cell Models Employed to Study mtDNA Defects

There are three types of cell models that have been employed to study the mechanisms of respiratory chain diseases associated with mtDNA defects: primary cultures, immortal cell lines lacking mtDNA (rho zero cells; r0 cells), and transmitochondrial hybrids (cybrids). Primary cultures are usually developed from myoblast or ﬁbroblast tissue from patients harboring the relevant mtDNA mutation. Primary cultures may be cloned resulting in cell lines with varying proportions of mutant mtDNA in each clone. Such primary cloned cultures can be employed to analyze transcription, translation, threshold for expression, and segregation. One of the problems with such primary cell cultures is that they have a limited life span in culture. For this reason most workers have concentrated on transformed culture systems described below. Arguably, primary culture models mimic the in vivo situation more closely. Rho zero cells are usually derived from lung carcinoma (A549) or osteosarcoma (143B; King and Attardi, 1989) cells by long-term exposure to ethidium bromide (an inhibitor of mtDNA replication). This results in complete depletion and permanent loss of their mtDNA. Rho zero cells lack oxidative phosphorylation and need glucose-supplemented medium in order to proliferate. Human rho zero cells also require pyrimidine and pyruvate (King and Attardi, 1996). Their dependence on pyrimidine is explained by the fact that dihydrooratate dehydrogenease, an enzyme involved in the pyrimidine biosynthetic pathway, requires mitochondrial electron transport for normal function. The addition of pyruvate is essential as an oxidant to regenerate NAD from NADH since most NAD is reduced in the absence of a normal respiratory chain. The principle use of rho zero cells has been to generate transmitochondrial cybrids. Transmitochondrial cybrid cells are developed by the fusion of rho zero cells with donor cells from patients harboring mtDNA mutations (King et al., 1992; Chomyn et al., 1992). Mitochondrial donors are enucleated cells usually derived from myoblast, ﬁbroblast, or lymphoblast cells of patients whose mtDNA harbors pathogenic mutations. Enucleation in these cells is achieved by centrifugation in the presence of cytochalasin B (Bodnar et al., 1993). Transmitochondrial cybrids are grown in glucose medium where they remain capable of maintaining a membrane potential and of producing some ATP by glycolysis, but they are auxotrophic for pyrimidine and pyruvate. Therefore fused cells (cybrids) can be distinguished from cells that have remained unfused because the latter die in medium with no pyrimidine. The generated cybrids allow the study of the effects of different proportions of mtDNA mutations on transcription, translation, and respiratory

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chain biochemistry in a constant nuclear background. Blood platelets that represent anucleate derivatives of megakaryocytes also have been used as mitochondrial donors to rho zero cells (Chomyn et al., 1994). The plateletderived rho zero cell system is potentially more accessible due to the noninvasive nature of platelet isolation and the efﬁciency in mitochondrial transfer. This system allows the study of a pathogenic mechanism of mtDNA mutation and the role of the nuclear genetic background since it allows the generation of cell lines that contain pure mutant or wild-type genome. Figure 1 illustrates the steps for the generation of transmitochondrial cybrids. The mitochondria donor cells

143B osteosarcoma cells

enucleation

ethidium bromide

cytoplasts

mix, fuse, select

recipient rho zero cells

cybrids

0

50

100

FIG. 1. Diagram illustrating the generation of transmitochondrial cybrids (van den Ouweland et al., 1999). The mitochondrial donor cells, usually from patients harboring a speciﬁc mtDNA mutation, are enucleated by exposure to cytochalasin B, and the resulting cells are called cytoplasts. The recipient cells are depleted of mtDNA by exposure to ethidium bromide (or ddC), and the resulting cells are termed “rho zero.” Cytoplasts are fused with rho zero cells and the resulting transmitochondrial cybrids allow the study of the mutated mtDNA molecules at different proportions in a constant nuclear background. Alternatively, this system may be used to study the effect of nuclear background.

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disadvantage of using the transformed cell systems described is principally that the transformed nuclear background departs signiﬁcantly from the in vivo system. Recently, cells lacking mitochondria have been produced by exposure to the nucleoside analogue 2 ,3 -dideoxycytidine (ddC) instead of ethidium bromide (Nelson et al., 1997). This system has allowed the generation of patient-derived cells with different proportions of mtDNA. Furthermore, cybrids are produced by the fusion of enucleated cells with ddC-treated cells. Such cybrids can be generated from cell lines of patients with distinct phenotypes to investigate the contribution of nuclear genes on the expression of a given primary mtDNA mutation. This system is easier to manipulate and more ﬂexible compared to the rho zero cell derived cybrids since ddC is safer to use compared to ethidium bromide, and the mtDNA content in the host cells can be regulated by exposure to different concentrations of ddC. Furthermore, transformation of the primary cell culture prior to ddC exposure is not required.

IV. Cell Models of Respiratory Chain Disease Associated with Speciﬁc mtDNA Defects

In this section we describe the use of cell models to study the commonest mtDNA mutations. We have focused upon large-scale rearrangements and the commonest point mutations in tRNA and protein coding genes.

A. CELL MODELS OF RESPIRATORY CHAIN DISEASE ASSOCIATED WITH mtDNA REARRANGEMENTS Large-scale rearrangements of mtDNA result in the accumulation of truncated mtDNA molecules that lack a fragment due to a speciﬁc deletion or multiple deletions, and are associated with a variety of clinical phenotypes. These may range from the severe Pearson’s syndrome and KSS through PEO to mild symptoms such as diabetes and deafness (Cortopassi et al., 1992; Corral-Debrinski et al., 1992). The most common deletion is 4977 base pairs (bp) in length occurring between two 13-bp direct repeats in the mtDNA sequence between positions 13447–13459 and 8470–8482 (Schon et al., 1989; Shoffner et al., 1989). This deletion encompasses ﬁve tRNA genes and seven gene-encoding subunits of cytochrome c oxidase, complex I, and ATPase. In patients with PEO associated with the presence of the 4977-bp deletion in the mtDNA sequence, there is some degree of correlation between the

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proportion of deleted mtDNA in muscle and/or the site of deletion and the severity of the clinical phenotype (Pulkes and Hanna, 2001). In order to examine whether increasing proportions of the deleted molecules are associated with a decrease in the oxidative capacity of the mitochondrial respiratory chain, cell culture models have been employed. It was shown by culturing skin ﬁbroblasts and Epstein-Barr virus-transformed lymphocyte cultures from a patient with Pearson syndrome harboring the 4977-bp deletion that different heteroplasmic proportions of the deletion correlated with the changes in respiratory chain activity (Bourgeron et al., 1993). Whereas in skin ﬁbroblasts there was a decrease in respiratory chain activity when the proportion of deleted mtDNA molecules was greater than 60% in lymphoblastoid cells, the respiratory chain defect was apparent only at proportions greater than 80%. These ﬁndings suggested that the threshold for biochemical expression is related to tissue speciﬁcity and therefore to nuclear background. Cybrid technology has also been used to evaluate the bioenergetic consequences in relation to different proportions of the deleted mtDNA. In one such study, mtDNA derived from ﬁbroblast cells of a patient with PEO bearing a 5196-bp mtDNA deletion was transmitted to clonal HeLa cells lacking mtDNA (Hayashi et al., 1991). In the generated cybrids the deleted mtDNA molecules were selectively propagated in the clones and accumulation of the deletion to proportions greater than 60% resulted in inhibition of translation and impaired COX activity. Evidence was obtained that in clones with less than 60% of the truncated molecule translational complementation occurred between the deleted and normal mtDNA. In another study, cybrid clones generated by the fusion of a rho osteosarcoma cell line with enucleated ﬁbroblast cells from a patient with PEO bearing the 4977-bp deletion were used to evaluate the membrane potential and rate of ATP synthesis (Porteous et al., 1998). It was indicated that signiﬁcant defects were present at proportions of the mutant greater than 50%, conﬁrming that a deﬁciency in the respiratory chain activity is linked to the pathogenic deletion. Furthermore, this value is consistent with the threshold value of heteroplasmy that causes a clinically recognizable phenotype in vivo that is in the range between 20 and 80% (Holt et al., 1989). Such cell studies have provided convincing evidence that deletions of mtDNA are pathogenic. They have shown that impaired translation of mtDNA (due to loss of tRNA genes) and loss of protein-coding genes contribute to impaired respiratory chain function. Cell models have shown that thresholds for expression may differ between cell types, although the mechanisms for this variation are not yet determined. The imprecise relationship between proportion of deleted mtDNA and clinical severity indicate that

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further factors must inﬂuence the relationship between genotype and phenotype. Nevertheless, it still remains to be understood how a defect in the bioenergetic capacity of the cells gives rise to clinical symptoms and whether variables other than ATP synthesis and membrane potential are affected by the accumulation of the truncated mitochondrial DNA molecules.

B. CELL MODELS OF RESPIRATORY CHAIN DISEASE ASSOCIATED WITH POINT MUTATIONS IN THE MITOCHONDRIAL tRNA GENE FOR LEUCINE (UUR) There have been extensive cell model studies of the tRNA leucine (UUR) gene mutation at nucleotide position (np) 3243. It has been shown in vivo that the relationship between the percentage of the mutant mtDNA molecule and tissue dysfunction is not simple, and an imprecise correlation exists with disease severity (Ciafaloni et al., 1992). For example, large amounts of mutant mtDNA above the critical threshold for respiratory dysfunction have been observed not only in affected but also in unaffected tissues of patients with the np3243 tRNA leucine gene mutation (Shiraiwa et al., 1993). Furthermore, in a patient bearing a heteroplasmic proportion of the np3243 mutation of only 6%, the bioenergetic state of his muscle was signiﬁcantly reduced (Chinnery et al., 2000). In contrast in another patient with 32% of the mutation there was no evidence of mitochondrial dysfunction (Chinnery et al., 2001). The np3243 mutation is also associated with diverse phenotypes ranging from ocular myopathy alone through diabetes with deafness to overlapping MELAS/MERRF syndrome ( Jean-Francois et al., 1994; Hammans et al., 1995). Such differences may be partly attributed to variable distribution of mutant and wild-type mtDNA molecules and to the different thresholds of tissue vulnerability to oxidative impairment (Liou et al., 1994; Matthews et al., 1994). It is also noteworthy that different phenotypes arise as a result of point mutations at distinct positions in the same tRNA leucine (UUR) gene. For example, the G3256T mutation has been associated with PEO, the A3260G mutation with cardiomyopathy and myopathy, and the T3271G mutation with MELAS. It is likely that other factors, in addition to the proportion of mutant molecule, contribute to the great variety of clinical phenotypes associated with the np3243 mutation. In cybrid cells containing mtDNA from patients suffering from MELAS, it was found that for respiratory chain dysfunction and translational defects to become apparent, proportions above or equal to 95% of the np3243 mutation are required (King et al., 1992). Interestingly, it was also found that this mutation results in increased production of a novel RNA molecule resulting from abnormal transcription that may

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interfere with mitochondrial translation (Schon et al., 1992). These observations suggest that despite the direct relationship in cybrid cell lines between the tRNA leucine (UUR) mutation and the degree of translational inhibition of the respiratory chain complexes, abnormal RNA processing might change this correlation by altering translation. This may be an explanation of the indirect correlation between disease severity and mutational load. In contrast to nuclear tRNA genes whose function is limited to translational processes, the mitochondrial human tRNA genes appear to be involved in transcription by being positioned between rRNA and proteincoding genes, thereby acting as signals for the endonucleolytic cleavage of the polycistronic mitochondrial transcript (Ojala et al., 1981). The increased complexity in the pathogenetic mechanisms arising from molecular alterations in the tRNA leucine (UUR) gene may be attributed to the positioning of the tRNA molecule within a conserved tridecamer sequence that binds the mtDNA transcription termination factor, mTERF, and is therefore involved in the regulation of rRNA:mRNA synthesis. This has been studied in vitro, where it was shown that the A3243G mutation inhibits 16S rRNA transcription termination(Hess et al., 1991). However, it was not conﬁrmed in cybrid cell lines harboring the mutation or in vivo. In the cybrid cells there was no signiﬁcant change in the amounts of the two rRNA species encoded upstream of the mTERF binding site (Chomyn et al., 1992). Also, it was shown in vivo that in two patients heteroplasmic for the A3243G mutation there was no correlation between the biochemical defects and transcription termination of the 16S rRNA (Hammans et al., 1992). An alternative explanation for the phenotypic variability associated with the np3243 mutation is that variable intrinsic molecular mechanisms exist that compensate for the defects in mitochondrial translation by the np3243 mutation. Using lung carcinoma cybrid cells harboring the np3243 mutation, it was demonstrated that a second site suppressor mutation at np12300 may decode the codon UUR, thus restoring respiration (El Meziane, 1998). A change at np12300 was not found to inﬂuence the A3243G-associated phenotype in vivo. However, there is evidence that a change at position 12308 may have an inﬂuence (Pulkes and Hanna, 2001). Alternatively, it is possible that a suppressor effect is exhibited by structural modiﬁcation of some other mitochondrial tRNA that naturally inﬂuences the decoding properties of the leucine (UUR) codon (Abdellatif et al., 1998). Temporal analysis in cybrid clones derived from fusion with the osteosarcoma rho zero cell line has revealed that the proportion of mutant mtDNA molecule increases over time (Yoneda et al., 1992). The pattern of mitochondrial segregation may be inﬂuenced by different cellular backgrounds. For example, whereas the mutant mtDNA molecules for the np3243 mutation

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are maintained in the osteosarcoma transformed cell line (Yoneda et al., 1992; Dunbar et al., 1995), they are lost in the lung carcinoma cell line to be replaced by a normal complement (Dunbar et al., 1995). This may explain the variable clinical features associated with MELAS, and the same principle applies for other mitochondrial disorders. It is possible that polymorphic variation in the nuclear genetic background may differentially inﬂuence mutant and wild-type mtDNA molecules to result in variable genotypes for any given tissue and consequently to distinct clinical phenotypes. Such cell studies of the A3243G mutation have provided strong evidence that it is the cause of many respiratory chain diseases. Most studies indicated that translational failure is the key pathogenetic mechanism. The precise molecular explanation of the clinical phenotypic diversity observed is not clear. Further cell studies examining the effect of nuclear genetic background and mitochondrial haplotype may elucidate this.

C. CELL MODELS OF RESPIRATORY CHAIN DISEASE ASSOCIATED WITH POINT MUTATIONS IN THE MITOCHONDRIAL tRNA GENE FOR LYSINE In contrast to the np3243 point mutation that exhibits marked phenotypic heterogeneity, the A–G transition at position 8344 in the tRNA gene for lysine has been most commonly described in patients with MERRF (Hammans et al., 1993) and is relatively phenotypically speciﬁc. For the np8344 mutation there is some degree of correlation between disease severity and the proportion of mutant mtDNA. However, there are exceptions— for example, the mutant molecule has been observed at proportions greater to 96% in both clinically affected and unaffected tissues of a patient with MERRF and Leigh’s syndrome (Sweeney et al., 1994). Various cell culture systems have been used to study threshold levels for restoration of respiratory capacity associated with the np8344 MERRF mutation. The translational alteration induced by the np8344 tRNA lysine mutation that causes MERRF has been attributed to decrease in the tRNA lysine aminoacylation capacity (Enriquez et al., 1995). Using two distinct rho zero cell lines, it was shown that virtually 100% mutant mtDNA from the myoblast cells of a MERRF patient with the tRNA lysine defect is required for respiratory dysfunction (Chomyn et al., 1991). In primary myotubes formed by fusion of mutant and wild-type myoblasts, translation is restored with 15% of wild-type mtDNA, whereas in primary cultures of human myoblasts higher proportions of wild-type molecule are required to restore translation (Boulet et al., 1992). The differences between the ﬁndings in primary cultures and in rho zero cells may be attributed to the loss of the ability in the transformed

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cells to accumulate mitochondria that would increase their translational efﬁciency (Bourgeron et al., 1993). The relatively small proportion of mutant to wild-type molecules that is sufﬁcient in myoblast cultures to impair translation in contrast to myotubes has been attributed to differences in the ratio of mtDNA copy number required for normal translational activity to actual mtDNA copy number being lower in myotubes (Hanna et al., 1995). This is supported by the observation that in platelet-derived rho zero cell lines, where the mtDNA copy number is low (average of one mtDNA molecule per mitochondrion; Shuster et al., 1988), only 37% 8344 mutant mtDNA is sufﬁcient to impair respiration (Chomyn et al., 1994). There also may be differences in the complementation capacities of mitochondria between different cell types and derived transformants (Hanna et al., 1995). The tRNA lysine mutation also has been analyzed for its effects on transcription processes. In myoblast primary cultures it was observed that the tRNA lysine mutation inhibits translation, but there is no evidence for abnormal RNA processing (Hanna et al., 1995). The loss of translational efﬁciency in the myoblast cells was more dramatic for the COX subunits than the remaining peptides, which is consistent with the observation that COX-negative ﬁbers are more common in MERRF than in MELAS patients (Hammans et al., 1992). However, a study that used ﬁbroblast-derived transmitochondrial cybrid cells from a MERRF patient with a generalized COX deﬁciency demonstrated that there were no changes in the size or the content of COX in muscle mitochondria, but the defect was rather in the activity of COX that was selectively reduced (Antonicka et al., 1999). It has been proposed that this is due to premature termination of translation that results in the accumulation of truncated mitochondrial peptides forming complexes that are functionally impaired (Enriquez et al., 1995). The threshold of COX activity that affects synthesis of ATP was found to be 30%, in agreement with the threshold level for COX activity for the respiration of muscle mitochondria in MERRF patients (Larsson et al., 1992). It has been shown by histochemical methods that in cell cybrids harboring a speciﬁc mutation there are clusters of both COX-positive cells and COX-negative cells in the same clone after several passages (Antonicka et al., 1999). Cell models for the np8344 mutation have conﬁrmed the pathogenicity of this mutation, which correlates to some extent with the degree of disease severity. Nevertheless, there is variability in the biochemical proﬁles and thresholds produced in various studies from clones of the same mutation. This may be attributed to differences in the innate patterns of segregation of speciﬁc mutations, or it may be that the nuclear genetic background may inﬂuence differentially segregation of different pathogenic mutations of mtDNA as is discussed below.

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D. CELL MODELS OF RESPIRATORY CHAIN DISEASE ASSOCIATED WITH POINT MUTATIONS IN PROTEIN-CODING GENES The molecular and cellular mechanisms that underlie the pathology of mitochondrial respiratory chain diseases associated with mutations in protein-coding genes have also been studied using cell culture systems. The most common disorder of this type is Leber’s Hereditary Optic Neuropathy (LHON), a maternally inherited disease characterized by progressive central vision loss due to optic nerve atrophy. The disease is highly genetically heterogeneous with at least 17 mtDNA sequence variants suggested to be linked to the disease (Brown and Wallace, 1994). Among these, the most common transitions are the G3460A, G11778A, T14484C, and T14459A in the genes encoding protein subunits of complex I [NADH dehydrogenase (ND)]. In general, these four commonest LHON mutations that have been termed “primary” are present in homoplasmic proportions in affected individuals. The pathogenetic signiﬁcance of the np11778 primary mutation in the ND4 subunit has been demonstrated in cybrid cell lines. The presence of the mutation caused a signiﬁcant decrease in the respiratory chain capacity, which was further impaired by the presence of the secondary mutations 4216 and 13705 in the ND1 and ND5 subunits, respectively (Smith et al., 1994; Vergani et al., 1995). In another study, the pathogenicity of the common LHON mutations at nucleotide positions 3460, 11778, 14484, and 14459 was examined by measuring their effects on complex I enzyme activity and respiratory function using cell cybrid clones from lymphoblastoid cell lines of LHON patients (Brown et al., 2000). It was demonstrated that all mutations were pathogenic with variable severity: the np3460 in the ND1 subunit resulted in a 79% reduction in complex I enzyme activity and a mild defect in respiratory capacity; the np11778 resulted in a 20% impairment in the activity of the enzyme and alteration in the interaction between complex I and coenzyme Q (CoQ), the electron acceptor; the np14484 mutation resulted in a mild impairment in respiratory capacity and no defects in enzyme activity. It was suggested that the biochemical severity conferred by the np14484, the np11778, and the np3460 mutations roughly correlates with clinical severity. It has been shown that visual recovery in LHON patients harboring the np14484 mutation is much more likely than in those with the np3460 mutation or the np11778 mutation ( Johns et al., 1993). Although cell models have shown that the LHON mutation impairs respiratory chain function, the factors leading to onset of blindness remain unclear. While the mtDNA mutations that cause the LHON phenotype are most commonly found in respiratory chain genes (complexes I–IV), mutations in

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the ATP synthase (complex V) gene are associated with maternally inherited Leigh’s syndrome (Tatuch et al., 1992) as well as with the complex neurological disorder NARP (Holt et al., 1990). The most common mtDNA mutation is the G to C transition at np8993 that converts a highly conserved leucine to arginine in the gene encoding the ATP synthase subunit 6 (ATPase 6). It has been demonstrated that cybrid clones homoplasmic for the mutant nucleotide exhibit a signiﬁcant reduction in coupled respiration consistent with a defect in the proton channel and ADP phosphorylation of ATPase 6 (Trounce et al., 1994). The relationship between degree of heteroplasmy of the np8993 mutation and clinical phenotype has been examined in cell culture experiments from patients with a spectrum of disease severity including retinal degeneration alone, NARP, and Leigh’s syndrome (Shoffner et al., 1992; Ciafaloni et al., 1993; Santorelli et al., 1993). These studies suggested that the different clinical presentations associated with the mutation correlate with the heteroplasmic proportions of the mutation and consequently with the proportional decrease in ATP synthesis. In another study, the segregation pattern of the np8993 mutation was investigated in osteosarcoma and lung carcinoma rho zero cells fused with mitochondria harboring the mutation derived from a NARP patient (Vergani et al., 1999). It was revealed that the segregation pattern of the mutation was not random, and while the mutated molecule was selectively maintained in both nuclear backgrounds the wild-type mtDNA molecule was not propagated in either of the two cybrid cultures. The inability to propagate the mtDNA molecule was speciﬁc for the NARP mtDNA as no such abnormality was noted with other mtDNA donors. A different pattern of segregation exists for the np3243 mutation associated with MELAS (described earlier in this chapter) whereby selection of mutant and wild-type mtDNA is different between the two distinct cellular backgrounds(Dunbar et al., 1995). Taken together, these ﬁndings suggest that determination of mtDNA segregation is non-random but may be dependent on the complex interactions between the nuclear and mitochondrial genomes that encode the numerous factors involved in the control of mtDNA copy number. In summary, cell studies have provided clear evidence that the commonest mutations associated with LHON (G3460A, G11778A, T14484C, T14459C) do induce respiratory chain dysfunction. However, it remains unclear why only a proportion of individuals that are homoplasmic for one of these mutations develop blindness. Furthermore, the trigger to the onset of blindness in adult life is not elucidated. Cell studies in relation to the NARP mutation at position 8993 in the ATPase gene have provided evidence for pathogenicity. Furthermore, such studies have conﬁrmed that there is good correlation between proportion of mutant mtDNA, degree of biochemical dysfunction, and clinical severity.

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V. Classiﬁcation of Nuclear DNA Defects Causing Respiratory Chain Disease

The nuclear genes involved in the function of the respiratory chain are classiﬁed into those encoding 1. 2. 3. 4.

structural components of the respiratory chain complexes; factors involved in the assembly of the respiratory chain complexes; proteins involved in intergenomic communication; and mitochondrial proteins indirectly related to the function of the respiratory chain.

The identiﬁed pathogenic nuclear genes are listed in Table I.

A. DEFECTS IN NUCLEAR GENES ENCODING SUBUNITS OF THE RESPIRATORY CHAIN COMPLEXES A pathogenic 5-bp deletion was identiﬁed in a nuclear-encoded subunit of complex I, namely the NDUFS4, that was present in a complex I deﬁcient patient suffering from a progressive fatal multisystem disorder (van den Heuvel et al., 1998). Subsequently, mutational analysis of the NDUFS7 subunit in a family with symptoms of Leigh’s syndrome with autosomal-recessive mode of inheritance revealed the presence of a highly conserved point mutation in two siblings (Triepels et al., 1999). A point mutation causing a fatal leukodystrophy with myoclonic epilepsy was identiﬁed in NDUFV1 ﬂavoprotein gene (Schuelke et al., 1999) and a point mutation in the NDUFS8 caused Leigh’s syndrome (Loeffen et al., 1998). Since the total of complex I subunits encoded by nuclear genome is at least 36, it is likely that many more pathogenic mutations in nuclear DNA genes of this complex will be identiﬁed. The completion of the human genome project and the development of DNA chip arrays will allow considerable progress to be made in the identiﬁcation and the chromosomal localization of yet other nuclearencoded genes involved in the structure and assembly of the mitochondrial OXPHOS system.

B. DEFECTS IN NUCLEAR GENES ENCODING PROTEINS INVOLVED IN THE ASSEMBLY OF THE RESPIRATORY CHAIN COMPLEXES In addition to genes of the respiratory chain components (the above four in complex I and one in complex II) (Sue and Schon, 2000), genes encoding regulatory proteins of the respiratory chain have been investigated. These

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TABLE I CLASSIFICATION OF NUCLEAR GENETIC DEFECTS ASSOCIATED WITH MITOCHONDRIAL ENCEPHALOMYOPATHIESa Gene

Phenotype

Reference

A. Mutations in genes encoding respiratory chain protein subunits • Complex I deﬁciencies NDUFS4

Leigh-like syndrome

NDUFS7

Leigh syndrome

van den Heuvel et al. (1998) Trieples et al. (1999)

NDUFS8

Leigh syndrome

Loeffen et al. (1998)

NDUFV1

Leukodystrophy and myoclonic epilepsy

Schuelke et al. (1999)

• Complex II deﬁciencies SDHA

Leigh syndrome

Bourgeron et al. (1995) Leckschat et al. (1993)

B. Mutations in genes encoding assembly proteins • Complex IV deﬁciencies SURF1

Leigh syndrome

Tiranti et al. (1998)

SCO2

Cardioencephalomyopathy

Papadopoulou et al. (1999)

SCO1

Hepatic failure and encephalopathy

Valnot et al. (2000a)

COX 10

Leigh and Toni-Fanconi-Debre syndrome

Valnot et al. (2000b)

C. Mutations in genes of intergenomic communication Unknown

MtDNA depletion syndrome

Moraes et al. (1991)

TP

MNGIE

Hirano et al. (1994)

Unknown

arPEO with multiple mtDNA deletions

Bohlega et al. (1996)

ANT1

adPEO with multiple mtDNA deletions

Kaukonen et al. (2000)

Twinkle

adPEO with multiple mtDNA deletions

Spelbrink et al. (2001)

POLG

adPEO with multiple mtDNA deletions

Van Goethem et al. (2001)

D. Mutations in genes encoding factors indirectly related to respiratory chain DDP1

X-linked deafness-dystonia

Koehler et al. (1999)

ABC7

X-linked ataxia/sideroplastic aneamia

Allikmets et al. (1999)

Frataxin

Friedreich’s ataxia

Cavadini et al. (2000)

Paraplegin

Hereditary spastic paraplegia

Casari et al. (1998)

a

SDHA = ﬂavoprotein subunit of succinate dehydrogenase; COX = cytochrome c oxidase; TP = thymidine phosphorylase; MNGIE = mitochondrial neurogastrointestinal encephalomyopathy; ANT1 = adenine nucleotide translocator; ar = autosomal recessive; ad = autosomal dominant; PEO = progressive external opthalmoplegia; POLG = mtDNA polymerase γ ; DDP = deafness dystonia peptide; ABC = ATP-binding cassette.

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include the cytochrome oxidase assembly genes SURF1, SCO2, and COX10 (Tiranti et al., 1998; Zhu et al., 1998; Papadopoulou et al., 1999; Jaksch et al., 2000; Sue et al., 2000; Huckriede and Agsteribbe, 1994; Koehler et al., 1999; Puccio and Koenig, 2000). Mutations in these genes have largely been associated with Leigh’s syndrome. The mechanisms by which they inﬂuence the pathogenesis of the respiratory chain disorders are not fully elucidated.

C. DEFECTS IN NUCLEAR GENES ENCODING FACTORS FOR INTERGENOMIC COMMUNICATION Defects in nuclear genes that affect the integrity of mtDNA have recently been identiﬁed and cause respiratory chain dysfunction as a result of secondary mtDNA defects induced by the primary nuclear genetic defect. Mutations in the gene encoding thymidine phosphorylase (TP) are thought to lead to impaired replication of mtDNA and are the cause of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), an autosomal-recessive disorder associated with both mtDNA depletion and multiple deletions (Hirano and Vu, 2000). This gene was identiﬁed by linkage studies followed by sequence analysis that indicated the mutations. The pathogenicity of the mutations was conﬁrmed in functional studies on patients’ leukocytes and identiﬁed that the mutation was causing a dysmetabolism of thymidine (Nishino et al., 2001). Another genetic defect of intergenomic communication is a point mutation in the adenine nucleotide translocator 1 (ANT1) gene that causes autosomal-recessive and autosomaldominant forms of PEO with multiple mitochondrial deletions in muscle (Kaukonen et al., 2000). Further nuclear gene mutations were identiﬁed in patients suffering from autosomal-dominant PEO (adPEO) with multiple deletions. One of these genes codes for a mitochondrial protein with high similarity to the phage T7 primase/helicase protein localized to mitochondrial nucleoids. It is located on chromosome 10 and is named “Twinkle” (Spelbrink et al., 2001). Another gene located on chromosome 15 found to carry mutations in association with adPEO codes for the DNA polymerase γ , and is essential for mtDNA maintenance (Van Goethem et al., 2001). Other candidate genes of this type that have been screened for pathogenic mutations include the mtTFA mapped to chromosome 10q, the mitochondrial RNA polymerase mapped to chromosome 19q, DNA polymerase γ on chromosome 15q, and mitochondrial single-stranded binding protein on chromosome 7q (reviewed by Taanman, 1999). For these genes no pathogenic mutations have yet been identiﬁed.

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D. DEFECTS IN NUCLEAR GENES ENCODING FACTORS INDIRECTLY RELATED TO RESPIRATORY CHAIN It has been suggested that in addition to the group of disorders commonly referred to as mitochondrial encephalomyopathies, other disorders may also be attributed to mutations in genes encoding proteins involved in cell respiration (Zeviani and Klopstock, 2001). The human X-linked genetic deafness dystonia syndrome has been attributed to a truncation of a protein called DDP1 ( Jin et al., 1996). This protein was identiﬁed by homology to the yeast Tim8p/9p protein and plays a role in the import of protein molecules through the intermembrane space and the insertion of cytosolic chaperones to the inner mitochondrial membrane (Koehler et al., 1999). The pathogenesis of an autosomal-recessive form of hereditary spastic paraplegia is also related indirectly to the function of the respiratory chain (Casari et al., 1998). This is because the identiﬁed disease causing mutation is located in a gene encoding the protein paraplegin that is localized to the mitochondria. Histological examination of muscle biopsy specimens in patients carrying the mutation revealed signs of OXPHOS defect—namely, the appearance of ragged-red ﬁbers that stained intensely for succinate dehydrogenase (Casari et al., 1998). Another disorder relevant to a defect in the mitochondria is the X-linked sideroplastic anemia and ataxia. This disorder has been associated with a point mutation in the ABC7 gene that encodes a protein of the ATP-binding cassette (ABC) superfamily of iron transporters across the mitochondrial membrane (Allikmets et al., 1999). Finally, Friedreich’s ataxia is another genetic disorder caused by a mutation in a gene encoding an iron transport protein—namely, frataxin (Rotig et al., 1997; Cavadini et al., 2000). This mutation was shown to result in an overload of mitochondrial iron, instability of mtDNA, and oxidative stress (Foury and Talibi, 2001).

VI. Cell Models of Respiratory Chain Disease Associated with Nuclear DNA Defects

A. APPLICATION OF CELL MODELS FOR THE IDENTIFICATION OF NUCLEAR GENE DEFECTS ASSOCIATED WITH RESPIRATORY CHAIN DISEASES The nuclear genome contributes a great deal to respiratory chain function since the majority of the polypeptides that constitute the OXPHOS system and all the proteins involved in its assembly as well as in mtDNA transcription, translation, and replication are encoded by nuclear genes. The involvement of nuclear genetic factors in the development of respiratory

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chain disorders is evident from the inheritance patterns of certain mitochondrial encephalomyopathies. One approach to the identiﬁcation of nuclear genes is to perform linkage studies. However, this method is hampered by the difﬁculty in obtaining a homogeneous population due to the great degree of genetic heterogeneity. An alternative method used is the identiﬁcation of candidate genes by screening the human databases for expressed sequence tags (EST) with protein or circular DNA (cDNA) sequences of yeast involved in the mitochondrial respiratory chain. In a recent investigation starting with 340 yeast protein sequences as templates, 102 human homologous genes were cloned and mapped to human chromosomes (Rotig et al., 2000). Candidate genes isolated from the DNA of patients are then screened for pathogenic mutations. Cell culture systems may not be used only to study the molecular mechanisms by which known genetic defects inﬂuence expression of phenotype but may also be applied to identify unknown nuclear genes using strategies based on complementation by chromosome transfer. The SURF1 gene encoding a mitochondrial protein that acts at the third stage in the four-step process of COX assembly (Tiranti et al., 1999) was identiﬁed by the use of functional complementation techniques in cell cultures. First, the chromosome containing the complementing gene was identiﬁed using microcellmediated chromosome transfer (Killary and Fournier, 1995). In this way it was shown that transfer of chromosome 9 into COX-defective cells restored COX activity, and the genetic defect was localized by deletion mapping to a 4.5 cM region of chromosome 9q34, containing SURF1. Sequencing of SURF-1 in patients revealed ﬁve different pathogenic mutations, all of which predicted a truncated protein (Zhu et al., 1998). A similar approach based on fusion of COX-defective cell lines with rodent/human rho zero cell lines was used later to identify ﬁve more mutations in the same gene (Tiranti et al., 1998). Biochemical analysis suggested that SURF-1 mutations are responsible for the loss of function in a major complementation group of COX-negative Leigh’s disease (Tiranti et al., 1999).

B. CELL MODELS OF RESPIRATORY CHAIN DISEASE ASSOCIATED WITH NUCLEAR DNA DEFECTS CAUSING THE mtDNA DEPLETION SYNDROME The mtDNA depletion syndrome was identiﬁed in 1991 in infants with marked depletion in mtDNA and defective oxidative phosphorylation in affected tissues (Moraes et al., 1991). Since then more than 30 patients have been described, and they present with infantile-onset muscle weakness and hepatic or renal failure associated with tissue-speciﬁc depletion of

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mtDNA, that may be as high as 99%. A correlation exists between age of onset and severity of depletion. No pathogenic mutations have been identiﬁed in mtDNA, and this defect is inherited as an autosomal-recessive (Moraes et al., 1991) or autosomal-dominant trait with incomplete penetrance (Mazziota et al., 1992), suggesting the presence of nuclear DNA mutations. The differential tissue expression may be explained by the fact that the nuclear genetic defect interferes with mtDNA replication or causes failure of resumption in mtDNA replication early in embryogenesis, resulting in mtDNA depletion in individual stem cell populations (Moraes et al., 1991). The latter assumption could explain the tissue-speciﬁc depletion of mtDNA and also the variable tissue expression among members of the same family. The nuclear origin of mtDNA depletion has been demonstrated in a patient whose ﬁbroblasts (that exhibited respiratory chain defects in tissue culture, and only grew in medium containing uridine and pyruvate) were enucleated and fused with human lung carcinoma derived rho zero cell line (Bodnar et al., 1993). The cybrids regained normal mtDNA concentration and respiratory chain function showing that the nucleus of the rho zero cell line had replaced the defective factor in the patient’s mtDNA depleted cells. A defect in the patient’s mtDNA was ruled out since his mtDNA could replicate and function in a normal nuclear environment. Replication of mtDNA is controlled by factors encoded by nuclear DNA; however, most of these factors have not yet been deﬁned. The human mitochondrial transcription factor A (mtTFA) had been considered as a likely candidate because it primes the transcription and replication of mtDNA. However, no mutations have been found to support the concept that defects in mtTFA cause depletion. Similarly, no mutations were found in another candidate gene, polymerase γ . A second experiment examined the mechanisms of mtDNA depletion using a primary myoblast cell culture from a patient with mtDNA depletion syndrome. The cultured cells initially had normal levels of mtDNA, but showed a progressive decrease of mtDNA at later passages (Taanman et al., 1997). Transfer of patient mitochondria with residual mtDNA levels to rho zero cells resulted in restoration of mtDNA levels, and therefore supported the results of the previous study that a nuclear factor is involved in the depletion.

VII. Application of Cell Models for the Development of Therapeutic Strategies in mtDNA Disease

A potential strategy for the treatment of mtDNA respiratory chain diseases is gene therapy. One way gene therapy may be achieved is by

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transferring mtDNA to patient cells using cybrid technology. The principle is based on in vitro experiments showing that when enucleated cells (cytoplasts) are fused with rho zero cell lines or nucleated cells they transfer their mitochondria to these cells (cybrids). In the cybrids that were made by introducing wild-type cytoplasts to the rho zero cells, the respiratory activity was restored, whereas in those produced from mutant mtDNA from patients the activity remained as in the original rho zero cells (Kagawa et al., 2001). Theoretically, in the in vivo situation patient cells that are rich in wild-type mtDNA may be selectively proliferated by the addition of speciﬁc translation factors into their medium. These cells may then be converted to cytoplasts and fused into the mutant cells with cell-fusion assisting proteins or polyethylene glycol (Kagawa et al., 2001). It is also possible to achieve mitochondrial gene transfer using as vectors newly described vesicles made of dequalinium, called DQAsomes (Weissig and Torchilin, 2000). An alternative therapeutic method to gene therapy that is based on results from cell culture experiments also has been suggested. The observation that a patient with respiratory chain disease attributed to a rare mutation in the tRNA gene for leucine at np12320 had the pathogenic mutation only in mature muscle led investigators to examine the segregation patterns of the mutation in primary cultures (Clark et al., 1997). It was conﬁrmed that despite the proportion of mutant mtDNA molecules being 94% in mature muscle, cultured myoblasts derived from satellite cells contained no mutated molecules even after a small number of cell divisions. Therefore the experimental results suggested the potential usefulness of muscle regeneration (which promotes satellite cell division) as a therapeutic strategy and led investigators to examine the situation in vivo. It was demonstrated that by inducing necrosis in a small muscle region there was a marked increase in the proportion of COX-positive ﬁbers in the regenerated muscle. Single-ﬁber polymerase chain reaction (PCR) conﬁrmed the reduction in the proportion of the mutant mtDNA molecules in the regenerated region, which also became COX positive. However, a subsequent trial in patients with chronic progressive external ophthalmoplegia (CPEO) that aimed to restore the levator muscle function of the eye failed to provide satisfactory improvement of ptosis (Andrews et al., 1999). A very similar result has been achieved in a patient with a KSS phenotype due to a heteroplasmic point mutation in the tRNA gene for leucine (CUN) at position 12,315 (Shoubridge et al., 1997). Three weeks after chemical induction of necrosis in mature muscle there was complete reversal of the mutant mtDNA molecules in the regenerating skeletal muscle ﬁbers. The investigators suggested that a realistic therapeutic strategy based on these ﬁndings would be to induce satellite cell incorporation into existing myoﬁbers through microscopic damage to the muscle produced by eccentric

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contractions (muscle contractions at full muscle extension). It was then shown that short-term aerobic training could improve exercise and oxidative capacity in patients with mitochondrial myopathy (Shoubridge et al., 1997). However, this study did not assess the mechanisms that accompanied this improvement. It was not assessed whether there was increased proliferation of the wild-type vs the mutant mtDNA molecules, or whether there were changes in the activity of the respiratory chain enzymes. These biochemical and genetic mechanisms that could underlie the increase in skeletal muscle oxidative capacity were examined in a subsequent study. The ﬁndings were that increased levels of respiratory chain enzymes accompanied increased oxidative capacity, but this occurred despite an increase in the proportion of the mutant rather than the wild-type mtDNA molecules (Taivassalo et al., 1999). This raises concerns about recommending such a therapy to patients. In the longer term an increase in mutant mtDNA load may be detrimental. It is possible to inhibit replication of mtDNA by using peptide nucleic acids (PNAs) that bind with great afﬁnity to the single-stranded mtDNA complementary strand during replication (Taylor et al., 1997). The feasibility of this as a treatment strategy has been demonstrated in an in vitro run-off assay. Two DNA templates were generated one mutant and the other wild type for the common 4977-bp deletion and for the npA8344G MERRF point mutation. PNAs were synthesized complementary to the deletion breakpoint sequence or to the MERRF mutant sequence. The results indicated that the replication of the mutant mtDNA molecule was successfully inhibited by the PNAs and was accompanied by the formation of a truncated product, while for the wild-type molecule it remained intact. When varying proportions of wild-type and mutant templates were used in the run-off assay to reﬂect the situation in vivo, there was a concomitant increase in the formation of truncated replication product (Taylor et al., 1997). These ﬁndings suggest it may be possible to treat heteroplasmic patients by inhibiting replication of the mutant mtDNA molecules. The development of strategies to introduce the PNAs into cells is being studied (Taylor et al., 1997).

VIII. Animal Models of Respiratory Chain Disease

A. ANIMAL MODELS OF RESPIRATORY CHAIN DISEASE ASSOCIATED WITH mtDNA DEFECTS Recently, an approach to the study of the pathogenetic mechanisms of mitochondrial encephalomyopathies due to mutations in genes encoded by the mitochondrial or the nuclear genomes has been the use of animal

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models. Generating mouse models for disease attributed to mtDNA defects has been difﬁcult because until recently it was not possible to stably introduce mutagenized mtDNA into the mitochondria of mammalian cells using available vectors. This is because the commonly used vectors were retroviruses that would require DNA replication for their insertion into genomic DNA. This is not possible with myoblasts, which like neurons cease DNA synthesis before fusion (Kagawa et al., 2001). In contrast, after the formation of transformed cybrids, it is possible to achieve stable mtDNA replication. Based on this, Inoue et al. (2000) used the rho zero cell system to develop a mouse model. Mitochondria that contained the deleted mtDNA molecules were isolated in synaptosomes (resealed vesicles from nerve endings) from aged mouse brain. The synaptosomes were fused with rho zero cells and the resulting cybrids were screened by polymerase chain reaction (PCR) for the presence of mtDNA deletions. The cells in identiﬁed cybrid clones were enucleated and then electrofused with zygotes before implantation to pseudopregnant female mice (Fig. 2). The speciﬁc mtDNA deletion in mice was carried into both the somatic cells and the germ line, and it accumulated in successive generations until a threshold of 90% was reached for mitochondrial dysfunction to become apparent in various tissues. This “mito-mouse” is potentially useful for the study of the mechanisms of transmission and tissue-speciﬁc expression for mtDNA partial duplications and deletions. It represents a good model for human mitochondrial encephalomyopathies attributed to large-scale rearrangements in mtDNA (Inoue et al., 2000). However, there are ﬁve important differences in the features of disease in humans and the condition induced in mice, and these have been reviewed by Shoubridge (2000): First, RRFs that are a characteristic feature of mtDNA disease in affected human muscle are not seen in mice. Second, the threshold for respiratory dysfunction is higher in the mouse than in humans. Third, the predominant phenotypic features in mice include anemia and renal failure, which are uncommon in humans. Fourth, whereas germ line transmission of mtDNA deletions has rarely been reported in humans, mtDNA deletions are transmitted in the mice through the germ line and it is possible that they rearrange later in somatic cells. Finally, whereas there are marked differences in the degree of heteroplasmy in human tissues, mice showed similar proportions of deleted mtDNAs in all tissues. Another mouse model of mtDNA disease was developed by the introduction of the “chloramphenicol (CAP)-resistant” mtDNA mutation into the embryonic stem cells of the mice. The mutation that results from an A to T substitution at np2379 in the 16S rRNA gene of mtDNA was originally derived from mouse 3T3 cells that are resistant to CAP due to the presence of the mutation. Chimeric mice were generated by eliminating CAP-sensitive mtDNAs from the embryonic stem cells and then by fusing

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synaptosome

451

rho° cell

cybrid

enucleate, fuse with donor embryo

embryo with mutant mtDNA

transfer to pseudopregnant mice with different proportions of mutant mtDNA FIG. 2. Steps for the generation of a mouse model harboring mutant mtDNA (Shoubridge, 2000). Synaptosomes from mouse brain (resealed cytoplasmic vesicles) containing mitochondria with heteroplasmic proportions of mutant mtDNA are fused with rho zero cells. The generated cybrids are enucleated and fused with a donor mouse embryo. The resulting embryos are implanted into pseudopregnant females, and offspring are screened for containing heteroplasmic proportions of mtDNA. Reprinted with permission from Shoubridge, E. A. (2000).

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them with enucleated CAP-resistant 3T3 mouse cells. The resulting cell lines had a proportion of mutant mtDNA of 90%, and examination by cytochemical staining revealed reduced cytochrome oxidase activity (Marchington et al., 1999). These cells were then injected into the blastocyst to generate chimeric heteroplasmic mice (transmitochondrial mice) with a percentage of mutant mtDNAs that varied from 0 to 50% in the tissues analyzed (Levy et al., 1999; Marchington et al., 1999). The symptoms in the transmitochondrial mice included cataracts and retinal changes as well as mitochondrial cardiomyopathy and myopathy (Levy et al., 1999; Sligh et al., 2000). It has been suggested that phenotypes in humans that could potentially be studied with the CAP-resistant mouse model include: (a) nerve deafness because it is attributed to an rRNA gene, speciﬁcally the 12S rRNA gene in the mtDNA sequence; (b) the np3243 MELAS mutation because it causes a translational defect of similar severity to the CAP-resistant mtDNA mutation; and (c) the NARP np9883 ATPase6 mutation because it is associated with similar alterations in retinal function (Marchington et al., 1999; Sligh et al., 2000). The development of the CAPresistant mouse is a good model for the study of segregation and transmission of mtDNA since it was demonstrated that the ability of the embryonic cells to differentiate into tissues is not lost and the distribution of mutant mtDNA into different tissues has common features with human mitochondrial disease (Marchington et al., 1999). Finally, the method used may be applied to the generation of mouse mutants for different mtDNA mutations. This is especially important since there is no method of site-directed mutagenesis for the mitochondrial genome. A further animal model of mtDNA disease involves mice with severe combined immunodeﬁciency (SCID) containing human mtDNA mutations in their muscle (Clark et al., 1998). To develop this model human myoblasts were injected into the tibialis anterior muscle of SCID mice, and it was conﬁrmed that the regenerated muscle ﬁbers express the human mitochondrial genome. Next it was demonstrated that the regenerated ﬁbers contained mutant mtDNA when myoblasts were derived from patients with NARP and MERRF syndromes. Therefore the mice could express heteroplasmic proportions of human mtDNA pathogenic mutations. Table II summarizes the animal models of mitochondrial disease.

B. ANIMAL MODELS OF RESPIRATORY CHAIN DISEASE ASSOCIATED WITH NUCLEAR DNA DEFECTS Mouse models for nuclear DNA defects that lead to dysfunction of the OXPHOS system or to abnormalities in mtDNA replication and transcription

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TABLE II IN VIVO MODELS OF MITOCHONDRIAL ENCEPHALOMYOPATHIESa Model

Symptoms

Reference

A. Animal models of respiratory chain disease associated with mtDNA defects Mouse model with mtDNA deletion

Renal failure and premature death

Inoue et al. (2000)

CAP-resistant mouse with mutation in 16S rRNA

Cataracts, retinal changes, cardiomyopathy, myopathy

Marchington et al. (1999)

SCID mouse

Not described

Clark et al. (1998)

B. Animal models of respiratory chain disease associated with nuclear DNA defects MtTFA knockout mice with mtDNA depletion

Growth retardation and death at embryonic stage

Larsson et al. (1998)

ANT1 knochout mouse with multiple deletions

Exercise intolerance and cardiac defects

Graham et al. (1997)

SOD2 knockout mouse

Cardiomyopathy, lactic acidosis, fat accumulation in liver

Li et al. (1995)

C. Other models of respiratory chain disease P183 (or tam) mutant (Drosophila M)

Reduction in eye development and locomotive deﬁcit

Iyengar et al. (1999)

Sluggish-A (Drosophila M)

Locomotive defects

Hayward et al. (1993)

Knockout (Drosophila M)

Locomotive defects

Royden et al. (1987)

Stress-sensitive (Drosophila M)

Locomotive defects

Zhang et al. (1998)

AAC2 mutant (S. Cerevisiae)

Defective respiration

Kaukonen et al. (2000)

R. Capsulatus

Complex I defect

Lunardi et al. (1998)

a CAP = chloramphenicol resistant; SCID = severe combined immunodeﬁciency; MtTFA = mitochondrial transcription factor A; ANT1 = adenine nucleotide translocator; SOD = superoxide dismutase; Drosophila M = Drosophila melanogaster; S. Cerevisiae = Saccharomyces cerevisiae; R. Capsulatus = Rhodobacter capsulatus.

processes may be generated by the use of knockout gene technology, whereby speciﬁc genes are inactivated or bases within a gene are modiﬁed. MtTFA is a nuclear-encoded protein that binds upstream of the lightand heavy-chain promoters of mtDNA to promote its transcription, and it may also regulate mtDNA replication processes as studies in vitro have suggested (Parisi and Clayton, 1991). In vivo evidence of the importance of mtTFA in maintaining the mtDNA levels has been obtained from studies in knockout mice that can be generated by introducing loxP sites in embryonic stem cells. This allowed excision of exons 6 and 7 of the Tfam gene

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by cre -mediated recombination resulting in knockout allele Tfam− (Larsson et al., 1998). The TfamloP TfamloP mice were normal, while after speciﬁc matings, offspring that inherited one knockout allele had reduced mtDNA copy numbers while offspring homozygous for the mutant allele exhibited severe mtDNA depletion and died at embryonic stage (Larsson et al., 1998). It also was found that speciﬁc tissues were more sensitive to changes in the levels of mtTFA implying that the gene–dosage effect may contribute to tissue-speciﬁc differences in respiratory chain capacity in humans (Larsson et al., 1998). This model demonstrated that the mtTFA is necessary for the regulation of mtDNA copy number and for embryonic development. Furthermore, it indicated that variation in mtTFA gene sequence leads to mild defects in the respiratory chain capacity, which may be additive to the effect of other alterations in the mtDNA sequence (Larsson et al., 1998). A genetic defect of the ANT1 gene of the inner mitochondrial membrane is of direct relevance to the development of respiratory chain diseases, and such defects have been associated with autosomal-dominant PEO (Kaukonen et al., 2000). The development of a knockout mouse deﬁcient in the heart/muscle isoform of ANT (ANT1) has provided in vivo evidence that ANT1 induces multiple mtDNA deletions and causes reduced rates of mitochondrial ADP-stimulated respiration giving rise to a clinical phenotype. The symptoms of the ANT1 mutants included exercise intolerance and cardiac hypertrophy. In addition, histological examination revealed a dramatic proliferation of mitochondria and the presence of ragged-red muscle ﬁbers. Therefore, the ANT1 knockout mouse exhibits similar phenotypic features to those of patients with mitochondrial myopathies and represents a good animal model for mitochondrial disease (Graham et al., 1997). The same knockout mouse model has also been analyzed in relation to the production of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) (Esposito et al., 1999). This study suggested that the levels of antioxidant defense enzymes induced by the increased production of ROS in the ANT1deﬁcient mice are higher in skeletal muscle than the hear; consequently the accumulation of mtDNA damage is lower in skeletal muscle (Esposito et al., 1999). Therefore, reduced ATP synthesis as a result of ANT1 inactivation and dysfunction in the capacity of the OXPHOS system causes increased levels of ROS and increased damage to the mtDNA with tissue differences in severity. Another defect in the nuclear genome that has been associated with reduced ATP synthesis, and with increased oxidative stress is the mutational alterations in the genes for superoxide dismutase (SOD) an enzyme that reduces the superoxide anion (produced during the OXPHOS process) to H2O2. The superoxide anion gives rise to further toxic ROS (Giulivi et al., 1995). There are three genes for SOD, and their effects have been examined

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in knockout mice inactivated for the genes through homologous recombination (Melov et al., 1999). The most deleterious phenotypic effects in mice were exhibited by inactivation of the gene for SOD2, and symptoms included cardiomyopathy, lactic acidosis, and fat accumulation in the liver (Li et al., 1995). Biochemical analysis revealed severe deﬁciencies in the respiratory chain enzymes such as complexes I and II, and the tricarboxylic acid cycle enzymes causing severe inhibition of ATP synthesis and the accumulation of oxidative mtDNA damage (Melov et al., 1998; Melov et al., 1999). Furthermore, it has been demonstrated that the SOD mimetic and synthetic antioxidant manganese 5,10,15,20-tetrakis (4-benzoic acid) porphyrin (MnTBAP) may prevent the development of cardiomyopathy and the accumulation of lipid in the liver (Melov et al., 1998). Therefore, the SOD2 knockout mice constitute a good model to examine the efﬁcacy of antioxidants and to study the mechanisms of oxidative damage that may be associated with mitochondrial disease.

IX. Other Models of Respiratory Chain Disease

Other models that have been developed for the study of mitochondrial diseases employ the invertebrates Drosophila melanogaster andCaenorhabditis elegans as well as the yeast Saccharomyces cerevisiae and the photosynthetic bacterium Rhodobacter capsulatus (Table II). Genetic studies in invertebrates have been widely applied due to the relative simplicity of these organisms and the availability of advanced molecular and genetic techniques. A mutant strain of the D. melanogaster, the P183 mutant, shows reduction in compound eye development and failure to respond to light that is due to a locomotory deﬁcit (Iyengar et al., 1999). Genetic mapping of the region of the eliminated gene in the P183 strain and sequence analysis indicated that this gene that was renamed to “tam” is homologous to the human catalytic subunit of mtDNA polymerase. It has been suggested that the tam mutant displays similar features to the mTFA mouse mutant. Therefore, the tam mutant constitutes a model for the study of the mechanisms of mtDNA replication, and as suggested it may give an insight into the identiﬁcation of mutations in the pathway that modify the tam phenotype (Iyengar et al., 1999). There are three more Drosophila M mutants for genes encoding mitochondrial proteins. These are the sluggish-A (slgA) mutants, and the knockout (tko) and stress-sensitive B (sesB) mutants—all of which exhibit defects in locomotion (Hayward et al., 1993; Royden et al., 1987; Zhang et al., 1998).

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Despite the differences in the features of the OXPHOS system between yeast and human, yeast S. cerevisiae was the ﬁrst living organism used for the characterization of the OXPHOS system. This was partly due to the advancement of the molecular genetics of yeast and also due to the accessibility of this organism. This system has allowed the identiﬁcation of a number of nuclear-factors involved in mitochondrial biogenesis (Grivell, 1989) and will help to understand nuclear–mitochondrial interactions. For example, it has become apparent in yeast that, although mtDNA is under the inﬂuence of nuclear genes that encode all the factors responsible for mtDNA replication and transcription, the converse is also true. Hence, the quality and quantity of mtDNA modulates the levels of nuclear DNA expression (Parikh et al., 1987). Due to the evolutionary structural and functional conservation between human and yeast genes, studies in yeast have enabled the elucidation of the homologous human genes involved in the mitochondrial respiratory chain (Foury and Cazzalini, 1997). This will help our understanding of human disease-associated gene functions. Indeed, 30% of human ESTs in the databases match yeast genes, and the fraction of positionally cloned yeast genes matching human genes is comparable to that of random collection of human cDNAs. A yeast model has been developed for the study of the functional significance of the ANT1 nuclear gene mutation at position 114 that changes a conserved alanine for proline causing PEO (Kaukonen et al., 2000). Using two different S. cerevisiae strains lacking the AAC2 gene corresponding to the human ANT1 gene, it was shown that the equivalent to human A114P mutation was pathogenic since it induced defective respiratory chain activity by disrupting ADP/ATP translocation (Kaukonen et al., 2000). This is not the case in humans, however, where ANT1 causes secondary accumulation of mutant mtDNA molecules by unknown mechanisms. Nevertheless, the results in the yeast have suggested the ANT1 mutation is likely to interfere with mammalian enzymes that regulate intramitochondrial dATP concentrations important for DNA synthesis. Homology between the human mitochondrial genes encoding subunits of complex I enzyme with bacterial genes encoding NADH:ubiquinone oxidoreductases is also notable. An enzymatic model has been developed in Rhodob. capsulatus for the characterization of the mutations in relation to complex I deﬁciency (Lunardi et al., 1998). In this way the respiratory defects caused by the common np11778 point mutation in the ND4 subunit of complex I that is associated with LHON were studied in R. capsulatus that was speciﬁcally mutagenised at the equivalent position. The mutant exhibited biochemical features similar to the ones observed in cell cultures of the human mitochondria cells harboring the mutation. There was impairment in oxidative phosphorylation capacity and a preserved proton

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pump activity. Therefore, R. capsulatus represents a model for the study of the mechanisms involved in the pathogenesis of complex I deﬁciencies (Lunardi et al.,1998).

X. Conclusions

Cell culture experiments have been used to study the effects of speciﬁc known transitions and rearrangements in the mtDNA sequence in relation to biochemical processes in the mitochondrion such as respiratory capacity, membrane potential, rate of ATP production, and enzyme activity. The effects on translation, replication, and transcription of mtDNA have also been analyzed. These studies have revealed which identiﬁed mutation is pathogenic. In addition, information regarding the proportion of mutant DNA required for respiratory dysfunction, the tissue speciﬁcity of the threshold values, the degree of correlation between respiratory dysfunction and extent of heteroplasmy, and the suppressor effects of coexisting mutations in the mtDNA has been obtained. The variations in respiratory capacity observed among transformants obtained by the fusion of different mitochondria-less recipient cells with variable mitochondrial donors suggest that not only the mtDNA haplotype but also the nuclear genetic background inﬂuence the pathogenicity of mutations. Further studies will be required to identify other factors inﬂuencing the of transmission of pathogenic mtDNA mutations and inﬂuencing the genotype–phenotype relationships observed in patients. Experiments in cell culture systems have also enabled investigators to identify whether a genetic defect causing respiratory chain dysfunction is of nuclear or mitochondrial genome origin. For nuclear genetic defects it has been possible to identify the chromosomal location of the defect in complementation studies and to isolate the causative gene. It is expected that more research will focus on the identiﬁcation of nuclear-encoded genes that play a role in the functioning of the respiratory chain. Studies in cell culture systems cannot always be directly related to the human phenotype. The generation of animal models offers a greater potential for the study of the pathogenesis of the mitochondrial encephalomyopathies caused by mtDNA or nuclear gene defects. The use of embryonic stem cells for the development of mutant mice in the mitochondrial genome has aided the study of the mechanisms of transmission and segregation of mtDNA. They also have the potential to be manipulated to generate mutant animals that harbor different heteroplasmic proportions of the mutation. Knockout mouse models have been generated for the study of mitochondrial diseases

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attributed to nuclear genome defects. Some of the animal models develop symptoms that are similar to the human disease. Combining the results from both cell and animal studies has advanced understanding of the mechanisms of pathogenesis of the mtDNA disorders, and the relationships between genotype and phenotype. The nuclear gene disorders are now beginning to emerge. It is likely that they will ultimately comprise a larger group than mtDNA associated respiratory chain diseases. Cell and animal models will be important in elucidating and developing treatments, such as effective drugs or gene therapy, for these nuclear gene disorders.

Acknowledgments

Research performed in our laboratory is funded by the MRC as part of the University College London MRC Cooperative “Mitochondria in Health and Disease.” We acknowledge Dr. Eric Shoubridge and the Nature Genetics Group as well as Prof. Ton Maassen and the editors of Diabetologia for permitting copyright of the ﬁgures used in this chapter.

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van den Heuvel, L., Ruitenbeek, W., Smeets, R., Gelman-Kohan, Z., Elpeleg, O., Loeffen, J., Trijbels, F., Mariman, E., de Bruijn, D., and Smeitink, J. (1998). Demonstration of a new pathogenic mutation in human complex I deﬁciency: A 5-bp duplication in the nuclear gene encoding the 18-kD (AQDQ) subunit. Am. J. Hum. Genet. 62, 262–268. van den Ouweland, J. M., Maechler, P., Wollheim, C. B., Attardi, G., and Maassen, J. A. (1999). Functional and morphological abnormalities of mitochondria harboring the tRNA (Leu)(UUR) mutation in mitochondrial DNA derived from patients with maternally inherited diabetes and deafness (MIDD) and progressive kidney disease. Diabetologia 42, 485–492. Van Goethem, G., Dermaut, B., Lofgren, A., Martin, J. J., and Van Broeckhoven, C. (2001). Mutation of POLG is associated with progressive external ophthalmoplegia characterized by mtDNA deletions. Nat. Genet. 28, 211–212. Vergani, L., Martinuzzi, A., Carelli, V., Cortelli, P., Montagna, P., Schievano, G., Carrozzo, R., Angelini, C., and Lugaresi, E. (1995). MtDNA mutations associated with Leber’s hereditary optic neuropathy: studies on cytoplasmic hybrid (cybrid) cells. Biochem. Biophys. Res. Commun. 210, 880–888. Wallace, D. C. (1992). Mitochondrial genetics: A paradigm for aging and degenerative diseases? Science 256, 628–632. Wallace, D. C., Singh, G., Lott, M. T., Hodge, J. A., Schurr, T. G., Lezza, A. M., Elsas, L. J., 2nd, and Nikoskelainen, E. K. (1888a). Mitochondrial DNA mutation associated with Leber’s hereditary optic neuropathy. Science 242, 1427–1430. Wallace, D. C., Zheng, X. X., Lott, M. T., Shoffner, J. M., Hodge, J. A., Kelley, R. I., Epstein, C. M., and Hopkins, L. C. (1988b). Familial mitochondrial encephalomyopathy (MERRF): Genetic, pathophysiological, and biochemical characterization of a mitochondrial DNA disease. Cell 55, 601–610. Weissig, V., and Torchilin, V. P. (2000). Mitochondriotropic cationic vesicles: A strategy towards mitochondrial gene therapy. Curr. Pharm. Biotechnol. 1, 325–346. Yoneda, M., Chomyn, A., Martinuzzi, A., Hurko, O., and Attardi, G. (1992). Marked replicative advantage of human mtDNA carrying a point mutation that causes the MELAS encephalomyopathy. Proc. Natl. Acad. Sci. USA 89, 11164–11168. Zeviani, M., and Klopstock, T. (2001). Mitochondrial disorders. Curr. Opin. Neurol. 14, 553–560. Zeviani, M., Moraes, C. T., DiMauro, S., Nakase, H., Bonilla, E., Schon, E. A., and Rowland, L. P. (1998). Deletions of mitochondrial DNA in Kearns-Sayre syndrome. Neurology 51, 1525–1533. Zhang, Y. Q., Roote, J., Brogna, S., Davis, A. W., Barbash, D. A., Nash, D., and Ashburner, M. (1999). Stress sensitive B encodes an adenine nucleotide translocase in Drosophila melanogaster. Genetics 153, 891–903. Zhu, Z., Yao, J., Johns, T., Fu, K., De Bie, I., Macmillan, C., Cuthbert, A. P., Newbold, R. F., Wang, J., Chevrette, M., Brown, G. K., Brown, R. M., and Shoubridge, E. A. (1998). SURF1, encoding a factor involved in the biogenesis of cytochrome c oxidase, is mutated in Leigh syndrome. Nat. Genet. 20, 337–343.

SECTION VII DEFECTS OF β-OXIDATION INCLUDING CARNITINE DEFICIENCY

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DEFECTS OF β-OXIDATION INCLUDING CARNITINE DEFICIENCY

K. Bartlett1 and M. Pourfarzam∗ Department of Child Health∗ ,1 and Department of Clinical Biochemistry University of Newcastle upon Tyne Newcastle upon Tyne NE1 4LP, United Kingdom

I. Introduction II. Background Biochemistry A. Fuel Physiology B. Enzymology and the Control of Mitochondrial β-Oxidation C. Tissue Differences III. Inherited Disorders of Mitochondrial β-Oxidation A. Primary Carnitine Deficiency (Carnitine Uptake Defect) (OMIM 212140) B. CPT I Deficiency (OMIM 600528) C. Carnitine Acylcarnitine Translocase Deficiency (OMIM 212138) D. CPT II Deficiency (OMIM 600650) E. VLCAD Deficiency (OMIM 201475) F. MCAD Deficiency (OMIM 201450) G. SCAD Deficiency (OMIM 201470) H. Trifunctional Protein Deficiencies (OMIM 600890) I. Short-Chain 3-Hydroxyacyl-CoA Dehydrogenase Deficiency (SCHAD) (OMIM 201470) J. Glutaric Aciduria Type II (Multiple Dehydrogenase Deficiency) (OMIM 231680) IV. Conclusions References

I. Introduction

Inherited disorders of long-chain fatty acid mitochondrial β-oxidation are an important group of diseases that collectively comprise a group of commoner genetic disorders. Since fatty acids are an important source of energy, particularly under fasting conditions, it is not surprising that if the pathway ﬂux is attenuated by deﬁciency of one of the constituent enzymes there are far-reaching consequences that, if not recognized and treated 1 To whom correspondence should be addressed at Department of Child Health, Sir James Spence Institute of Child Health, Royal Victoria Inﬁrmary, Queen Victoria Road, Newcastle upon Tyne NE1 4LP, United Kingdom.

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promptly, may result in death, brain damage, and a range of other pathologies depending on the enzyme affected. Although fatty acid oxidation also occurs in the peroxisomal compartment, this homologous pathway is genetically, biochemically, and functionally quite distinct, and although genetically determined disorders of the peroxisomal pathway are also known, the present review is only concerned with the mitochondrial pathway and its disorders. Similarly, fatty acids may also undergo oxidation of the terminal methyl group (ω-oxidation) or of the ﬁrst methylene group at C2 (α-oxidation); neither of these pathways are considered here, other than their role as detoxiﬁcation mechanisms when β-oxidation ﬂux is compromised. Since carnitine, together with three enzymes (carnitine palmitoyltransferase I, carnitine palmitoyltransferase II, and carnitine acylcarnitine translocase), are required for the transport of long-chain fatty acids across the mitochondrial inner membrane, deﬁciency of any one of these four results in impaired fatty acid oxidation. Accordingly, they can be considered to be functionally part of the β-oxidation pathway and deﬁciencies of these components are also considered here. The literature concerned with these disorders and associated biochemistry is considerable, and space precludes an exhaustive review. However, where appropriate early reports, subsequent reviews and the more recent literature is cited. For each of the disorders the commonly accepted acronymns is used and the EC number, OMIM, and genetic database acquisition numbers are given.

II. Background Biochemistry

A. FUEL PHYSIOLOGY In normal subjects under fasting conditions, the insulin/glucagon ratio is low with resultant stimulation of lipolysis. Fatty acids are mobilized from fat depot stores into the circulation, and are taken up and oxidized by most tissues with the notable exception of the CNS. In liver there is almost quantitative conversion of fatty acids to ketone bodies, which are in turn exported for oxidation by extrahepatic tissues. Simultaneously, glycogenolysis occurs, and in liver, and to a lesser extent kidney, glucose is mobilized for extrahepatic utilization. Skeletal muscle has substantial glycogen reserves, but these are utilized endogenously particularly during exercise. Thus the net affect of fasting or indeed any stress leading to counterregulation of insulin, is a switch from a carbohydrate-based fuel economy to one in which a greater proportion of energy is derived from the oxidation of lipid with a

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concomitant lowering of the respiratory quotient. The resultant sparing of glucose allows the direction of glucose to those tissues with an obligatory requirement such as CNS. This, in brief, is the conventional view of the whole body response to fasting and is mediated by regulatory mechanisms, which is not discussed further here. It is clear from the above that impaired activity of any of the enzymes of β-oxidation or of the auxiliary systems concerned with fatty acid transport, with disposal of reducing equivalents, with disposal of acetyl coenzyme A (acetyl CoA), or with the degradation of polyunsaturated fatty acids, is likely to have a major impact on glucose sparing during periods of counterregulation. Furthermore, gluconeogenesis may well be attenuated due to lowered availability of reducing equivalents. This is particularly apparent in patients with disorders of the long- and medium-chain speciﬁc enzymes. However, in patients with the short-chain disorders, in milder variants, and in older patients, in whom exercise intolerance and muscle and heart involvement are the predominant presenting features, hypoglycemia and an inappropriate ketotic response to fasting may not be present. The concentrations of intermediary metabolites from patients with medium-chain acyl-CoA dehydrogenase deﬁciency and from patients with another causes of hypoketotic hypoglycemia, hyperinsulinism, are shown in Table I. Whether or not hypoglycemia is accompanied by an appropriate ketonemia is clearly of importance. In order to distinguish an appropriate ketotic response to hypoglycemia, particularly in the context of impaired βoxidation, it is helpful to relate log ([acetoacetate] + [3-hydroxybutyrate]) to the concentration of non-esteriﬁed fatty acids (NEFA) (Bartlett et al., 1991). Most patients with disorders of β-oxidation have high concentrations of free fatty acids but inappropriately low concentrations of ketone bodies for that degree of lipolysis. Figure 1 shows the sequential change in the relationship between free fatty acids and ketone bodies during the progression of the starvation provocation test in three children with medium-chain acyl-CoA dehydrogenase deﬁciency. It is clear that while the relationship is normal at the onset of the starvation, with increasing starvation-induced stress the relationship rapidly becomes abnormal. The sequential changes in children in whom there was no evidence of metabolic disease (Fig. 1— continuous lines) are also shown, and it is apparent that these data points stay within the 95% conﬁdence limits derived from cross-sectional data. It is instructive to compare these children with hyperinsulinemic children who have a relationship which falls within the 95% conﬁdence limits (Bartlett et al., 1991). Although these children had an inappropriately low concentration of ketone bodies for the degree of glycemia, the relationship with free fatty acids was appropriate. Thus, the hypoketonemia arose from decreased free fatty acid release as a result of the antilipolytic effect of insulin on adipose cells.

TABLE I CONCENTRATIONS OF INTERMEDIARY METABOLITES IN THE BLOOD OF NORMAL SUBJECTS, PATIENTS WITH MCAD DEFICIENCY AND PATIENTS a WITH HYPERINSULINISM

Analyte

Lactate (mmol/L)

Pyruvate (mmol/L)

Alanine (mmol/L)

3-Ohbutyrate (mmol/L)

Acetoacetate (mmol/L)

Glucose (mmol/L)

NEFA (mmol/L)

Glycerol (mmol/L)

Insulin (mU/L)

Controls (fasted for 24 h; n = 19) Mean SD

1.26 0.56

0.11 0.07

0.20 0.06

1.98 1.38

0.74 0.56

3.6 0.60

1.58 0.39

0.16 0.04

<1.0 —

Hyperinsulinemics (n = 13) Mean SD

1.07 0.53

0.09 0.04

0.24 0.08

0.29 0.47

0.14 0.17

2.5 0.9

0.58 0.45

0.74 0.11

9.8 7.6

MCAD deﬁciency Mean SD n

1.24 0.39 8

0.09 0.04 3

0.15 0.01 3

0.40 0.20 8

0.23 0.24 3

2.54 0.65 8

2.28 0.42 7

0.28 0.17 3

<1.0 — 5

a

Modiﬁed from Bartlett et al. (1991), with permission.

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FIG. 1. The relationship of plasma NEFA concentrations to (log) blood ketone body concentrations. The line of best ﬁt and the 95% conﬁdence limits are shown for 46 control subjects. Also shown are data from patients with medium-chain acyl-CoA dehydrogenase deﬁciency [•] and normal children during the course of a fasting provocation stress test. (Modiﬁed from Bartlett et al., 1991, with permission.)

B. ENZYMOLOGY AND THE CONTROL OF MITOCHONDRIAL β-OXIDATION Figure 2 shows the overall pathway of mitochondrial β-oxidation. Much of the control of pathway ﬂux is determined by rate of import of acyl groups into the mitochondrial matrix (CPT I), but there are also multiple intramitochondrial sites of control, which are indicated by the dashed lines in Fig. 2. The enzymes involved are listed in Table II and their properties are described brieﬂy below. 1. Carnitine Palmitoyltransferases and the AcylCarnitine-Carnitine Translocase The carnitine palmitoyltransferase (CPT) system for the import of longchain fatty acids into the mitochondrial matrix has been known for some

FIG. 2. The pathway of mitochondrial β-oxidation. ETF, electron transfer ﬂavoprotein; UQ , ubiquinone; ETF:QO, electron transfer ﬂavoprotein:ubiquinone oxidoreductase, CoA, coenzyme A. The dotted red lines indicate points of feedback conrol.

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TABLE II ENZYMES AND TRANSPORTERS OF MITOCHONDRIAL β-OXIDATION

Enzyme

Structure

MW (kDa)

Gene name

Chromosome location

Carnitine transporter

Monomer

63

OCTN2

5q33.1

Carnitine palmitoyl transferase I (liver) (muscle)

Unknown

82

CPT1A

11q13

Unknown

88

Carnitine acylcarnitine translocase

Unknown

32.5

CACT

3p21.31

Carnitine palmitoyl transferase II

Unknown

68

CPT2

1p32

Very-long-chain acyl-CoA dehydrogenase

Homodimer

150

ACADVL

17p11.2–p11.13

Long-chain acyl-CoA dehydrogenase

Homotetramer

180

ACADL

2q34–2q35

Medium-chain acyl-CoA dehydrogenase

Homotetramer

180

ACADM

1p31

Short-chain acyl-CoA dehydrogenase

Homotetramer

168

ACADS

12q22–qter

Long-chain 3-hydroxyacyl-CoA dehydrogenase (α subunit)

Heterooctamer

460

HADHA

2p23

Long-chain 3-oxoacyl-CoA thiolase (β subunit) (trifunctional protein)

HADHB

2p23

Short-chain 3-hydroxyacyl-CoA dehydrogenase

HADHSC

4q22–q26

Long-chain 2-enoyl-CoA hydratase (α subunit)

Short-chain 2-enoyl-CoA hydratase

Homohexamer

164

ECHS1

10q26.2–q26.3

Short-chain 3-oxoacyl-CoA thiolase

Homotetramer

169

ACAA2

18q21.1–q21.2

Medium-chain (“general”) 3-oxoacyl-CoA thiolase

Homotetramer

200

MCKAT

Electron transfering ﬂavoprotein (ETF)

Heterodimer

57

α Subunit

ETFA

15q23–q25

β Subunit

ETFB

19q13.3

ETFDH

4q23–qter

ETF dehydrogenase

Monomer

68

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years. Hepatic CPT I has been puriﬁed and shown to be immunologically distinct from CPT II (Kolodziej et al., 1992). It has been cloned and sequenced, allowing expression in yeast and demonstration of malonyl-CoA binding by the catalytic polypeptide (Esser et al., 1996). An isoform of CPT I immunologically distinct from that in liver is present in skeletal muscle, heart, and adipose tissue (Kolodziej et al., 1992; Esser et al., 1996) and McGarry and co-workers have demonstrated, on the basis of inhibitor binding studies, that two isoforms of CPT I, the liver isoform (CPT IA) and the skeletal muscle isoform (CPT IB), are simultaneously expressed in heart tissue (Weis et al., 1994b). They have subsequently shown, using the differential sensitivity of the two isoforms to dinitrophenyl-etomoxir (DNPetomoxir) (Weis et al., 1994a), that the contribution of the liver form to total heart CPT I activity decreases from 25% in the neonatal period to 2–3% in adult rats (Brown et al., 1995), and they have suggested that the markedly different kinetic characteristics of the two isoforms with respect to carnitine and to malonyl-CoA inhibition overcome the low perinatal carnitine levels in the heart. The subcellular location of CPT I has also been debated. Various groups have suggested either the inner face of the outer mitochondrial membrane or the outer face of the inner mitochondrial membrane. Recently, strong evidence for the latter location has been published (Hoppel et al., 1998). The carnitine acylcarnitine translocase has been puriﬁed and found to catalyze a slow unidirectional transport of carnitine as well as the translocase activity (Indiveri et al., 1990, 1991) and is embedded in the innermitochondrial membrane. Carnitine palmitoyl transferase II, the ﬁnal protein involved in the importation of long-chain fatty acids, is located on the inner face of the inner mitochondrial membrane (Fig. 3). 2. Acyl-CoA Dehydrogenase (ACD) There are multiple enzymes for each of the constituent steps of the pathway that vary in their chain-length speciﬁcity. In the case of acyl-CoA dehydrogenation there are four enzymes: short-chain acyl-CoA dehydrogenase (SCAD, active with C4 and C6), medium-chain acyl-CoA dehydrogenase (MCAD, C4 to C12), long-chain acyl-CoA dehydrogenase (LCAD, active with C8 to C20) and very-long-chain acyl-CoA dehydrogenase (VLCAD, active with C12 to C24). Each of these enzymes catalyzes the formation of 2-enoyl CoA from the corresponding saturated ester. SCAD, MCAD, and LCAD are homotetramers located in the matrix. The VLCAD, however, is a homodimer, and it is located on the inner face of the inner mitochondrial membrane. From early studies, it had been assumed that there are only three acyl-CoA dehydrogenases involved in mitochondrial β-oxidation: SCAD, MCAD, and LCAD. However, the isolation and puriﬁcation of VLCAD

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FIG. 3. Schematic of the organization of mitochondrial β-oxidation. CPT1 and 2, carnitine palmitoyltransferase 1 and 2; ETF:QO, electron transfer ﬂavoprotein:ubiquinone oxidoreductase; CoA, coenzyme A; ETF, electron transfer ﬂavoprotein; VLCAD, very-long-chain acyl-CoA dehydrogenase; LCEH, long-chain enoyl-CoA hydratase; LCHAD, long-chain 3-hydroxyacylCoA dehydrogenase; LCOAT, long-chain 3-oxoacyl-CoA thiolase; MC/SCAD, medium-chain and short-chain acyl-CoA dehydrogenases; MC/SCOAT, medium-chain (“general”) and shortchain thiolases; SCHAD, short-chain 3-hydroxyacyl-CoA dehydrogenase; short-chain enoyl-CoA hydradatase (“crotonase”). The red lines indicate the paths of acyl-CoA oxidation. The black lines indicate the role of co-factors.

(Izai et al., 1992) and the demonstration that patients previously thought to have inherited deﬁciencies of LCAD were in fact suffering from VLCAD deﬁciency (Yamaguchi et al., 1993) has shown that there are in fact four enzymes. The MCAD is the best characterized of the ACD family, and the structure of the protein at 3-A˚ resolution, a dimer of dimers, was reported some years ago (Kim and Wu, 1988). The mechanism of action of this group of ﬂavoproteins appear to be very similar with the concerted removal of the pro-R-α-hydrogen from the acyl CoA as a proton and elimination of the corresponding pro-R-β-hydrogen to the N-5 position of the ﬂavin as a hydride equivalent (Thorpe and Kim, 1995).

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3. Enoyl-CoA Hydratase (EH) There are two 2-enoyl-CoA hydratases. One, a soluble matrix enzyme, is most active toward short-chain substrates, although it will act on substrates up to C16 at a much slower rate and with higher K m values (Hass and Hill, 1969; Waterson and Hill, 1972). This was the ﬁrst enzyme of mammalian mitochondrial β-oxidation to be puriﬁed (crotonase, short-chain enoyl-CoA hydratase, EC 4.2.1.17). The long-chain enzyme (EC 4.2.1.74) is most active with C6 –10 substrates, and it is virtually inactive with crotonyl CoA (C4), the preferred substrate of crotonase (Wit-Peeters et al., 1971; Fong and Schulz, 1977). It is now apparent that the long-chain enzyme is infact a constituent of the trifunctional enzyme described below. 4. 3-Hydroxyacyl-CoA Dehydrogenase (HAD) The third step of the pathway, L-3-hydroxyacyl-CoA dehydrogenation, is catalyzed by two enzymes with overlapping chain-length speciﬁcities. The short-chain enzyme (SCHAD, EC 1.1.1.35) is a soluble matrix enzyme that will act on substrates of chain-length C4–C16 although, as with crotonase, the shorter chain length substrates are preferred (Bradshaw and Noyes, 1975; Osumi and Hashimoto, 1980; He et al., 1989). A long-chain HAD was ﬁrst demonstrated by El Fakhri and Middleton (1982). This enzyme is ﬁrmly associated with the inner mitochondrial membrane and active with medium- and long-chain substrate, C16 being the preferred substrate. As with the long-chain enoyl-CoA hydratase described above, the long-chain 3-hydroxyacyl-CoA dehydrogenase is a constituent of the trifunctional protein. 5. 3-Oxoacyl-CoA Thiolase (OAT) The ﬁnal step of the pathway, thiolytic cleavage of 3-oxoacyl CoA to yield acetyl CoA and a chain-shortened intermediate is catalyzed by three enzymes. Two soluble activities have been identiﬁed. One is speciﬁc for acetoacetyl CoA and 2-methylacetoacetyl CoA (EC 2.3.1.9; Middleton, 1972, 1973; Middleton and Bartlett, 1983). The second thiolase, the mediumchain or “general” thiolase, is active with all substrates from C6 to C16 to an approximately equal extent (Staack et al., 1978). Seubert et al., 1968; The third activity is a constituent of the trifunctional enzyme, which also comprises the long-chain 2-enoyl-CoA hydratase and long-chain 3-hydroxyacylCoA dehydrogenase activities described above. This complex, a heterooctamer made up of four α-units with long-chain enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities and four β-units with longchain 3-oxothiolase activity is closely associated with the inner mitochondrial membrane (Uchida et al., 1992; Carpenter et al., 1992; Luo et al., 1993). Evidence for the existence of such a complex was ﬁrst suggested from our studies of a child with an inherited disorder of β-oxidation.

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Analysis of acyl-CoA and acyl-carnitine esters derived from incubations of mitochondrial fractions with [U-14C]hexadecanoate revealed the presence of 3-oxo-, 3-hydroxy-, and 2-enoyl-derivatives. Subsequent enzyme measurement demonstrated total absence of long-chain 3-oxoacyl-CoA thiolase activity and markedly diminished long-chain enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities ( Jackson et al., 1992). The αunit is required for membrane binding and a mutant trifunctional protein with a totally absent α-unit resulted in mislocation of β-unit 3-oxothiolase activity in the matrix (Weinberger et al., 1995). A complex of activities associated with CPT II was isolated by Kerner and Bieber (1990). However, the constituent activities were not characterized with any precision, and it is difﬁcult to know if Bieber’s complex also involved the trifunctional enzyme. In any event CPT II, VLCAD, and the trifunctional protein are associated with the inner mitochondrial membrane together with complex I, ETF:CoQ oxidoreductase, and the remainder of the respiratory chain. 6. Auxiliary Enzymes of Polyunsaturated Fatty Acid Oxidation It is now generally accepted that mitochondrial oxidation of polyunsaturated fatty acids proceeds via the 2,4-dienoyl-CoA reductase/3,2-enoylCoA isomerase dependent route. However, a further enzyme, 3,5,2,4dienoyl-CoA isomerase, has been puriﬁed, and it has been shown to be necessary for the metabolism of fatty acids containing 5 double bonds rather than a direct reduction of the double bond (Smeland et al., 1992; Chen et al., 1994; Tserng et al., 1996), and this appears to be the major route operative in intact mitochondria (Tserng et al., 1996). 7. Organization of the Enzymes of Mitochondrial β-Oxidation In view of the presence of membrane-bound enzymes of β-oxidation and the close relationship between β-oxidation and the respiratory chain, together with the “leaky-hosepipe model,” we proposed a model of mitochondrial β-oxidation illustrated in Fig. 3. In this model, long-chain fatty acids enter the mitochondrion and are acted on by membrane-bound enzymes of β-oxidation. Reducing equivalents ETFH2 and NADH are channeled to ETF:QO and complex I respectively, and the concentrations of the CoA ester intermediates are kept low either by substrate channeling between the active sites of the enzymes or because of the lipophilic nature of long-chain acyl-CoA esters. The long-chain CoA esters, together with chainshortened CoA esters, are “leaked” from this pool of rapidly turning over intermediates, and in the case of the chain-shortened intermediates, are acted on by medium and short-chain enzymes in the matrix, or loosely associated with the inner mitochondrial membrane as suggested by Sumegi and Srere (1984). When the pathway is attenuated by the lack of one or more

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of the constituent enzyme activities, as occurs in inherited disorders of the pathway, substrates accumulate and undergo a number of detoxiﬁcation reactions such as hydrolysis or formation of the glycine or carnitine esters. The function of these reactions is to prevent sequestration of CoA and to allow the removal of acyl intermediates. C. TISSUE DIFFERENCES On a whole body basis, muscle, heart, liver, and kidney are the major tissues that utilize fatty acids. However, there is a growing body of evidence that glial cells have the capacity to utilise fatty acids and are ketogenic (Edmond et al., 1987; Auestad et al., 1991; Guzman and Blazquez, 2001). However, some of these observations must be interpreted with caution, since much of the data have been derived from studies of cultured neonatal brain preparations, and it is difﬁcult to be certain to what extent they can be extrapolated to the in vivo situation—particularly in adults. Astrocytes constitute approximately 50% of brain mass, and they are functionally and anatomically interposed between neurones and the blood supply. In terms of energy metabolism, glial cells resemble hepatocytes. They can take up glucose and store glycogen; however, they have a high glycolytic capacity and export lactate, as well as glucose, to neurones. Thus glucose entering the astrocytic compartment may simply traverse it to the neuronal compartment, may undergo glycolysis and be exported as lactate, or may be stored as glycogen. The balance between these routes is under ﬁne and rapid control in order to respond to local neuronal activity. All the enzymes of mitochondrial β-oxidation are detectable in brain, and with the exception of the general oxoacyl-CoA thiolase, are present at activities comparable to those found in hepatocytes (4–50%) (Yang et al., 1987). However, the general thiolase is present at very low activities (<1%), and it appears that it is the activity of this enzyme that limits β-oxidation ﬂux in brain.

III. Inherited Disorders of Mitochondrial β-Oxidation

A. PRIMARY CARNITINE DEFICIENCY (CARNITINE UPTAKE DEFECT) (OMIM 212140) 1. Clinical Presentation Systemic carnitine deﬁciency was ﬁrst reported in 1973 (Engel and Angelini, 1973). Subsequently, the term was used to describe a number of

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patients with reduced levels of carnitine and clinical symptoms consistent with impaired fatty acid oxidation (Angelini et al., 1987). Later it became evident that many of these patients did not have primary carnitine deﬁciency, and their condition was actually caused by other fatty acid oxidation enzyme defects (Coates et al., 1984; Hale et al., 1985b). The criteria for the diagnosis of primary carnitine deﬁciency was therefore proposed as (1) severe reduction of plasma or tissue carnitine levels, (2) evidence that low carnitine levels impair fatty acid oxidation, (3) correction of the disorder when carnitine levels are restored towards normal, and (4) absence of other primary defects in fatty acid oxidation (Treem et al., 1988). A defect in the plasma membrane carnitine tranporter OCTN2(OCT: organic cation transporter) has now been recognized and represents the only currently known cause of primary carnitine deﬁciency (Eriksson et al., 1988; Treem et al., 1988; Tein et al., 1990). The onset of the clinical symptoms in patients with primary carnitine deﬁciency ranges from one month to seven years, with different types of presentation: progressive and potentially fatal dilated and hypertrophic cardiomyopathy, myopathy, and hypoketotic hypoglycemic encephalopathy. In some cases all forms of presentation may exist (Tein et al., 1990; Garavaglia et al., 1991; Stanley et al., 1991). Progressive cardiomyopathy is the most common and usually occurs in older patients (Pierpont et al., 2000). Myopathy, manifesting in hypotonia or slowly progressive proximal weakness, is commonly associated with cardiomyopathy or encephalopathy. Acute encephalopathy associated with hypoketotic hypoglycemia is more commonly seen in younger patients (<2 years), and it is often triggered by a catabolic stress, before cardiomyopathy becomes apparent (Stanley et al., 1991; Pons and De Vivo, 1995). Several cases have been reported with sudden and unexpected infant death (Rinaldo et al., 1997). Heterozygotes can manifest heart involvement including benign cardiac hypertrophy (Garavaglia et al., 1991; Koizumi et al., 1999). The ethnic distribution includes Caucasian, Mexican, Japanese, Chinese, African-American and North African Arab. 2. Diagnosis Plasma free and total carnitine values are extremely low and associated with increased urinary fractional excretion of free carnitine. There is no abnormal urinary organic acid excretion, and dicarboxylic aciduria is mild or absent even during crises. Heterozygotes have been reported to have reduced plasma carnitine levels (Scaglia et al., 1998). The oxidation of labeled long-chain fatty acids is impaired in fresh lymphocytes and ﬁbroblasts cultured in carnitine-depleted medium. Addition of carnitine to the medium enhances ﬁbroblast fatty acid oxidation in patients, whereas there is little or no effect in control cells. The diagnosis is conﬁrmed by in vitro studies of carnitine uptake in cultured skin ﬁbroblasts (Eriksson et al., 1989; Tein et al., 1990; Stanley et al., 1991), leukocytes,

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cultured lymphoblasts (Tein and Xie, 1996), and cultured muscle cells (Pons et al., 1997). Homozygote patients have very low rates of carnitine uptake (<10% of control values), which precludes the calculation of K m and Vmax. Fibroblasts from heterozygotes have intermediate rates of carnitine uptake (normal Km values but reduced Vmax values). A lowered rate of carnitine uptake was shown in cultured amniocytes of an affected fetus (Christodoulou et al., 1996). 3. Genetics/Mutation Analysis (GenBank AB015050) Kinetic studies in different tissues and cultured cells suggest the involvement of several transporters in carnitine transport across the cell plasma membrane (Kerner and Hoppel, 1998). It is not known whether these transport components are the result of the expression of different transporters or correspond to differential activity states of a single transporter. Recently a sodium-ion-dependent, high-afﬁnity carnitine transporter OCTN2, a new member of the human OCT family, has been cloned (Wu et al., 1998; Tamai et al., 1998). The OTCN2 complementary DNA (cDNA) codes for a polypeptide of 557 amino acids with a calculated MW of 63 kDa. It is an integral membrane protein and is predicted to have 12 transmembrane domains and 3 potential sites for glycosylation, and both C- and N-terminal ends are oriented intracellularly. The OCTN2 is strongly expressed in various human tissues including kidney, skeletal muscle, heart, and placenta. The OCTN2 gene [∼30 kilobase (kb)] maps to chromosome 5q31, and consists of 10 exons and 9 introns. The mature messenger RNA (mRNA) is 3500 nucleotides long with an open reading frame of 1674 nucleotides. Mutations in the OTCN2 gene have been described in several patients with systemic carnitine deﬁciency (Lamhonwah and Tein, 1998; Koizumi et al., 1999; Nezu et al., 1999; Tang et al., 1999; Vaz et al., 1999; Wang et al., 1999). But they appear to be heterogenous with no prevalent mutation apparent for the condition. In a recent study Koizumi et al. (1999) screened 973 unrelated individuals for low serum-free carnitine level, and found 14 individuals with consistently low values (<5th percentile). Among these, OCTN2 mutations were identiﬁed in 9. This study predicts a carrier frequency of about 1% in this subpopulation of Japan. 4. Treatment There is clear indication for carnitine therapy in primary carnitine deﬁciency. Dramatic improvement in cardiomyopathy and skeletal myopathy is reported in patients treated with a high dose of carnitine within the ﬁrst few weeks, and most patients were essentially normal within three to six months (Tein et al., 1990; Stanley et al., 1991; Pierpont et al., 2000). Eighteen of

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19 patients treated with carnitine supplementation for periods of 1–10 years continue to be healthy. It also corrects any impairment in hepatic ketogenesis that may be present (Stanley et al., 1991). In addition, three children with signiﬁcant failure to thrive before therapy showed a marked improvement in growth after therapy (Tein et al., 1990). Oral carnitine supplementation at doses of 100 mg/kg/day should maintain the plasma carnitine levels in the low to normal range. 5. Pathogenesis Primary carnitine deﬁciency results from the failure of the speciﬁc highafﬁnity carnitine uptake into several tissues including skeletal muscle, heart, gut, and kidney but not liver or brain. Carnitine concentrations in tissues are generally 20-fold to 50-fold higher than plasma (Bremer, 1983). A defect in carnitine transport means that a normal tissue gradient cannot be maintained. This leads to insufﬁcient carnitine in skeletal muscle and heart to support fatty acid oxidation. Impaired intestinal absorption and failure to reabsorb carnitine in the kidney results in very low plasma carnitine levels, which, in turn, diminishes uptake of carnitine by passive diffusion in the liver. Hence ketogenesis may also become impaired. Carnitine administration may restore plasma and liver carnitine levels to normal whereas muscle carnitine levels may show no detectable rise and remain below 10% of control levels. Nevertheless cardiomyopathy and muscle weakness are corrected, thus indicating that the threshold for impairing fatty acid oxidation occurs at tissue carnitine levels less than 5% of normal (Treem et al., 1988; Stanley et al., 1991).

B. CPT I DEFICIENCY (OMIM 600528) Since the ﬁrst description of the disorder in 1981 (Bougneres et al., 1981), approximately 25 cases have been reported in the literature or are known to the authors (Bonnefont et al., 1999; Brown et al., 2001). Although CPT I is known to exist in at least two isoforms (liver and muscle), only the hepatic form of the disease has been documented to date. The ethnic distribution includes Caucasian, American Indian, Asian Indian, Arab, and Inuit. 1. Clinical Presentation Patients with liver CPT I (L-CPT I) deﬁciency usually present in early life with recurrent episodes of nonketotic hypoglycemia triggered by fasting or intercurrent infection that resembles Reye’s syndrome, or with hepatomegaly occasionally associated with acute liver failure. Cardiac or skeletal

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muscle involvement is not typically associated with this condition; however, slight cardiomegaly and hypotonia have been noted in a few cases (Tein et al., 1989; Schaefer et al., 1997). Recently an adult patient with L-CPT I was reported (Brown et al., 2001) with primarily muscle symptoms (cramps and pain) but no hypoglycemia. In addition, elevated serum creatine kinase has been reported in two families, indicating that muscle damage can occur in this condition. Renal tubular acidosis has been documented in three patients. Some patients have signiﬁcant neurological sequelae, presumably as a result of initial recurrent insults (Bonnefont et al., 1999). 2. Diagnosis Abnormal organic acid excretion is not observed in this condition, except for an occasional mild nonketotic dicarboxylic aciduria. In contrast to other fatty acid oxidation disorders, patients with CPT I deﬁciency have elevated concentrations of plasma free and total carnitine. This is due to an unusually high renal threshold for free carnitine in these patients (Stanley et al., 1992b). The acylated fraction consists primarily of acetyl carnitine. The blood acyl-carnitine proﬁle we examined in six patients by tandem mass spectrometry show almost complete absence of long-chain (i.e., C16, C18, and C18:1) acylcarnitines with high concentrations of free carnitine and acetylcarnitine (Fig. 4). Cultured ﬁbroblasts incubated with [U-14C]plamitate show accumulation of large amounts of palmitoyl CoA with no, or very small amounts of, palmitoylcarnitine in CPT I deﬁcient cells (Pourfarzam et al., 1994a). The overall oxidation of long-chain fatty acids by ﬁbroblasts and fresh lymphocytes is low, at 5–20% of control rates (Bonnefont et al., 1999). The deﬁnitive diagnosis of CPT I deﬁciency is made by enzyme activity measurement in the relevant tissue (i.e., ﬁbroblasts, lymphocytes, and liver in hepatic form CPT I deﬁciency). Carnitine Palmitoyltransferase I activity is measured in the absence of detergent and is identiﬁed by its sensitivity to inhibition by malonyl CoA (see Section III.D). Patients show activities in the range of 2–20% of control values.

FIG. 4. Blood acylcarnitine proﬁles from a control subject and patients with deﬁciencies of CPT I, Carnitine acylacarnitine translocase, CPT II, and VLCAD. Ion signals at representative m/z values correspond to free carnitine (m/z 218), free carnitine internal standard (m/z 221), C2 carnitine (m/z 260), C2 carnitine internal standard (m/z 263), C3 carnitine (m/z 274), C3 carnitine internal standard (m/z 277), C4 carnitine (m/z 288), C8 carnitine internal standard (m/z 353), C12 carnitine (m/z 400), C6DC carnitine (m/z 402), C14:1 carnitine (m/z 426), C14 carnitine (m/z 428), C8DC carnitine (m/z 430), C16:1 carnitine (m/z 454), C16 carnitine (m/z 456), C16 carnitine internal standard (m/z 456), C18:2 carnitine (m/z 480), C18:1 carnitine (m/z 482), C18 carnitine (m/z 484).

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3. Genetics (GenBank L39211, Y08683) Carnitine Polymitoyltransferase I exists in at least two tissue-speciﬁc isoforms, a liver type (L-CPT I or CPT IA) and a muscle type (M-CPT I or CPT IB), encoded by genes on human chromosome 11q13 and 22q13.3, respectively (McGarry and Brown, 1997; Britton et al., 1997). The structure of human L-CPT1 gene is partially characterized. It contains an open reading frame of 2219 base pairs (bp) with a 5 untranslated region of 76 and 35 bp of 3 untranslated material. The predicted protein contains 773 amino acids with molecular weight of 88.4 kDa (Britton et al., 1995). The gene encoding human M-CPT I is well characterized. It consists of two 5 -noncoding exons, 18 coding exons, and one 3 -noncoding exon spanning approximately 10 kilobase pairs (kbp) (Yamazaki et al., 1997). The M-CPT I cDNA has an open reading frame of 2316 bp and encodes a protein of 772 amino acids (Yamazaki et al., 1996). Molecular analysis of hepatic CPT I deﬁciency has been described in several patients. These include A1361G transition (changing aspartate at position 454 into glycine, D454G) (Ijlst et al., 1998), C1069T transition (R357W), T1451C transition (L484P), C1069T transition (R357W), G823A transition (A275T), C912G transversion (C304W), C367T transition (R123C), C1436T transition (P479L), 1183–1185 del CGT (del R395) (Brown et al., 2001), and G2129A transition (G710E) (Prip-Buus et al., 2001). The latter mutation has been reported in several patients belonging to the North American inbred Hutterites (Prasad et al., 2001). 4. Treatment Although this disorder may have a very severe and potentially lifethreatening presentation it seems that, once the diagnosis is made, the disorder is relatively easy to manage. Episodes of metabolic decompensation are treated with infusion of glucose and have been prevented by avoidance of fasting. Frequent feeding and replacement of dietary long-chain fat with medium-chain triglycerides (MCT) have been beneﬁcial. Early recognition of the condition and prompt management of the episodes may prevent the neurological sequelae. 5. Pathogenesis The metabolic consequences of L-CPT I deﬁciency is the impaired formation of long-chain acylcarnitines from the corresponding acyl-CoA esters. Hence, the entry of long-chain fatty acids into mitochondria for β-oxidation is diminished. In addition, this can lead to the accumulation of long-chain acyl-CoA esters in the cytoplasm that are potent inhibitors of adenine nucleotide translocase and could inhibit oxidative phosphorylation, further exacerbating the energy deﬁcit.

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C. CARNITINE ACYLCARNITINE TRANSLOCASE DEFICIENCY (OMIM 212138) Carnitine acylcarnitine translocase (CACT) (also known as the carnitine acyl-carnitine carrier) deﬁciency was ﬁrst described in 1992 (Stanley et al., 1992a), and some 27 additional cases have been reported. The ethnic distribution includes European, Turkish, Algerian, Hispanic, Japanese, Vietnamese, English-Chinese, and Pakistani. 1. Clinical Presentation There appear to be two phenotypes for this disorder: a severe neonatal onset and a milder phenotype. Patients with severe phenotype (about two thirds of the reported cases) all presented within four days of birth and died before three years of age with cardiomyopathy and skeletal myopathy or multiorgan failure (Stanley et al., 1992a; Pande et al., 1993; Niezen-Koning et al., 1995; Ogier et al., 1995; Chalmers et al., 1997; Invernizzi et al., 2000). At least 20 siblings have been reported with sudden unexplained neonatal death who were most probably also affected. The age of the presentation in the milder (surviving) cases ranges from two days to 17 months (Morris et al., 1998; Olpin et al., 1997; Huizing et al., 1997). The clinical presentation varies in both groups with no obvious features unique to any phenotype. Hypoglycemia is not always present. Hyperammonemia, resistant to dietary changes, seems to be a common feature. Cardiac involvement (cardiomyopathy, cardiac dysrhythmia, heart block) and skeletal myopathy have been reported in most patients. Hepatomegaly and liver failure are also commonly observed. 2. Diagnosis There is no characteristic abnormal organic aciduria or acidemia associated with this condition. Excretion of acyl glycines is normal and there is a normal proﬁle of acylcarnitine excretion. Dicarboxylic aciduria could be mild or even absent. However, an important diagnostic feature is the severe plasma free carnitine deﬁciency associated with an markedly elevated acylcarnitines fraction. Acylcarnitine analysis by tandem mass spectrometry shows highly elevated hexadecanoylcarnitine (C16) with the concentration of C16 greater than C18 and C18:1 species. The proﬁle can be distinguished from that seen in patients with severe CPT II deﬁciency in whom the level of C18 species is usually greater than that of C16 (Fig. 4). Analysis of the β-oxidation intermediates following the incubation of cultured ﬁbroblasts with [U-14C]palmitate shows accumulation of palmitoylcarnitine with very little chain shortening and is a useful diagnostic tool (Pourfarzam et al., 1994a; Ogier et al., 1995). Oxidation of long-chain fatty acids in ﬁbroblasts and lymphocytes is markedly reduced, although mild cases seem to have slightly higher residual value (Morris et al., 1998). The

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activity of CACT can be measured in selectively permeabilized ﬁbroblasts and lymphocytes (Pande et al., 1993). The translocase activity is related to the rate of [14C]acetylcarnitine efﬂux through the mitochondrial membrane following the mitochondial oxidation of [2-14C]pyruvate with the citric acid cycle inhibited. Since the assay requires functional integrity of pyruvate dehydrogenase and carnitine acetylcarnitine translocase, these two steps are assessed separately. A simpliﬁed assay based on the formation of [14C]CO2 from [14C]acetylcarnitine by premeabilised ﬁbroblasts has been described (Ijlst et al., 2001). Prenatal diagnosis is possible by in vitro fatty acid oxidation studies or enzyme assay in cultured ﬁbroblasts, chronic villus, or amniocytes (Chalmers et al., 1997; Ogier et al., 1995; Yang et al., 2001). 3. Genetics (GenBank NT026437) The human CACT gene spans about 16.5 kb, consists of nine exons separated by eight introns (Iacobazzi et al., 1998), and is assigned to chromosome 3p21. 31 (Viggiano et al., 1997). The translocase protein is a member of the mitochondrial carrier family. The human protein has a length of 301 amino acids (32.9 kDa) and contains a three fold repeated sequence of about 100 amino acids. It is proposed that each domain is folded into two transmembrane α-helices forming a structure with six α-helices in total connected by ﬁve hydrophilic loops (Indiveri et al., 1997; Iacobazzi et al., 1998). The molecular defect in the CACT gene was ﬁrst reported in a mild case, a cytosine insertion resulting in a frame shift and elongation of the protein by 21 amino acids at the C-terminal region (Huizing et al., 1997). Several other mutations, including deletion, insertion, splicing, missense, and point mutations have been reported (Huizing et al., 1998; Costa et al., 1999; Invernizzi et al., 2000; Hsu et al., 2000; Ogawa et al., 2000; Yang et al., 2001). These appear to be heterogenous with no prevalent mutation recognized for this disorder. 4. Treatment Management of acute illnesses involves provision of enough glucose to stimulate insulin secretion and block lipolysis as well as maintaining normal glucose homeostasis. For long-term management it is essential to avoid fasting, which stimulates the use of fatty acids as a source of energy. Some patients have been managed with frequent feeding (every 3 h) whereas others required continuous nocturnal intragastric feeding. The use of free carnitine supplementation has been reported in some patients, and as in other long-chain fatty acid oxidation defects, remains controversial. Its efﬁcacy should be evaluated on an individual basis. The usefulness of MCT administration in CACT deﬁciency seems to be partial and the possible

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adverse effects should be carefully assessed. There is evidence that oxidation of C10 and C12 fatty acids is also reduced in CACT deﬁciency (Chalmers et al., 1997; Parini et al., 1999) and a patient supplemented with MCT showed accumulation of C8 and C10 acylcarnitines (Nuoffer et al., 2000). Administration of large quantities of MCT may therefore lead to the accumulation of nonoxidized C10 and C12 fatty acids and the corresponding acyl-CoA and acylcarnitine esters. 5. Pathogenesis Deﬁciency of CACT leads to the accumulation of long-chain acylcarnitines along with their acyl-CoA counterparts, particularly in tissues active in the mitochondrial oxidation of long-chain fatty acids. The cytotoxic effect of these compounds, the inability of the tissue to derive energy from fatty acid oxidation, and the lack of alternative fuels, glucose, or ketone bodies leads to compromised organ function. This is indicated by multiorgan derangement often associated with the condition. The prominent cardiac involvement seen in CACT deﬁciency, as in other defects of longchain fatty acid oxidation, relates to the major requirement of the heart for mitochondrial fatty acid oxidation for energy. The hyperammonemia observed in some patients has been suggested to be due to defective N-acetyl-glutamate synthesis as a result of low concentrations of acetyl-CoA. However, treatment with carbamylglutamate (Ogier et al., 1995) or administration of medium-chain triglycerides (MCT) (Stanley et al., 1992a) did not appear to ameliorate the hyperammonemia.

D. CPT II DEFICIENCY (OMIM 600650) 1. Clinical Presentation Three distinct clinical phenotypes of CPT II deﬁciency have been recognized: a. Adult Muscular Form (OMIM 255110). The “classic” and most common form of CPT II deﬁciency is the adult myopathic form, ﬁrst described in 1973 (Di Mauro and Di Mauro, 1973). This was also the ﬁrst inherited fatty acid oxidation disorder to be described. Muscle CPT II deﬁciency is probably the most common inherited disorder of lipid metabolism affecting skeletal muscle and the most common fatty acid oxidation disorder after MCAD deﬁciency. Patients with this condition generally present as adolescents or young adults with recurrent episodes of muscle pain and rhabdomyolysis triggered by prolonged exercise. Occasionally these episodes are precipitated by fasting, infection, or exposure to cold or fever. Myoglubinuria may occur after

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severe episodes, occasionally leading to renal failure. Symptoms are usually restricted to skeletal muscle with no hepatic or cardiac involvement. The clinical presentation may be highly variable within a family ranging from asymptomatic to lethal (Handig et al., 1996). Although the disorder is inherited in an autosomal recessive manner, most of affected patients are males. Possibly because they undertake more prolonged exercise than females. b. Infantile and Neonatal Forms. A severe multiorgan and often lethal form of CPT II deﬁciency has been identiﬁed in infants and children (Demaugre et al., 1991; Hug et al., 1991; Elpeleg et al., 1993). Patients present with episodes of hypoketotic hypoglycemia resulting in coma and seizures. Hepatomegaly, cardiomyopathy, and peripheral myopathy is present in most patients. The neonatal onset form of the disease is more severe than the infantile form, with death occurring usually within the ﬁrst month of life. In addition to multiorgan involvement, dysmorphic features and renal dysgenesis has been reported in some patients (Hug et al., 1991; North et al., 1995; Brivet et al., 1999). 2. Diagnosis As in the other carnitine transport disorders, abnormal organic acid excretion is not associated with this condition. Plasma-free and total carnitine levels are often normal in the adult form of the disease, whereas patients with the early-onset multiorgan forms present with low free and total carnitine levels associated with a high acylation ratio. The ﬁrst clue to the diagnosis is obtained from the acylcarnitine analysis in blood specimens by tandem mass spectrometry. The proﬁles of patients with the early-onset form are characterized by markedly elevated long-chain acylcarnitines (i.e., C18, C18:1, C16) combined with very low acetylcarnitine concentrations (Fig. 4). Long-chain acylcarnitines may not be elevated in the adult myopathic form; however, even these patients show persistently low levels of acetylcarnitine with an elevated long-chain acylcarnitines/acetylcarnitine ratio. In the absence of a blood specimen for conﬁrmation, the accumulation of fatty acid oxidation intermediates can be demonstrated in ﬁbroblasts incubated with a labeled long-chain fatty acid (Pourfarzam et al., 1994a; Yang et al., 1998). Cells from CPT II deﬁcient patients incubated with palmitate show accumulation of large amounts of palmitoylcarnitine but very little palmitoyl CoA. The overall oxidation rate of labeled long-chain fatty acids in ﬁbroblasts or lymphocytes from patients with early-onset CPT II deﬁciency is usually <25% of control values (Demaugre et al., 1991; Bonnefont et al., 1996), whereas cells from adult-onset patients may show normal or only slightly reduced rates of long-chain fatty acid oxidation (Demaugre et al., 1988; Pourfarzam et al., 1994a). Carnitine Polymitoyltransferase II activity is

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determined by monitoring the reaction of palmitoyl-CoA with L-carnitine to produce palmitoylcarnitine and CoA or vice-versa. A number of different assays and assay conditions have been described; however, the isotope exchange method is widely used for clinical diagnosis (Zierz and Engel, 1985; Demaugre et al., 1988). In CPT II deﬁciency the enzyme defect has been demonstrated in a variety of tissues such as skeletal muscle, heart, liver, ﬁbroblasts, and lymphocytes irrespective of the clinical severity and presentation of the disease. In one study patients with the adult-onset form had ﬁbroblast CPT II residual activity about 15–25% of control values while those with the more severe early-onset form had values less than 10% of control activity (Bonnefont et al., 1996). It is debatable, however, whether a clear relationship exists between the severity of symptoms and the level of residual enzyme activity, since others have reported signiﬁcant overlap of CPT II residual activities between adult-type and infantile-type patients (Taroni et al., 1993; Wataya et al., 1998). It was also noted that abnormal ﬂux values were only found in patients whose CPT II residual activity was less than 15% of control values. Prenatal diagnosis of infantile CPT II is performed by mutation analysis, enzymatic assay, and/or in vitro fatty acid oxidation studies using chorionic villi or cultured amniocytes (Bonnefont et al., 1999). In one fetus the diagnosis was established by ultrasonography revealing enlarged echogenic kidneys (Witt et al., 1991). 3. Genetics (GenBank AF237952) The human CPT II cDNA contains an open reading frame of 1974 bp that encodes a protein of 658 amino acid residues including 25 residues of an NH2-terminal leader peptide that is cleared upon mitochondrial import to yield a mature protein of approximately 70 kDa (Finocchiaro et al., 1991). The human CPT II genomic structure has been well described. The gene is approximately 20 kb in size and is composed of ﬁve exons ranging from 81 to 1305 bp, separated by four introns ranging from 1.5 to 8.0 kb (Verderio et al., 1995). The gene is mapped to chromosome 1p32 (Gellera et al., 1994). It appears that in humans, as in rats, there is a single gene in operation, and the same enzyme is expressed in all tissues of the body (McGarry and Brown, 1997). The molecular basis of many patients with various forms of CPT II deﬁciency has been elucidated with more that 25 mutations described to date (reviewed in Bonnefont et al., 1999). The most common mutation in the adult myopathic form is a missense mutation (a C to T transition at mucleotide 439 in the cDNA) leading to a serine to leucine substitution at position 113 (S113L) of the mature protein (Taroni et al., 1993). This mutation has an allele frequency of between 53 and 60% in European and North

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American patients (Taroni et al., 1993; Kaufmann et al., 1997; Martin et al., 1999; Taggart et al., 1999) and authors personal data. Simple heterozygosity for certain CPT II mutations such as S113L may be sufﬁcient to predispose an individual to clinical symptoms (Taggart et al., 1999). The distinct clinical phenotypes of CPT II deﬁciency correlates to some degree with distinct genotype. Some “mild” mutations such as S113L, P50H, and D553N (Verderio et al., 1995) are associated with the adult form of the disease, whereas others such as F383Y (Yamamoto et al., 1996) or Y628S (Bonnefont et al., 1996) are associated with the severe neonatal form. However, clear exceptions exist. For example, a homozygous R631C mutation gives a different expression of the disorder in unrelated patients (Bonnefont et al., 1999). In addition, differences exist in the severity of the clinical symptoms exhibited by family members with the same mutation (Handig et al., 1996). Therefore, additional factors must inﬂuence the severity of the disease. 4. Treatment In the muscular type of CPT II deﬁciency, the long-term treatment is aimed at preventing episodes of rhabdomyolysis. Patients are generally advised to take frequent meals and avoid fasting. During intercurrent infection or before and during prolonged exercise, extra carbohydrate intake is beneﬁcial. During a severe rhabdomyolysis attack the risk of acute renal failure can be minimized by maintaining a good ﬂuid intake. The management of the severe type is similar to that for translocase deﬁciency. 5. Pathogenesis In CPT II deﬁciency long-chain acylcarnitines are transported into the mitochondrial matrix but are not efﬁciently β-oxidized since their conversion to the corresponding acyl-CoA esters is impaired. In the severe form of the disease there is marked elevation of long-chain acylcarnitines in blood, and it has been speculated that the increased level of these compounds may promote cardiac arrhythmia (Demaugre et al., 1991; Hug et al., 1991). In addition, since long-chain acylcarnitines have higher afﬁnity for the carnitine transporter than does free carnitine (Stanley et al., 1991), secondary carnitine deﬁciency may follow due to impaired renal re-absorption.

E. VLCAD DEFICIENCY (OMIM 201475) The ﬁrst patients with VLCAD deﬁciency were among the earliest reports of inherited disorders of mitochondrial β-oxidation, and were initially

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thought to be defects of LCAD (Hale et al., 1985a). Subsequently it became apparent, with the discovery of VLCAD, that these and other described patients had in fact deﬁciencies of VLCAD (Aoyama et al., 1993; Bertrand et al., 1993). 1. Clinical Presentation Three clinical phenotypes have been distinguished: a severe neonatal form characterized by hypoglycemia, cardiomyopathy, and a high rate of mortality (∼75%); a milder later-onset form of the disease characterized by a lower mortality rate, the absence of cardiomyopathy, but the presence of hypoketotic hypoglycemia, and a third muscle form in which patients present with progressive and exercise-induced rhabdomyolysis (Ogilvie et al., 1991; Vianey-Saban et al., 1998; Andresen et al., 1999; Minetti et al., 1998). Unlike SCAD and MCAD, there appears to be a clear correlation between genotype and phenotype (Andresen et al., 1999). Patients with the severe childhood phenotype have mutations that result in no residual activity, whereas the milder and later-onset phenotypes have mutations that give rise to some residual enzyme activity. Notwithstanding this clinical heterogeneity, all patients should be regarded as being at risk of hypoglycemia. 2. Diagnosis All patients with VLCAD deﬁciency are at risk of hypoglycemia. Urinary organic acids are characterized by the presence of saturated and unsaturated dicarboxylic acids. Blood acylcarnitines are also abnormal (Fig. 4), and typically 5-cis-tetradecenoyl-carnitine is present, which arises from the partial oxidation of oleoyl CoA (Brown-Harrison et al., 1996). Investigation of ﬁbroblast biochemistry conﬁrms the diagnosis either by direct enzyme assay (Vianey-Saban et al., 1998) or by analysis of intermediates by means of radio high performance liquid chromatography (HPLC) (Pourfarzam et al., 1994b) or tandem mass spectrometry (Nada et al., 1996). 3. Genetics (GenBank L46590) Very-long-chain acyl-CoA dehydrogenase deﬁciency is inherited as an autosomal-recessive condition. The VLCAD gene is located on chromosome 17p11.2-p11.1, consists of 20 exons, and codes a precursor protein of 655 amino acids, which includes an N-terminal leader sequence for mitochondrial import of 40 amino acids. The N-terminal sequence has a high degree of homology with other acyl-CoA dehydrogenases, but the C-terminal 180 amino acids do not, perhaps reﬂecting the dimer rather tetramer structure of the mature active enzyme and its subcellular localisation to the inner face of the inner mitochondrial membrane. In the largest series published, Andresen et al., document 58 mutations in 55 unrelated patients (Andresen

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et al., 1999). This included 45 mutations not previously documented and in excess of 60 disease-causing mutations have been recorded. 4. Treatment The avoidance of fasting, a high carbohydrate diet, and medium-chain triglycerides are the mainstay of long-term treatment. However, it is clearly crucial to be certain of the diagnosis before MCT treatment is commenced, and in particular, to exclude MCAD deﬁciency and glutaric aciduria type II. 5. Pathogenesis As with the other disorders of long-chain acyl-CoA oxidation, the predominant effect is energy deﬁciency. In tissues such as skeletal muscle and the heart, fat oxidation is the preferred substrate and resting muscle obtains approximately three quarters of its energy requirement from the oxidation of long-chain fatty acids. Thus a disorder involving attenuation of the ﬁrst step of β-oxidation, and moreover, the step with highest intramitochondrial control strength, will have a profound affect on pathway ﬂux. Clearly, rates of ketogenesis will be markedly inhibited and the switch in fuel selection, which is the hallmark of the fasted state, will not be possible. It is also highly probable that the accumulation of long-chain acyl-CoA esters will result in secondary inhibition of other processes, such as the adenine nucleotide translocase, which will further exacerbate the energy deﬁcit (Shug et al., 1971; Ho and Pande, 1974, Shug et al., 1975; Filippova et al., 1979; Woldegiorgis and Shrago, 1979; Bell, 1980a,b; Lochner et al., 1981; Shug and Subramanian, 1987). If, as now seems probable, glial cells are capable of β-oxidation (Edmond et al., 1987; Auestad et al., 1991; Guzman and Blazquez, 2001), glial ATP production may be compromised and contribute to the neurological phenotype.

F. MCAD DEFICIENCY (OMIM 201450) Since its ﬁrst description in 1982 (Kolvraa et al., 1982), MCAD deﬁciency has become the most well characterized of the inherited disorders of mitochondrial β-oxidation as well as the most prevalent. Space precludes a full review of the literature and the reader is directed to a very recent comprehensive review of this condition (Roe and Ding, 2001). 1. Clinical Presentation Until the advent of tandem mass spectrometric analysis of acylcarnitines, it could be rather difﬁcult to diagnose because of the episodic nature of the

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disorder. However, although there is some degree of clinical heterogeneity, even within a family, most cases present as a consequence of metabolic “stress” such as prolonged fasting, which may be associated with an otherwise trivial intercurrent illness. Typically, children present comatose with hypoglycemia that, if not treated promptly, can result in death. Although cases of MCAD deﬁciency presenting in the neonatal period and in later childhood have been documented, most cases present in the ﬁrst two years of infancy. The combination of the withdrawal of nocturnal feeding and an intercurrent infection will frequently precipitate a metabolic crisis that may be fatal. Indeed, MCAD deﬁciency has been implicated in sudden infant death syndrome because of the catastrophic consequences in some cases. Furthermore, in the largest clinical review published, in which 94 families were reported (Iafolla et al., 1994), 20% of the families had experienced one or more unexplained deaths in infancy. Moreover, in some of the surviving children (∼20%) there was signiﬁcant neurological and behavioral morbidity. 2. Diagnosis Under fasting conditions, MCAD deﬁciency results in the excretion of a range of abnormal metabolites including dicarboxylic acids (arising from ω-oxidation) and glycine conjugates. However, even during periods of remission when the urinary organic acids appear largely normal, the formation of medium-chain acylcarnitines still occurs and the presence of octanoylcarnitine in blood is diagnostic of the condition. The appreciation that all inborn errors of metabolism resulting in the accumulation of acyl-CoA esters (i.e., the majority of the β-oxidation disorders and organic acidemias) will result in the formation of the corresponding acylcarnitines that may be detected in dried blood spots by means of tandem mass spectrometry, has revolutionized the biochemical investigation of MCAD deﬁciency and other conditions characterized by the accumulation of acyl-CoA esters. Indeed, in some countries neonatal screening for these disorders has been introduced using this technology. The case for the introduction of neonatal screening for MCAD deﬁciency seems unassailable; the prevalence approaches that of phenylketonuria, delayed diagnosis leads to signiﬁcant mortality and morbidity, automated and robust analytical procedures for neonatal diagnosis have been developed, and therapy is inexpensive and consists of the avoidance of fasting and provision of a high carbohydrate diet during periods of illness. We have demonstrated in a retrospective study of stored blood spots that tandem mass spectrometry is highly speciﬁc (100%) and sensitive technique for the identiﬁcation of MCAD deﬁciency (Fig. 5 and Table III) (Pourfarzam et al., 2001).

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FIG. 5. Neonatal blood spot octanoylcarnitine concentrations plotted against the octanoylcarnitine:hexanoylcarnitine ratio, showing that the combination of raised values for both is highly speciﬁc for MCAD deﬁciency. ( ) MCAD deﬁcient patients; (䊊) premature neonates; (䊉) normal neonates.

3. Genetics (GenBank M60505) Medium-chain acyl-CoA dehydrogenase deﬁciency is inherited as an autosomal condition. The gene is located on chromosome 1p31, approximately 13 kb in length and consists of 12 exons coding a precursor with a molecular weight of 46.6 kDa. The primary transcript includes a 25 amino acid leader sequence for mitochondrial targeting. Medium-chain acyl-CoA dehydrogenase deﬁciency is notable in that approximately 90% of cases are due to a single point mutation A985G in exon11 resulting in a missense mutation K329E, although a number of other rare disease-causing mutations, deletions, insertions, and splicing mutations have been documented. The A985G mutation is exclusive to Caucasians of north European descent. This amino acid substitution is remote from any catalytically important part of the protein but is thought to compromise tetramer formation. The detailed genetics and population genetics of MCAD deﬁciency have been extensively studied, and the reader is referred to Roe and Ding (2001) and the citations therein.

DEFECTS OF β-OXIDATION

497

TABLE III BIOCHEMICAL, MOLECULAR, AND CLINICAL FEATURES IN SUBJECTS WITH RAISED NEONATAL BLOOD SPOT OCTANOYLCARNITINE LEVELS DETECTED BY TANDEM MASS SPECTROMETRIC ANALYSIS OF 100,600 GUTHRIE BLOOD SPOTS FROM A COHORT OF CHILDREN BORN BETWEEN JANUARY 1, 1991, a AND JULY 20TH 1993, IN THE NORTH OF ENGLAND Pt

C8Cn

C8Cn : C6Cn

985 status

Age at diagnosis

Clinical features

1

1.06

10.3

G/G

1.4 years

Hypoglycemia and death precipitated by gastroenteritis

2

0.87

18.1

G/A

3 years

Hypoglycemic encephalopathy at 3 years

3

1.45

15.8

G/G

3 years

Encephalopathic episodes at 2 and at 3 years

4

1.46

14.1

G/G

7.9 years

Neonatal apnoea due to hypoglycemia

5

1.88

15.9

G/G

7.4 years

Hypoglycemic encephalopathy at 6, 12, and 16 months

6

2.86

12.3

G/G

8.5 years

Drowsy with infections

7

1.24

15.2

G/G

7.5 years

Asymptomatic

8

1.37

9.8

G/G

9 years

Learning difﬁculties, no episodes of encephalopathy

0.68

2.1

A/A

Preterm 27 weeks gestation, asymptomatic

0.53

1.5

A/A

Preterm 29 weeks gestation, asymptomatic

0.51

0.5

A/A

Preterm 29 weeks gestation, tachyarrhythmias, bronchopulmonary dysplasia, died aged six months

0.37

0.8

A/A

Preterm 29 weeks gestation, asymptomatic

0.35

1.1

A/A

Preterm 29 weeks gestation, asymptomatic

0.34

3.4

A/A

Asymptomatic

Cases of MCAD deﬁciency diagnosed before retrospective study

Cases of MCAD deﬁciency diagnosed during retrospective study

Cases with no evidence of MCAD deﬁciency

a

Modiﬁed from Pourfarzam et al. (2001) with permission C8Cn = octanoylcarnitine concentration (mcmol/L); C8Cn:C6Cn = ratio of octanoylcarnitine: hexanoylcarnitine concentrations; 985 status = residue at position 985 of cDNA: A, wild type; G, common mutation.

498

BARTLETT AND POURFARZAM

4. Treatment Until neonatal screening for MCAD is univerally adopted, any neonate or child presenting with impaired conciousness associated with fasting or a vomiting illness should have a blood sample withdrawn for acylcarnitine analysis and glucose measurement, after which therapy with intravenous glucose should commence immediately. Delay in the correction of the hypoglycemia associated with MCAD deﬁciency has led to a number of deaths. Management of the condition consists of the avoidance of fasting and a high carbohydrate diet. 5. Pathogenesis The biochemical aetiology of the condition is now well understood. During periods of remission, with a normal caloric intake, there is little clinical indication that an individual suffers from MCAD deﬁciency. Under these circumstances, the ﬂux through β-oxidation is low, particularly in the liver, and due also in part to overlapping chain-length speciﬁcity of the acyl-CoA dehydrogenases, urinary organic acids are unremarkable. However, under conditions of counterregulation when insulin is low, lipolysis is stimulated with concomitant increased rate of delivery of fatty acids to liver and skeletal muscle. In normal individuals this would result in increased ketogenesis, a lowering of the respiratory quotient, glucose sparing, and on a whole body basis, a switch in fuel selection to fat oxidation with glucose being used selectively by the CNS. In MCAD deﬁciency, and many other inherited disorders of mitochondrial β-oxidation, this metabolic adaption to fasting is compromised. The result is that those tissues that would otherwise select fat as a fuel cannot do so and continue to oxidize carbohydrate. Since glycogen reserves are limited, hypoglycemia ensues. The situation is compounded by the accumulation of toxic intermediates and by the inhibition of gluconeogenesis consequent upon a failure to produce the necessary reducing equivalents through β-oxidation. Numerous fasting studies have demonstrated that the hypoglycemia associated with MCAD deﬁciency is accompanied by an inappropriately blunted ketogenic response and hyperfatty acidaemia. In addition, tissue steatosis is frequently observed. It is presumed that multiple episodes of hypoglycemia in early life cause the neurological and behavioral impairment observed in children who are diagnosed late. Since ketogenesis is also impaired in these children, the energy deﬁcit is compounded as ketone bodies synthesized by the liver are not available as an alternate fuel. Furthermore, if, as now seems probable, glial cells are also capable of ketogenesis, this source of neuronal oxidative substrate will also be attenuated in MCAD-deﬁcient patients. It is also

DEFECTS OF β-OXIDATION

499

possible that the accumulation of medium-chain CoA and their secondary metabolites may have direct neurotoxic effects.

G. SCAD DEFICIENCY (OMIM 201470) Short-chain acyl-CoA dehydrogenase deﬁciency was ﬁrst reported in 2 patients by Amendt et al., since which time a further 14 patients with enzyme-proven deﬁciency have been described (see Corydon et al., 2001 and citations therein). The biochemical hallmark of the disorder has been assumed to be the excretion of ethylmalonic acid, arising from the carboxylation of the accumulating butyryl-CoA, although other pathogenomic metabolites such as butyrylglycine and butyrylcarnitine may also be present. The early cases had extremely variable phenotypes including hypoglycemia, acidosis, hypotonia, developmental delay, progressive skeletal muscular weakness, and microcephaly. However, in the later series of 10 patients reported by Corydon et al. (2001), it appears that hypotonia (8/10) and developmental delay (7/10) are consistent features of the disorder. Diagnosis is complicated by the ﬁnding that the excretion of ethylmalonic acid is extremely variable—indeed, in some of the cases described by Corydon et al. (2001), the levels of ethylmalonate observed would be classed as normal by many laboratories. Furthermore, the excretion of ethylmalonic acid is frequently observed in patients presenting with the signs and symptoms suggestive of metabolic disease but in whom there is no other evidence of SCAD deﬁciency, or in whom other inborn errors are demonstrated. This rather confusing picture is compounded by the mutational analysis, and it now appears that SCAD deﬁciency can present with a wide range of clinical severity with severe mutations at one end of the spectrum and patients homozygous for “common” mutations at the other. Whether or not a particular patient comes to clinical attention probably depends on the coincidence of a number of genetic, metabolic, and environmental factors. Recently, it has been suggested that temperature-sensitive folding mutations may contribute to this group of patients, further complicating investigation (Corydon et al., 1998). Finally, from the perspective of the analysis of body ﬂuids for abnormal metabolites, which is normally the mainstay of biochemical investigation, it is worth pointing out the very close similarity of this disorder to the milder variant of glutaric aciduria type II—“ethylmalonic/adipic aciduria.” 1. Genetics (GenBank AF237952) Small-chain acyl-CoA dehydrogenase deﬁciency is inherited as an autosomal condition. The gene is located on chromosome 12q22-qter,

500

BARTLETT AND POURFARZAM

approximately 13 kb in length and consists of 10 exons coding a precursor of 412 amino acids with a molecular weight of 44.3 kDa. The primary transcript includes a 24 amino acid leader sequence for mitochondrial targeting. There is a high degree of homology with MCAD and other acyl-CoA dehydrogenases. There appear to be a number of disease causing mutations as well as common variations (G625A; C511T). In the largest series published, 10 patients had either one of the rare mutations and/or one of the common mutations in each allele (Corydon et al., 2001). 2. Treatment The majority of SCAD-deﬁcient patients have a progressive neurological presentation characterized by hypotonia, developmental delay, and failure to thrive, although an acute “MCAD-like” presentation has also been documented (Bhala et al., 1995; Corydon et al., 2001). In these circumstances it is difﬁcult to delineate general recommendations for treatment; however, avoidance of fasting and administration of a high carbohydrate diet during intercurrent infections would be logical. Acute episodes are managed by restriction of fat and therapy with glucose and carnitine. Long-term treatment with carnitine may be warranted. 3. Pathogenesis Unlike the disorders of long-chain mitochondrial oxidation, the pathogenesis of SCAD deﬁciency is most probably related to the toxicity of butyric acid rather than to energy deﬁciency. Moreover, butyric acid is an inhibitor of histone deacetylase and a known initiator of apoptosis (Salminen et al., 1998) as well as having direct toxic effects on astrocytes in culture (Bruce et al., 1992).

H. TRIFUNCTIONAL PROTEIN DEFICIENCIES (OMIM 600890) Patients with inherited disorders of the trifunctional protein fall into two groups: those in whom only the long-chain L-3-hydroxyacyl-CoA dehydrogenase activity is absent and those in whom all three constituent activities are attenuated. 1. Clinical Presentation and Diagnosis Isolated deﬁciency of long-chain L-3-hydroxyacyl-CoA dehydrogenase activity is a relatively common inborn error of mitochondrial β-oxidation and many patients have been described (see Roe and Ding, 2001; and citations therein). The disorder is clinically heterogeneous and patients have presented with either predominantly hepatic disease, cardiomyopathy, or

DEFECTS OF β-OXIDATION

501

myopathy. Patients may present in the neonatal period with hypoglycemia and most present within the ﬁrst two years of life (Tyni et al., 1997). However, there appear to be patients with a later presenting adolescent or adult variant of the disorder characterized by higher residual enzyme activity (Miyajima et al., 1997; Ibdah et al., 1998). These patients appear to have a predominantly myopathic presentation. Urinary organic acids are usually abnormal, particularly in the patients with the isolated disorder, and are characterized by the presence of 3-hydroxydicarboxylic acids (C6-C14). Blood acyl carnitines are also abnormal and 3 -hydroxyacylcarnitines are present (C16:0, C18:1, C18:2). Although functional tests with ﬁbroblast preparations are helpful, it is necessary to perform direct enzyme measurements to distinguish the isolated disorder from the rarer combined defect of all three enzyme activities—trifunctional protein deﬁciency. An unusual characteristic of the isolated long-chain 3-hydroxyacyl-CoA dehydrogenase disorder is that obligate heterozygous mothers have an increased risk of HELLP (hemolysis, elevated liver enzymes, and low platelets) or the related condition, fatty liver, as a potentially lethal complication of pregnancy when carrying an affected fetus. This was ﬁrst reported by Wilcken et al. (1993), and has since been conﬁrmed by several groups. In a recently published series of 19 pregnancies in which the child was shown to be affected, 79% of the mothers developed either HELLP or fatty liver of pregnancy (Ibdah et al., 1999). 2. Genetics (GenBank D16480, D16481) Both the α and β subunit genes are located at chromosome 2p23 and are contiguous and coordinately expressed (Aoyama et al., 1997; Orii et al., 1999). Approximately 70% of cases of isolated 3-hydroxyacyl-CoA dehydrogenase deﬁciency are caused by a common mutation of the α subunit (G1528C; Ijlst et al., 1994). By contrast, there are a number of mutations of the gene encoding the β subunit that results in the combined deﬁciency. 3. Treatment As with the other disorders of long-chain fatty acid oxidation, treatment involves a high carbohydrate diet. Supplementation with medium-chain triglycerides is of beneﬁt.

I. SHORT-CHAIN 3-HYDROXYACYL-CoA DEHYDROGENASE DEFICIENCY (SCHAD) (OMIM 201470) Very few cases of this disorder have been described, and accordingly, it is difﬁcult discern common features. Patients have presented as sudden

502

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infant death (Treacy et al., 2000), and at the ages of 3 months with DiGeorge syndrome (Bennett et al., 1996), 13 months with seizures (Bennett et al., 1996) and 16 years with hypoketotic hypoglyacemia, hypertrophic cardiomyopathy, and myoglobinuria (Tein et al., 1991). Biochemical ﬁnding were similarly disparate. However, very recently, a case has been reported of a female infant who presented at four months with hypoglycemia in whom hyperinsulinism was noted. However, there were high blood concentrations of hydroxybutyryl carnitine, and a deﬁciency of SCHAD was demonstrated in cultured ﬁbroblasts (<5% control). This case is the only known instance of hyperinsulinism associated with a β-oxidation defect (Clayton et al., 2001). 1. Genetics (GenBank AF026853) The mode of inheritance of this condition has not been established with certainty, but available evidence suggests an autosomal recessive mode. However, the only case in which a mutation has been demonstrated is that described above asociated with hyperinsulinism. Genomic DNA analysis revealed a homozygous mutation (C773T) resulting in a P258L substitution. Heterozygosity was demonstrated in the parents. The gene is located at chromosome 4q22–26 and comprises at least 8 exons spanning 49 kb. It codes a precursor protein of 314 amino acids, including a 12-mer mitochondrial leader sequence (Vredendaal et al., 1998).

J. GLUTARIC ACIDURIA TYPE II (MULTIPLE DEHYDROGENASE DEFICIENCY) (OMIM 231680) 1. Clinical Presentation (GA II ) Glutaric aciduria type II or multiple acyl-CoA dehydrogenase deﬁciency is a disorder of ETF metabolism in which all ETF-dependent enzymes are affected, including those concerned with fatty acid metabolism. The condition was ﬁrst recognized in 1976 (Przyrembel et al., 1976). Since then at least a further 50 patients have been reported and it has become apparent that there are three clinical phenotypes (Frerman and Goodman, 2001). A neonatal phenotype associated with congenital anomalies usually presents during the ﬁrst two days of life with devastating hypoglycemia, metabolic acidosis, hepatomegaly, and hypotonia. The congenital anomalies include facial dysmorphism and renal cysts. These patients usually do not survive for more than a week. They also have a characteristic odor of “sweaty feet” due to the presence of isovaleric acid arising from deﬁciency of isovaleryl CoA—an ETF-dependent acyl-CoA dehydrogenase. A second phenoype, which also presents in the early neonatal period but without congenital anomalies, has been recognized. Most of these patients also succumb, usually within the ﬁrst

DEFECTS OF β-OXIDATION

503

few months of life. Patients with a milder or intermittent course constitute a third very variable phenotypic group. 2. Diagnosis As with many inborn errors of β-oxidation, the mainstay of diagnosis is the detection of characteristic abnormal urinary organic acids and glycine conjugates. This is usually quite unambiguous, particularly in patients presenting with the severe neonatal form of the disease associated with hypoketotic hypoglycemia. Typically, analysis of urinary organic acids reveals the presence of metabolites characteristic of the absence of the ETF-dependent enzymes, and includes isovaleric, isobutyric, and 2-methylbutyric acids. In addition, the dicarboxyic acids—glutaric (C5), ethylmalonic (branched C5; arising from the carboxylation of butyryl CoA), adipic (C6), suberic (C8), sebacic (C10), and dodecanedioic (C12)—and the glycine conjugates; isovalerylglycine, 2-methylbutyrylglycine and isobutyrylglycine, and the hydroxyacids; 3-hydroxyisovaleric, 2-hydroxyglutaric and 5-hydroxyhexanoic—are observed. However, the relative amounts of these substances is very variable. Blood acylcarnitine analysis by tandem mass spectrometry shows highly elevated saturated acylcarnitines with carbon chain-length of C4 to C18 in the severe form and moderate elevation in the milder cases. The milder, later presenting cases can be more problematic. In order to distinguish the two causes of GA II, i.e., ETF or ETF:QO deﬁciency, it is necessary to carry out more detailed biochemical studies on cultured ﬁbroblasts or tissues. 3. Genetics (GenBank S69232) All three phenotypes of GA II can arise from deﬁciency of either ETF or ETF:QO, although the severe neonatal form associated with renal cystic dysplasia is usually due to ETF:QO deﬁciency. Thus mutations of the genes coding for ETFα, ETFβ , or ETF-QO have been identiﬁed; all are inherited as autosomal recessive traits. These are summarized in Table IV. 4. Pathogenesis Since GA II results in attenuated activity of all ETF-dependent enzymes, it is probable that both energy deﬁciency and the accumulation of toxic intermediates play a role in pathogenesis. In common with the isolated longchain disorders, there is a failure of ketogenesis, and therefore the selection of alternative fuels in the fasted and stressed states cannot occur. The secondary effects of the disorder such as hyperammonemia and hypoglycemia are probably due to the failure of acetyl-CoA production, possibly in combination with competitive inhibition by short-chain acyl-CoA esters, with resultant failure of ureagenesis (lack of activation of carbamyl phosphate synthase) and hypoglycemia (lack of activation of pyruvate carboxylase).

TABLE IV MUTATIONS OF THE ETF AND ETFDH GENES GIVING RISE TO GLUTARIC ACIDURIA TYPE II Gene/exon

Mutation (gDNA)

Mutation (cDNA)

Polymorphism

Protein

Reference

ETFA/6

470T > G

V157G

(Indo et al., 1991)

ETFA/9

797C > T

T266M

(Freneaux et al., 1992)

ETFA/4

346G > A

G116R

(Freneaux et al., 1992)

ETFA/6

453 470del

N152 V157del

(Freneaux et al., 1992)

ETFA/10

817 882del

E273 K294del

(Freneaux et al., 1992)

ETFA/9

808 810del

V270del

(Freneaux et al., 1992)

ETFA/6

512C/T

ETFB/5 ETFB/5

IVS5 + 1G > C

T/I-171

(Freneaux et al., 1992)

491G > A

R164Q

(Colombo et al., 1994)

439 597del

G147 M199del

(Colombo et al., 1994)

T/M-154

(Colombo et al., 1994)

D128N

(Olsen et al., 2001)

461C/T

ETFB/5 ETFB/4

382G > A

382G > A

ETFDH/4

427 433del

427 433del

T143fsX148

(Beard et al., 1995)

ETFDH/12

IVS12 + 1G > T

1469 1690del

G490 A563del

(Beard et al., 1995)

ETFDH/1

2T > C

M1T

(Frerman and Goodman, 2001)

ETFDH/2

36delA

A12fsX19

(Frerman and Goodman, 2001)

ETFDH/2

47T > G

F16C

(Frerman and Goodman, 2001)

ETFDH/9

1001T > C

L334P

(Frerman and Goodman, 2001)

ETFDH/11

1392 1393delAT

V464fsX488

(Frerman and Goodman, 2001)

ETFDH/12

1623delT

D541fsX546

(Frerman and Goodman, 2001)

ETFDH/2

51 52insT

18AfsX22

(Frerman and Goodman, 2001) (Olsen et al., 2001)

ETFDH/2

121C > T

R41X

(Frerman and Goodman, 2001)

ETFDH/12

1685C > T

P562L

(Frerman and Goodman, 2001)

ETFDH/7

806A > T

Q269L

(Olsen et al., 2001)

806A > T

DEFECTS OF β-OXIDATION

505

IV. Conclusions

The inherited disorders of mitochondrial β-oxidation are a complex group of disorders. Those associated with hypoglycemia can be distinguished from other causes of hypoglycemia by simple tests such as the measurement of the major metabolic fuels. However, the availability of sophisticated analytical methods such as tandem mass spectrometry and gas chromatography– mass spectrometry has allowed the detection of disease-speciﬁc metabolites and patterns of metabolites in body ﬂuids and tissue preparations. Only when such analyses of body ﬂuids for abnormal metabolites has proven uninformative, is it necessary to proceed to more invasive tests, such as fasting provocation, to distinguish other causes of hypoglycemia. Furthermore, tandem mass spectrometry in particular, has led to the development of procedures for neonatal screening by means of which most of the disorders described above can be detected early in life. For some of the disorders direct enzyme measurement is necessary for precise diagnosis, supplemented by investigations at the gene level. In some instances, for example most cases of medium-chain acyl-CoA dehydrogenase deﬁciency, the identiﬁcation of the pathognomic metabolites (octanoyl-carnitine in blood, suberylglycine in urine) combined with the identiﬁcation of the common mutation, is sufﬁcient for unambiguous diagnosis and direct enzyme measurement is generally not necessary. Treatment may be simple, such as avoidance of fasting and the administration of slow-release carbohydrate in the case of MCAD deﬁciency, or carnitine supplementation in primary carnitine deﬁciency. Some of the mitochondrial β-oxidation disorders, however, are intractable to treatment such as the severe neonatal forms of glutaric aciduria type II.

Acknowledgments

We thank Dr. Rikke Olson for the provision of the data contained in Table IV, and Professor Niels Gregersen for his advice and discussion.

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SECTION VIII MITOCHONDRIAL INVOLVEMENT IN AGING

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THE MITOCHONDRIAL THEORY OF AGING: INVOLVEMENT OF MITOCHONDRIAL DNA DAMAGE AND REPAIR

Nadja C. de Souza-Pinto and Vilhelm A. Bohr1 Laboratory of Molecular Gerontology National Institute on Aging NIH Baltimore, Maryland

I. II. III. IV. V. VI.

Mitochondria: The Biological Clock? Oxidative Damage to Mitochondria Accumulation of Oxidative Damage to mtDNA DNA Repair in Mammalian Mitochondria Changes in mtDNA Repair with Age Conclusions References

I. Mitochondria: The Biological Clock?

Although the concept of aging is natural to all of us, the task of deﬁning it is a tricky one. Its appropriate deﬁnition depends on the species and on one’s perspective. From the evolutionary point of view one could argue that aging is the process that leads to loss of reproduction capability, since in that stage organisms are no longer subject to natural selection. However, in general terms aging could be simply deﬁned as a time-dependent degenerative process that ultimately leads to death. In multicellular organisms, it is clear that aging involves a series of complex alterations in the organism’s physiology. Thus it is reasonable to assume that more than one target play important roles in the sequence of events that lead to the aging phenotype. There are two major lines of thought to explain aging in higher organism. The ﬁrst one suggests that aging is a genetically programmed process that, in a similar fashion to development, occurs via differential gene expression. The recent discoveries of “aging genes” in the round worm Caenorhabditis elegans and in mice (de Haan et al., 1998; Guarante and 1 To whom correspondence should be addressed at 5600 Nathan Shock Drive, Baltimore, Maryland 21224.

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Kenyon, 2000) provide some experimental support for this theory. The second one proposes that aging is simply the result of random accumulation of cellular damage, or the error-catastrophe theory (Terman, 2001). In 1956, Denham Harman ﬁrst proposed the free radical theory of aging (Harman, 1956), which implied that oxidative damage to cellular components produced by free radicals, formed as by-products of the utilization of molecular oxygen as an electron acceptor, was the underlying cause of aging. This theory was later modiﬁed to propose that oxidative damage to mitochondria was the determining factor of life span, and therefore set mitochondria as the “biological clock” that governs longevity (Harman, 1972). In this chapter we explore the experimental evidences that support a pivotal role for mitochondrial dysfunction in aging. It is still unclear, however, in what manner damage to mitochondria leads to the aging phenotype.

II. Oxidative Damage to Mitochondria

In most cell types mitochondria are the main site for the production of reactive oxygen species (ROS). Although a variety of enzymes in the cytosol, such as oxygenases and oxidases, generate small amounts of ROS, it is estimated that more than 95% of superoxide anions produced during normal metabolism is generated at the electron transport chain in the inner mitochondrial membrane (Sohal and Brunk, 1995). Initial observations by Boveris and Chance suggested that up to 5% of the total molecular oxygen utilized by mammalian mitochondria was converted into reactive species (Boveris and Chance, 1977), but more recent analysis estimates about 1–2% to be converted into superoxide anion (O2·− ) (Cadenas and Davis, 2000). Reactive oxygen species are very reactive toward biomolecules. The hydroxyl radical (OH· ) will react with any molecule within 15 A˚ of its site of generation with a rate constant that is limited only by diffusion (Ward, 1985). Upon oxidative attack, a variety of different damages can be generated in biomolecules, such as (1) lipid peroxidation that leads to the production of highly reactive aldehydes such as malondialdehyde and 4-hydroxynonenal; (2) oxidation of SH groups, leading to the formation of protein aggregates; (3) oxidation of amino acids side chains forming carbonyl groups, which will target the protein to degradation; (4) oxidation of the deoxyribose in DNA, resulting in strand breaks; and (5) oxidation of the bases in nucleic acids, resulting in oxidized adducts or abasic sites (reviewed in Cadenas, 1989; Breen and Murphy, 1995).

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Membranes are particularly susceptible to oxidative damage because of the presence of carbon–carbon double bonds in the phospholipids. Oxidative damage to lipids can occur via the direct attack of ROS to unsaturated lipid side chains, or indirectly through the production of aldehydes. Those aldehydes can react, in turn, with other biomolecules. In fact, 4-hydroxynonenal (4HNE) has been shown to react with phospholipids and proteins in the mitochondrial membrane, causing a decrease in membrane ﬂuidity (Spitz et al., 1990). Another potential lipid target for oxidative damage in the mitochondrial membranes is cardiolipin because of its high degree of insaturation. This molecule is a major component of the inner membrane, and it plays a critical role in the function of proteins such as cytochrome oxidase and the adenine nucleotide translocator. The levels of cardiolipin have been shown to decrease with age in heart, liver, and brain mitochondria (Paradis et al., 1997). It is unclear, however, whether this is associated with oxidation. An increase in protein oxidation levels with age has been demonstrated in a variety of models such as human brain, many rodent tissues, cultured human cells, insects, and primates (Berlett and Stadman, 1997), and in some species there is a logarithmic increase in protein carbonyl contents with increasing age (Stadman, 1992). Once oxidized, proteins are recognized by a series of different proteases and completely degraded; entirely new proteins are then synthesized de novo. However, some experimental evidence suggests that the rate of degradation of oxidized protein declines with age (Agarwal and Sohal, 1994), which, in association with an increased rate of ROS production, could lead to loss of cellular function. Another mechanism by which ROS can inactivate proteins is by the direct oxidation of co-factors. Many of the proteins involved in oxidative metabolism in mitochondria have iron–sulfur clusters essential for enzymatic activity. Aconitase and succinate dehydrogenase are examples, and in fact recent results show that aconitase is a primary target by oxidative damage during aging (Yan et al., 1997). While most of the studies described so far have been associative, the actual functional relevance of oxidative damage at the mitochondrial level was evaluated in a recent study. It was found that oxidative damage accumulates in the mitochondrial DNA (mtDNA) (and lipids and proteins) of mice heterozygous for the mitochondrial form of superoxide dismutase (MnSOD−/+), and that mitochondrial function was decreased (Williams et al., 1998). In addition, there was an age-related decline in mitochondrial function culminating with elevated apoptosis (Kokoszka et al., 2001). Taken together, these results suggest that oxidative damage to mitochondria is indeed associated with dysfunction and aging, and that mitochondrially induced apoptosis may be involved in the aging process.

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III. Accumulation of Oxidative Damage to mtDNA

While any oxidative damage to biomolecules is likely to have harmful effects, modiﬁcations to DNA are particularly harmful to the organism since they may lead to permanent and heritable changes via the formation of mutations and other types of genomic instability. Reaction of DNA with ROS causes many potentially mutagenic or lethal lesions including strand breaks, abasic sites, and oxidized bases. At least two of the most common DNA lesions, 8-oxo-7-hydro-2 -deoxyguanosine (8-oxo-dG) and 5 -hydroxy2 -deoxycytidine (5-OH-dC), are mutagenic (Grollman and Moryia, 1993; Purmal et al., 1994). Based on discoveries showing that mtDNA is associated with the mitochondrial membrane (Shearman and Kalf, 1977), in close proximity to the electron transport chain, it was concluded that this DNA was likely to be the most critical target of oxidative attack. Therefore, the resulting loss of mitochondrial function would be an early event in a cascade that would lead, ultimately, to aging. Mitochondrial DNA has several unique properties that make this hypothesis attractive (Singh et al., 1992). First, it is contained in organelles that tend to sequester any positively charged species because of the charge gradient generated by the electron transport chain. Thus, any DNA-damaging agent with those characteristics would tend to accumulate in the mitochondria, preferentially attacking the mtDNA. Second, as noted above, mtDNA is closely associated with the mitochondrial electron transport chain, which makes it extremely susceptible to oxidation. Third, unlike nuclear DNA, which is packed into nucleosomes arranged into chromatin, mtDNA is not associated with structural proteins, making the DNA strands more accessible to damaging agents. Fourth, during replication, which occurs at the inner membrane (Shearman and Kalf, 1977), approximately one half of the H-strand is completed prior to initiation of L-strand replication. As a result, mtDNA exists partly in a single-stranded conformation, suggesting that it might be more vulnerable than nuclear DNA to attack. Finally, the presence of damage or mutations in mtDNA could lead to an altered function in the electron transport chain. In a vicious circle, decreased function in the electron transport may lead to an increase in oxygen radical production (Miquel et al., 1980; Bandy and Davison, 1990). One of the most well-studied lesions resulting from interactions between DNA and reactive oxygen species is 8-oxo-dG. The addition of an OH group at the eighth position in guanine by reducing agents in the presence of oxygen was ﬁrst described by Kasai and Nishimura (1983, 1984). The use of electrochemical detection (ECD) in conjunction with high performance liquid chromatography (HPLC) to detect 8-oxo-dG was pioneered by Floyd

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and co-workers (1986). ECD of 8-oxo-dG is approximately 1000-fold more sensitive than optical methods of detection, and it was the ﬁrst technique to allow detection and quantiﬁcation of an oxidatively modiﬁed DNA base at physiologically relevant levels. Since that time, hundreds of studies have been dedicated to this lesion, and more has been learned about it than about any of the other oxidative DNA lesions. 8-oxo-dG is a mutagenic lesion because it will often adopt the syn conformation (Kouchakdjian et al., 1991; McAuley-Hecht et al., 1994), and mispair with adenine during DNA replication and transcription. Most DNA polymerases fail to recognize this mismatch, and so 8-oxo-dG in the template strand results in a G:A mismatch and eventually, if the mismatch is not correctly repaired, in G → T transversions (Shibutani et al., 1991). The mitochondrial polymerase γ is no exception (Pinz et al., 1995). 8-oxo-dG is not the most mutagenic lesion, based on the mutation spectrum resulting from oxidative stress, which favors GC → AT transitions (Moraes et al., 1990; Tkeshelashvili et al., 1992). However, its biological relevance is demonstrated by the fact that enzymes for its repair and to prevent its incorporation into DNA are found in organisms ranging from Escherichia coli to humans. The measurement of damage in mtDNA faces many challenges. In most methods, the mitochondria must ﬁrst be isolated from the cells, and DNA is then isolated from this puriﬁed and concentrated suspension. Clearly, this procedure itself has the potential to induce oxidative damage. In 1988, HPLC/ECD was applied for the ﬁrst time to mtDNA. Using mitochondria isolated from rat liver, it was found that the DNA contained 117 8-oxo-dG per 106 dN, in comparison to only 7.2 8-oxo-dG per 106 dN in nuclear DNA from the same animals (Richter et al., 1988). This ﬁnding added a tremendous amount of support to the mitochondrial theory of aging. However, there is great variability in the values reported for 8-oxo-dG in mtDNA, as might be expected given the difﬁculties in measuring oxidative DNA. The reported values range from a low of 0.08 8-oxo-dG per 106 dN in HeLa cells (Higuchi and Linn, 1995) to an astonishing high of 4840 per 106 dN in heart tissue taken from a 100-week-old rat (Takasawa et al., 1993). It is noteworthy that the largest change with age is found within a single method, and indeed, by a single laboratory: using HPLC/MS(mass spectroscopy), values for mtDNA are reported to increase 250-fold with age, from 5.7 8-oxo-dG per 106 dN for human cardiac tissue from 30 year olds to 1430 8-oxo-dG per 106 dN for cardiac tissue from 90 year olds (Hayakawa et al., 1993). One problem in interpreting such results is that very few tissues have been examined by more than one laboratory. Thus, it is difﬁcult to say with certainty whether the observed 250-fold increase seen with age in human heart is due to the fact that it is an unusual tissue, or to technical problems with the HPLC/MS methodology. Although the former possibility is more

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likely in light of a report from the same laboratory showing that age-related increases are not seen in rat liver (Takasawa et al., 1993), a 250-fold increase is truly remarkable. As already noted, discrepancies due to methodology are of major concern in the measurement of 8-oxo-dG. For those tissues that have been examined by more than one laboratory, there is disagreement over both absolute levels and changes with age. Mitochondrial DNA from porcine liver has been reported to contain 2 8-oxo-dG per 106 dN (Hegler et al., 1993) or 682 8-oxo-dG per 106 dN (Zastawny et al., 1998). In our own laboratory, using mtDNA from rat liver mitochondria, values from young rats were found to be as low as 5 8-oxo-dG per 106 dN using HPLC/ECD, or as high as 135 8-oxo-dG per 106 dN using gas chromatography/MS (GC/MS) (Hudson et al., 1998; Anson et al., 1999, 2000). In order to improve the detection and minimize some of the difﬁculties in measuring 8-oxo-dG, our laboratory has been using the HPLC–Coularray system, which allows identiﬁcation of the measured species not only by retention time but also by ionization potential (Hudson et al., 1998). We also created standards by damaging DNA with photoactivated methylene blue to ascertain the sensitivity of the system. The levels of damage detected in the standards were linear over a wide range, including the lower levels, which were comparable to those observed in liver DNA. There was no change in the levels of 8-oxo-dG in the nuclear DNA from 6-month-old (young) and 23-month-old (senescent) rat liver DNA. However, at 6 months, the level of 8-oxo-dG in mtDNA was 5-fold higher than nuclear and increased to approximately 12-fold higher by 23 months of age. This is in agreement with other studies. In almost every case in which both nuclear and mitochondrial values were presented, mtDNA was more damaged than nuclear DNA and 8-oxodG levels increased with age. Further support for the age-associated increase in oxidative damage was provided by two studies, which also found that age-related increases in the level of mitochondrial 8-oxo-dG were lower in caloric restricted animals than in those fed ad libitum (Chung et al., 1992; Sohal et al., 1994). Other markers of oxidative damage were also reduced. The effect of caloric restriction is important since this is the only intervention proven to directly alter the rate of aging an extend life span in mammals (Masoro, 1995). The proper repair of 8-oxodG is fundamental for the maintenance of genome integrity. In bacteria, the loss of the mutM gene, which encode the Fapy-glycosylase (FPG) leads to a strong mutator phenotype. Additional losses of mutY and mutT cause even higher spontaneous mutation rates (Tajiri et al., 1995). Similar observations have been made recently in mammals; the development of a mouse with a null mutation at the ogg1 gene, which encodes for the main oxoguanine DNA glycosylase in the nucleus, permitted the demonstration that unrepaired 8-oxodG increases spontaneous

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mutation rates in mammalian cells as well (Klungland et al., 1999). The biological relevance of 8-oxodG can be demonstrated by two recent ﬁndings in cells from cancer patients. Polyak et al. (1998) found that mtDNA from colon cancer cells contained several homoplasmic mutations. The pattern of those mutations, mainly G → T substitutions, suggested that they might arise from unrepaired oxidative base damages. Analysis of mtDNA from head and neck, bladder, and lung cancer patients also suggested that there were deﬁciencies in oxidative damage repair of the mitochondrial DNA (Fliss et al., 2000). A higher number of mutations in mtDNA obtained from older subjects also have been detected, providing further support to the hypothesis that accumulation of oxidative damage in the mtDNA leads to mutations and abnormal gene expression. In a recent study, Michikawa et al. (1999) demonstrated that high copy number of point mutations exist in the control region for replication of human ﬁbroblast mtDNA from normal old subjects. In the same study, longitudinal analysis showed that most of those mutations appeared in a given individual only at an advanced age. Despite the experimental evidence suggesting a correlation between an increase in oxidative damage and the aging process, the biological consequences of such damage are still only partially understood. Does such damage actually cause aging? Evidence that mitochondrial ROS formation may be involved in aging comes from comparative studies between species of different maximum life span. For example, a pigeon lives approximately ten times as long as a rat, a hummingbird at least three times as long as a mouse. Recently, two groups have shown that mitochondrial ROS production is lower in the longer lived avian species (Ku and Sohal, 1993; Barja et al., 1994), and in addition, avian renal epithelial cells are extremely resistant to growth under 95% oxygen, and to treatment with hydrogen peroxide, paraquat, or γ irradiation. Herrero and Barja (1999), Barja and Herrero (2000) reported recently that mtDNA oxidative damage levels are lower in several long-lived species than in short lived species. Thus, it is possible that the rate of oxidative damage formation in the mitochondrial DNA functions as the “biological clock” in determining life span.

IV. DNA Repair in Mammalian Mitochondria

The initial observation that mitochondria were unable to remove UVinduced damage from their genomes (Clayton et al., 1974) led to the notion that mitochondria lack DNA repair capacity. Although it is true that mitochondria do not remove UV-induced DNA damage, subsequent studies

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clearly showed that mitochondria repair some types of damage from their DNA (Satoh et al., 1988; LeDoux et al., 1992; Driggers et al., 1993). One of the earliest reports on mitochondrial DNA repair showed that alkylation damage was removed from rat liver mtDNA, and that the kinetics was similar to that of a nuclear sequence (Satoh et al., 1988). Our knowledge about the DNA repair mechanisms in mitochondria is based on the identiﬁcation of speciﬁc enzymes and on studies of the repair of various lesions. Different DNA lesions are repaired via different repair pathways, and these have been identiﬁed in studies on nuclear or total DNA. The study of mitochondrial DNA repair was initially limited by difﬁcult in isolating enough mtDNA for repair measurements. This problem was circumvented by the use of the gene-speciﬁc repair assay, which utilizes speciﬁc repair enzymes to create strand breaks at the sites of damage and southern blotting to measure repair in deﬁned regions of the genome. Using this technique and a variety of damaging agents, efﬁcient repair of strand breaks and alkali-sensitive sites has been demonstrated in rodent and human mitochondrial DNA. Repair of fapy-glycosylase (FPG)-sensitive sites in mtDNA has been reported for rat (Driggers et al., 1993), CHO (LeDoux et al., 1992) and human cells (Anson et al., 1998). In the latter study, human cultured ﬁbroblasts were exposed to the photo-activated dye methylene blue, which generates mainly 8-oxodG. The removal of 8-oxodG from the mitochondrial genome was very efﬁcient: after 9 h, 47% of the initial lesion had been repaired. In addition, analysis of repair on both strands of the highly transcribed ribosomal region and nonribosomal regions showed that repair of 8-oxodG in mitochondria is without bias for the transcribed strand, as seen in nuclear DNA repair, and thus is not coupled to transcription. Further, the isolation of enzymes that can carry out base excision repair (BER) suggested that this repair pathway exists in mitochondria. In this regard, apurinic/apyrimidinic (AP) endonucleases class I and II, glycosylases, DNA ligase, and DNA polymerase have been identiﬁed in mammalian mitochondria (Croteau et al., 1999, and references therein). Base excision repair is one of the main pathways for the removal of small base modiﬁcation from DNA. Base excision repair is initiated by a glycosylase, which will speciﬁcally recognize a damaged base and cleave the N-glycosyl bond between the base and the sugar, generating an abasic (AP) site. The AP site is further processed by an AP endonuclease, which introduces a strand break 5 to the baseless sugar and generates a 5 -deoxyribose phosphate (dRP) terminus. This intermediate is a blocking end for polymerization, and removal of the dRP moiety is often the rate-limiting step during BER. A DNA polymerase, pol β in nuclei and pol γ in mitochondria, removes the dRP motif and introduces a new nucleotide. A DNA ligase then seals the gap (Demple and Harrison, 1994, and references therein).

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Whereas in vitro DNA repair assays have proven very useful in the investigation of the nucleotide excision repair (NER) mechanisms (Wood, 1997), no such assays had been adapted to study mitochondrial DNA repair. Using protein extracts obtained from Xenopus mitochondria, Ryoji et al. (1996) used a repair incorporation assay to demonstrate that plasmid DNA treated with H2O2 was repaired. In another approach, Pinz and Bogenhagen (1998) reconstituted in vitro the repair of abasic sites in an oligomer substrate using solely proteins isolated from Xenopus mitochondria, an AP endonuclease, DNA pol γ , and a mtDNA ligase, which was suspected to be related to the nuclear DNA ligase III. In fact, recently it was conﬁrmed that the human DNA ligase III gene encodes both nuclear and mitochondrial isoforms (Lakshmipathy and Campbell, 1999). In mammalian systems, we recently demonstrated that protein extracts from rat liver mitochondria support the repair of uracil containing substrates (Stierum et al., 1999). Using a repair incorporation assay and single lesion DNA constructs, we detected incorporation of radioactive dCTP in a double-strand oligonucleotide containing uracil opposite deoxyguanine and also in plasmid DNA. In the nucleus of mammalian cells there are two pathways for BER, the short patch or one nucleotide replacement, and the long patch, which results in the incorporation of 2–6 nucleotides. These two pathways involve different subsets of proteins and operate independently (Dianov and Lindahl, 1994; Frosina et al., 1996). The utilization of a single lesion construct in the repair incorporation assay allowed us to study the patch size for the repair of uracil by mitochondrial extracts. We found this repair event proceeds solely via the short patch, or single nucleotide replacement pathway (Stierum et al., 1999). The DNA glycosylases are a class of enzymes that recognize speciﬁc base modiﬁcations in DNA and attack the N-glycosyl bond, releasing the free damaged base. There are two classes of glycosylases: (a) monofunctional glycosylases, which only release the modiﬁed base leaving an abasic site as product, and (b) bifunctional glycosylases, or glycosylases/AP lyases. The latter release the damage base and attack the phosphodiester bond generating a single-strand break. The lyase activity usually proceeds through a β, δ elimination reaction (Krokan et al., 1997). Uracil DNA glycosylase (UDG) was one of the ﬁrst repair activities detected in mitochondrial extracts (Anderson and Friedberg, 1980). The enzyme, a 30 kD protein, was puriﬁed from human cells by afﬁnity chromatography (Domena and Mosbaugh, 1985) and was later named UDG1 to differentiate it from the nuclear uracil DNA glycosylase, UDG2. The same gene, ung, encodes both enzymes. The two different isoforms are generated via two different promoters and alternative splicing (Nilsen et al., 1997). Although both isoforms show a peak in expression during the S phase of the cell cycle, the two enzymes are expressed in a

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differential manner, and show different tissue abundance in humans (Haug et al., 1998). The enzyme UDG is a monofunctional glycosylase—in other words, it has no associated AP lyase activity. This enzyme recognizes the U:G mismatch and releases the uracil residue generating an abasic site. For the completion of the repair process, however, it requires the coupled activity of an AP endonuclease (Demple and Harrison, 1994). The observation that mammalian mitochondria efﬁciently removed FPGsensitive sites from their genomes suggested that these organelles possessed a glycosylase that speciﬁcally recognizes oxidative modiﬁed purines. Using rat liver mitochodria, an activity that recognizes and incises at 8-oxodG and abasic sites in duplex DNA was identiﬁed. This activity was puriﬁed and characterized, and called mitochondrial oxidative damage endonuclease (mtODE) (Croteau et al., 1997). It was shown to be speciﬁc for 8-oxoG, with preference for 8-oxoG:C base pairs. We demonstrated that this activity was an 8-oxodG glycosylase/lyase enzyme, because it was covalently linked to an 8-oxoG-containig oligonucleotide by sodium borohydride reduction. In addition, we conﬁrmed its glycosylase activity by measuring the release of 8-oxdG from oxidized DNA using HPLC-EC (Hudson et al., unpublished results). In the nuclear DNA, the oxoguanine DNA glycosylase 1 (OGG1) is the main enzyme for the removal of 8-oxodG lesions. Analysis of the ogg1 gene showed a mitochondrial localization signal upstream from the coding sequence, suggesting that this enzyme could be transported into the mitochondria. Further experiments using CHO cells transfected with an ogg1containing construct identiﬁed ﬁve different isoforms, from which three were localized to the mitochondria (Takao et al., 1998). We recently demonstrated that the major 8-oxodG glycosylase/AP lyase activity in mouse liver mitochondria is encoded, in fact, by the ogg1 gene, since liver mitochondria from ogg1−/− animals showed no detectable incision activity with a construct containing a single 8-oxodG (Souza-Pinto et al., 2001a).

V. Changes in mtDNA Repair with Age

A large body of experimental evidence supports the existence of a relationship between genomic instability, DNA damage, and aging. Over the past years, various attempts have been made to measure DNA repair capacity changes with age. Despite initial results demonstrating a linear correlation between the logarithm of life span and the DNA repair capacity in cells from different mammalian species (Hart and Setlow, 1974), the results from further studies have varied and there is no consensus on this matter

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TABLE I CHANGES IN OGG1 ACTIVITY WITH AGEa Increase in incision with age

Mouse liver mitochondria

Mouse liver nucleus

Rat liver mitochondria

Rat heart mitochondria

6 to 12–14 months

42%

−18%

101%

120%

6 to 23 months

77%

nd

63%

57%

a The OGG1 activity in liver and heart mitochondria was quantiﬁed as described in SouzaPinto et al. (1999), using the an 8-oxodG containing oligonucleotide and 100 μg of mitochondrial extracts. Results are presented as percentage of OGG1 activity at 6 months for each organ and species; n = 6 for each group.

(Bohr and Anson, 1995). Moreover, this subject was further complicated by the heterogeneity in repair between different regions of the genome. Just recently, Guo et al. (1998) reported that the removal of UV-induced damage in actively transcribed regions is lower in hepatocytes isolated from old than from young rats, suggesting a decrease in transcription coupled repair with age. Studies on DNA repair changes with aging have, so far, only been done in the nuclear DNA or in total cellular DNA. There has been no such analysis of DNA repair changes in the mitochondrial DNA with age, despite the observations that oxidative damage accumulation with age is remarkably higher in the mitochondrial than in nuclear DNA (see Section IV). Therefore, it is relevant to ask whether mitochondrial DNA repair efﬁciency changes with age. To answer that question, we compared mtOGG1 activity in liver and heart mitochondria from 6-, 12–14-, and 23-month-old animals. These three age groups represent young adult, middle age, and old, respectively, and cover the adult life span of rats and mice. The results from these studies are presented in Table I (Souza-Pinto et al., 1999, 2001b). Interestingly, we observed that mtOGG1 activity increases with age in both liver and heart. Similar results were obtained in both mouse and rat, suggesting that this is a common response associated with age in mammals. On the other hand, mtUDG activity did not change with age in any of the different organs or species examined (not shown). This speciﬁc increase in the 8-oxoG-incision activity suggests that the repair of oxidative DNA damage could be induced in mitochondria. In contrast, we found that the nuclear OGG1 activity decreased slightly with age in mouse liver. This differential change with age in the mitochondrial and nuclear 8-oxoG glycosylase activities suggest that the expression of these two isoforms may be differentially regulated. These results are consistent with the observations that the mtDNA is more prone to oxidative damage than nuclear DNA, and that BER initiated by OGG1 is the most important line of defense against 8-oxoG in mitochondria.

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Contrary to the common notion that DNA repair declines with age, our results show that mtDNA repair of 8-oxoG does not decline, but rather increases with age. This observation does not contradict that accumulation of 8-oxoG in the mtDNA increases with age. A steady-state accumulation of damage can be caused by an increased rate of formation or by a decrease in the rate of removal. It has been suggested that the rate of free radical production is the determinant factor in the aging process (Perez-Campo et al., 1998). Thus, it is possible that the rate of damage formation exceeds the mitochondrial DNA repair capacity leading to damage accumulation. In that case, the induction of the glycosylase activity may represent a cellular response in an attempt to counteract increased damage formation. VI. Conclusions

It is clear that damage to mitochondria play a critical role in the sequence of events that cause the aging phenotype. However, our comprehension of the molecular mechanisms involved in this process is still very fragmented, and most of the relationships found are solely based in correlation between events. It is our mission for the future to establish causal associations between the different kinds of damage to mitochondrial structures and functional degeneration, as well as the sequence of events that lead to cellular dysfunction. The greater understanding of these processes could, then, provide grounds for the development of interventions that could minimize or retard the progression of the aging phenotype.

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Souza-Pinto, N. C., Hogue, B. A., Eide, L., Stevnsner, T., Seeberg, E., Klungland, A., and Bohr, V. A. (2001a). Repair of 8-oxodG in mitochondrial DNA in mice depends on the OGG1 gene. Cancer Res. 61, 5378–5381. Souza-Pinto, N. C., Hogue, B. A., and Bohr, V. A. (2001b). DNA repair and aging in mouse liver: 8-oxodG glycosylase activity increase in mitochondrial but not in nuclear extracts. Free Rad. Biol. Med., 30, 916–923. Spitz, D. R., Malcolm, R. R., and Roberts, R. J. (1990). Cytotoxicity and metabolism of 4hydroxy-2-nonenal and 2-nonenal in H2O2–resistant cell lines. Do aldehydic by-products of lipid peroxidation contribute to oxidative stress? Biochem J. 267, 453–459. Stadman, E. R. (1992). Protein oxidation and aging. Science 257, 1220–1224. Stierum, R. H., Dianov, G. L., and Bohr, V. A. (1999). Single-nucleotide patch base excision repair of uracil in DNA by mitochondrial protein extracts. Nucleic Acids Res. 27, 3712–3719. Tajiri, T., Maki, H., and Sekiguchi, M. (1995). Functional cooperation of MutT, MutM and MutY proteins in preventing mutations caused by spontaneous oxidation of guanine nucleotide in Escherichia coli. Mutat. Res. 336, 257–267. Takao, M., Aburatani, H., Kobayashi, K., and Yasui, A. (1998). Mitochondrial targeting of human DNA glycosylases for repair of oxidative DNA damage. Nucleic Acids Res. 26, 2917–2922. Takasawa, M., Hayakawa, M., Sugiyama, S., Hattori, K., Ito, T., et al. (1993). Age-associated damage in mitochondrial function in rat hearts. Exp. Gerontol. 28, 269–280. Terman, A. (2001). Garbage catastrophe theory of aging: imperfect removal of oxidative damage? Redox Rep. 6, 15–26. Tkeshelashvili, L. K., McBride, T., Spence, K., and Loeb, L. A. (1992). Mutation spectrum of copper-induced DNA damage. J. Biol. Chem. 266, 6401–6.l. Ward, J. F. (1985). Biochemistry of DNA lesions. Radiat. Res. 104, S103–S111. Williams, M. D., Van Remmen, H., Conrad, C. C., Huang, T. T., Epstein, C. J., and Richardson, A. (1998). Increased oxidative damage is correlated to altered mitochondrial function in heterozygous manganese superoxide dismutase knockout mice. J. Biol. Chem. 273, 28510– 8515. Wood, R. D. (1997). Nucleotide excision repair in mammalian cells. J. Biol. Chem. 272, 23465– 23468. Yan, J.-L., Levine, R. L., and Sohal, R. S. (1997). Oxidative damage during aging targets mitochondrial aconitase. Proc. Natl. Acad. Sci. USA 94, 11168–11172. Zastawny, T. H., Dabrowska, M., Jaskolski, T., Klimarczyk, M., Kulinski, L., Koszela, A., Szczesniewicz, M., Seiwinska, M., Witkowski, P., and Olinski, R. (1998). Comparison of oxidative base damage in mitochondrial and nuclear DNA. Free Rad. Biol. Med. 24, 722–725.

INDEX

A AAA-proteases, HSP, 81–82 AAC, see ATP/ADP carrier ACD, see Acyl-CoA dehydrogenase Aceruloplasminemia, 177 N-Acetyl aspartate, Wilson’s disease, 184 3-Acetylpyridine, mitochondrial dysfunction, 262–264 Acylcarnitine–carnitine translocase, 473– 476 Acyl-CoA dehydrogenase, 476– 477 AD, see Alzheimer’s disease ADP, mitochondrial ETC, 27–28 ADP–ATP translocator, 44 – 45 adPEO, see Autosomal-dominant progressive external ophthalmoplegia Adult muscular carnitine palmitoyltransferase II deﬁciency, 489– 490 Afg3p, 194 –195 Age, mtDNA repair changes, 528–530 ALPERS’ disease, 220 ALS, see Amyotrophic lateral sclerosis Alzheimer’s disease amyloid cascade hypothesis, 342–344 amyloid precursor protein mutations, 394 –395 apolipoprotein E, 397 apoptotic cascades, 392–394 brain cytochrome oxidase deﬁciency, 355–356 calcium homeostasis dysregulation, 391–392 calorie intake, 397–399 cybrid studies, 371 cytochrome oxidase cybrid data, 366–370 defect as cause, 372–373 genetic component, 358–361 genetic study inconclusiveness, 361–366

non-CNS tissues, 356–358 overview, 346–355 folic acid, 400– 401 historical overview, 342–344 intellectual activities, 399– 400 α-ketoglutarate dehydrogenase complex, 346 metabolic dysfunction, 344 –345 morphological studies, 345 neurodegenerative cascades, 387–389 overview, 342 oxyradical production, 390–391 perturbed energy metabolism, 389–390 physical activities, 399– 400 presenilin mutations, 395–397 pyruvate dehydrogenase complex, 345 Amyloid cascade hypothesis, 342–344 Amyloid precursor protein historical overview, 343–344 mutations, 394 –395 Amyotrophic lateral sclerosis brain, 417 clinical features, 413 liver, 420 lymphocytes, 415 mitochondrial abnormalities, 414 – 415 muscle, 418– 419 overview, 411– 412 platelets, 415– 416 skin, 416– 417 spinal cord, 418 transgenic mouse models, 420– 423 Anasthetic agents, mitochondrial disorder treatment, 124 –125 Animal models amyotrophic lateral sclerosis, 420– 423 Friedreich’s ataxia, 152–154 Huntington’s disease, 326–328 mitochondrial dysfunction, 179

535

536

INDEX

mtDNA defect-associated respiratory chain disease, 449– 452 nuclear DNA defect-associated respiratory chain disease, 452– 455 ANT1 gene, defect, 454 Antioxidant therapy, Friedreich’s ataxia, 163–164 3-AP, see 3-Acetylpyridine Apolipoprotein E, Alzheimer’s disease, 397 Apoptosis cascades in Alzheimer’s disease, 392–394 Huntington’s disease, 323–324 Parkinson’s disease, 299–301 APP, see Amyloid precursor protein Ascorbate, mitochondrial disorder treatment, 127 ATP, mitochondrial ETC, 27–28 ATP/ADP carrier, mitochondrial preproteins, 66 F1F0-ATP synthase, complex V, 41– 44 Authophagy, Huntington’s disease, 323–324 Autosomal-dominant hereditary spastic paraplegia, 198–199 Autosomal-dominant progressive external ophthalmoplegia, 109–110, 223–224 Autosomal-recessive hereditary spastic paraplegia SPG5, SPG11, SPG14, 197 SPG7, 193–196 Autosomal-recessive progressive external ophthalmoplegia, 223–224 B Bacteria, Friedreich’s ataxia model, 152 Benign infantile myopathy, 218–219 β oxidation acylcarnitine–carnitine translocase, 473– 476 acyl-CoA dehydrogenase, 476– 477 carnitine acylcarnitine translocase I deﬁciency clinical presentation, 487 diagnosis, 487– 488 genetics, 488 pathogenesis, 489 treatment, 488– 489 carnitine palmitoyltransferase I deﬁciency clinical presentation, 483– 485, 489– 490 diagnosis, 485, 490– 491

genetics, 486, 491– 492 pathogenesis, 486, 492 treatment, 486, 492 carnitine palmitoyltransferases, 473– 476 enoyl-CoA hydratase, 477– 478 enzyme organization, 479– 480 glutaric aciduria type II clinical presentation, 502–503 diagnosis, 503 genetics, 503 pathogenesis, 503 3-hydroxyacyl-CoA dehydrogenase, 478 medium-chain acyl-CoA dehydrogenase deﬁciency clinical presentation, 494 – 495 diagnosis, 495 genetics, 496 pathogenesis, 498– 499 treatment, 498 overview, 469– 470 3-oxoacyl-CoA thiolase, 478– 479 polyunsaturated fatty acid oxidation, 479 primary carnitine deﬁciency clinical presentation, 480– 481 diagnosis, 481– 482 genetics/mutation analysis, 482 pathogenesis, 483 treatment, 482– 483 short-chain acyl-CoA dehydrogenase deﬁciency genetics, 499–500 pathogenesis, 500 treatment, 500 short-chain hydroxyacyl-CoA dehydrogenase deﬁciency, 501–502 tissue differences, 480 trifunctional protein deﬁciencies clincial presentation and diagnosis, 500–501 genetics, 501 treatment, 501 very long-chain acyl-CoA dehydrogenase deﬁciency clinical presentation, 493 diagnosis, 493 genetics, 493– 494 pathogenesis, 494 treatment, 494 L-BOAA, see β-N- Oxalylamino-L-alanine Brain, amyotrophic lateral sclerosis, 417

INDEX

C CACT, see Carnitine acylcarnitine translocase I deﬁciency CAG repeats diseases, 318–319 Huntington’s disease, 317–318 Calcium homeostasis Alzheimer’s disease, 391–392 MPTP administration, 248–249 Calorie intake, Alzheimer’s disease, 397–399 CAP-resistant mtDNA, see Chloramphenicol-resistant mitochondrial DNA Cardiomyopathy, 232 Carnitine, mitochondrial disorder treatment, 129 Carnitine acylcarnitine translocase I deﬁciency clinical presentation, 487 diagnosis, 487– 488 genetics, 488 pathogenesis, 489 treatment, 488– 489 Carnitine palmitoyl transferase deﬁciency β oxidation control, 473– 476 recurrent myoglobinuria, 107 Carnitine palmitoyl transferase I deﬁciency clinical presentation, 483– 485 diagnosis, 485 genetics, 486 mitochondrial fatty acid oxidation, 48 pathogenesis, 486, 492 treatment, 486, 492 Carnitine palmitoyl transferase II deﬁciency clinical presentation, 489– 490 diagnosis, 490– 491 genetics, 491– 492 mitochondrial fatty acid oxidation, 48 Carnitine uptake defect clinical presentation, 480– 481 diagnosis, 481– 482 genetics/mutation analysis, 482 pathogenesis, 483 treatment, 482– 483 Carrier proteins, mitochondrial preprotein import, 74–76 CBP, see CREB-binding protein Cell death, see Apoptosis

537

Cell models mtDNA disease therapeutic strategies, 447– 449 respiratory chain disease application, 445– 446 leucine (UUR) point mutations, 436– 438 lysine point mutations, 438– 439 model types, 432– 434 mtDNA depletion syndrome, 446– 447 mtDNA rearrangements, 434 – 436 protein-coding gene point mutations, 440– 441 Cellular iron, Friedreich’s ataxia, 155–156 Chelating therapy, Wilson’s disease, 185–186 Chloramphenicol, mitochondrial disorder treatment, 130 Chloramphenicol-resistant mitochondrial DNA, 450– 452 Chronic progressive external ophthalmoplegia characteristics, 95–96, 108 progressive limb myopathy without CPEO, 106–107 Citric acid cycle, see TCA cycle Coenzyme Q10 Friedreich’s ataxia therapy, 164 mitochondrial disorder treatment, 125–126 Coenzyme Q deﬁciency, 115 Complex I deﬁciency biochemical features, 113–114 idiopathic Parkinson’s disease, 286–287 mitochondrial ETC, 29–31 Complex I inhibitors L-BOAA, 253 MPTP/MPP+, 243–251 rotenone, 251–253 Complex II deﬁciency biochemical features, 114 mitochondrial ETC, 32–34 Complex II inhibitors malonate, 257–258 3-nitropropionic acid, 254 –257 Complex III deﬁciency, 34–37 Complex IV deﬁciency, 38– 41, 114 Complex IV inhibitors cyanide, 258–259 hydrogen sulﬁde, 259–260 sodium azide, 260

538

INDEX

Complex V deﬁciency biochemical features, 115 mitochondrial ETC, 41– 44 Copper absorption, Wilson’s disease therapy, 186 Copper transport health role, 175–177 Menkes’ Disease role, 177–178 Counseling, mitochondrial disorder treatment, 125 COX deﬁciencies, see Cytochrome c oxidase deﬁciency CPEO, see Chronic progressive external ophthalmoplegia CPT, see Carnitine palmitoyl transferase CPT I, see Carnitine palmitoyl transferase I CPT II, see Carnitine palmitoyl transferase II Cranial magnetic resonance imaging, Wilson’s disease, 183–184 Creatine, mitochondrial disorder treatment, 129–130 CREB-binding protein, Huntingtin function, 322 Cyanide, cytochrome c oxidase inhibition, 258–259 Cybrid lines Alzheimer’s disease cytochrome oxidase dysfunction, 366–370 Alzheimer’s disease issues, 371 mitochondrial defect models, 432– 433 Cytochrome b deﬁciency, 114 Cytochrome c oxidase deﬁciency autosomal-dominant PEO, 223–224 autosomal-recessive PEO, 223–224 biogenesis, 205–208 cell models, 439 COX10, 217 cyanide inhibition, 258–259 hydrogen sulﬁde inhibition, 259–260 infantile autosomal-recessive mitochondrial encephalomyopathy, 219–220 infantile autosomal-recessive muscle, 217–219 Kearns–Sayre syndrome, 232 Leigh syndrome chromosome 2p16, 214 Leigh syndrome SURF-1, 209, 213–214 maternally inherited myopathy and cardiomyopathy, 232 maternally inherited PEO, 231

MELAS, 230–231 MERRF, 230 mitochondrial neurogastrointestinal encephalomyopathy, 225 mtDNA defect overview, 229 mtDNA depletion syndromes, 221–223 SCO1, 214 –215 SCO2, 215–217 sodium azide inhibition, 260 subunit I mutations, 225–228 subunit II mutations, 228 subunit III mutations, 228–229 Cytochrome oxidase, Alzheimer’s disease brain deﬁciency, 355–356 cybrid data, 366–370 defect as cause, 372–373 genetic component, 358–361 genetic study inconclusiveness, 361–366 non-CNS tissues, 356–358 overview, 346–355 Cytosine, human mtDNA distribution, 6 D DA, see Dopamine DAB, see 3,3 -Diaminobenzidene DCA, see Dichloroacetate ddC, see 2 ,3 -Dideoxycytidine DDP1, see Deafnes–dystonia peptide 1 Deafnes–dystonia peptide 1, 78–80, 100 Deletions, COX deﬁciency, 229–232 Dentato-rubral pallido-luysian atrophy, 318–319 3,3 -Diaminobenzidene, Alzheimer’s disease, 351 Dichloroacetate, mitochondrial disorder treatment, 128–129 2 ,3 -Dideoxycytidine, mitochondrial defect induction, 434 D-loop region, human mtDNA, 7 DNA, see Mitochondrial DNA DNA polymerase γ , mtDNA replication, 10–11 Dopamine, toxicity in Huntington’s disease, 325–326 Drosophila melanogaster, respiratory chain disease model, 455 DRPLA, see Dentato-rubral pallido-luysian atrophy

INDEX

Drugs, mitochondrial disorder treatment, 124–125 Duplications, COX deﬁciency, 229–232 E EH, see Enoyl-CoA hydratase Electron microscopy mammalian mtDNA, 5 mitochondrial disorders, 120–121 Electron transfer ﬂavoprotein, 50 Electron transport chain defects ADP–ATP translocator, 44 – 45 Alzheimer’s disease, 347–348, 365–368 characterization, 96 clinical features, 103–105 complex I, 29–31 complex II, 32–34 complex III, 34 –37 complex IV, 38– 41 complex V, 41– 44 genetics, 98–101 overview, 27–28 Encephalomyopathies ALPERS’ disease, 220 Leigh-like syndromes, 219–220 MELAS, 104 –105, 111, 230–231, 436, 438 mitochondrial neurogastrointestinal encephalomyopathy, 225 types, 110–112 Energy metabolism, Alzheimer’s disease, 389–390 Enoyl-CoA hydratase, β oxidation control, 477– 478 ETC, see Electron transport chain defects ETF, see Electron transfer ﬂavoprotein Excitotoxicity Huntington’s disease, 327 Parkinson’s disease, 297–299 Exercise, mitochondrial disorder treatment, 123 F FAD, see Flavin adenine dinucleotide Fatal infantile myopathy, 218 Fatty acid oxidation carnitive transport, 48 fatty acid β-oxidation, 48–51

539

overview, 47– 48 polyunsaturated fatty acid enzymes, 479 Flavin adenine dinucleotide, complex II, 33 Flavoprotein succinate:ubiquinone oxidoreductase, see Complex II Folic acid, Alzheimer’s disease, 400– 401 Forced paternal inheritance, mitochondrial disorder therapy, 131–132 Frataxin protein, Friedreich’s ataxia gene mutations, 151–152 Free radical generation, MPTP administration, 248–249 Friedreich’s ataxia antioxidant therapy, 163–164 bacterial model, 152 cellular iron regulation, 155–156 clinical features, 147–148 frataxin protein, 151–152 gene therapy, 165 genetic features, 148–150 heme biosynthesis, 156–157 iron chelation therapy, 163 iron–sulfur cluster synthesis, 157–159 mitochondrial dysfunction, 181 mitochondrial function, 159–161 mitochondrial iron, 154 –155 mitochondrial-targeted therapy, 165 oxidative stress, 161–162 pathological features, 148 therapeutic intervention overview, 162–163 transcription, 150–151 transgenic mouse model, 152–154 yeast model, 152 FROA, see Friedreich’s ataxia Fuel, physiology, 470– 473 G Gene therapy Friedreich’s ataxia, 165 mitochondrial disorders, 130–132 Genetics Alzheimer’s disease COX dysfunction basic theory, 358–361 study inconclusiveness, 361–366 autosomal-dominant HSP, 198–199 autosomal-recessive HSP SPG5, SPG11, SPG14, 197 SPG7, 193–196

540

INDEX

carnitine acylcarnitine translocase deﬁciency, 488 carnitine palmitoyl transferase I deﬁciency, 486 carnitine palmitoyltransferase II deﬁciency, 491– 492 Friedreich’s ataxia, 148–150 glutaric aciduria type II, 503 Huntington’s disease, 317–318 medium-chain acyl-CoA dehydrogenase deﬁciency, 496 mitochondrial disorders, 98–102 primary carnitine deﬁciency, 482 short-chain acyl-CoA dehydrogenase deﬁciency, 499–500 short-chain hydroxyacyl-CoA dehydrogenase deﬁciency, 502 trifunctional protein deﬁciencies, 501 very long-chain acyl-CoA dehydrogenase deﬁciency, 493– 494 Glutaric aciduria type II clinical presentation, 502–503 diagnosis, 503 genetics, 503 pathogenesis, 503 Glutathione, Parkinson’s disease, 296 GSH, see Glutathione GTP, mitochondrial transcript translation, 16 Guanine, human mtDNA distribution, 6 H HAD, see 3-Hydroxyacyl-CoA dehydrogenase HD, see Huntington’s disease Health, copper transport role function, 175–176 transport, 176–177 Heat shock proteins, Alzheimer’s disease, 370 Heme biosynthesis, Friedreich’s ataxia, 156–157 Hereditary spastic paraplegia autosomal-dominant HSP, 198–199 autosomal-recessive HSP, 193–196 clincial features, 192–193 mitochondria, 200–201 mitochondrial inner membrane proteins, 81–82 neurodegeneration, 200–201

overview, 191 X-linked HSP, 199 HSP, see Heat shock proteins; Hereditary spastic paraplegia; H-strand promoter H-strand promoter, 12–13 Huntingtin function, 321–323 intracellular localization, 319–321 Huntington’s disease apoptosis, 323–324 authophagy, 323–324 dopamine toxicity, 325–326 epidemiology, 316 genetics, 317–318 Huntingtin function, 321–323 Huntingtin localization, 319–321 metabolic dysfunction, 324 –325 neuropathology, 316–317 3-nitropropionic acid, 254 –255 overview, 315–316 oxidative stress, 324 –325 symptomatology, 316 transgenic mouse models, 326–328 Hydrogen sulﬁde, COX inhibition, 259–260 3-Hydroxyacyl-CoA dehydrogenase, β oxidation, 478 I Idebenone, Friedreich’s ataxia therapy, 163–164 Idiopathic Parkinson’s disease, complex I deﬁciency, 286–287 Infantile autosomal-recessive mitochondrial encephalomyopathy ALPERS’ disease, 220 Leigh-like syndromes, 219–220 Infantile autosomal-recessive muscle, 217–219 Infantile carnitine palmitoyltransferase II deﬁciency, 490 Infantile myopathy benign infantile myopathy, 218–219 fatal infantile myopathy, 218 severe types, 111–112 Inner membrane proteins, mitochondria, HSP, 81–82 Intellectual activities, Alzheimer’s disease, 399– 400

INDEX

Intergenomic communication, nuclear genes, 444 Intestine, Wilson’s disease therapy, 186 In vitro fertilization, mitochondrial disorder therapy, 131 Iron chelation therapy, Friedreich’s ataxia, 163 Iron metabolism Friedreich’s ataxia cellular iron regulation, 155–156 heme biosynthesis, 156–157 iron–sulfur cluster synthesis, 157–159 mitochondrial iron, 154–155 Parkinson’s disease, 295–296 Iron–sulfur cluster synthesis, Friedreich’s ataxia, 157–159 Iron–sulfur protein, complex III, 34–37 K Kearns–Sayre syndrome, 108, 125, 232 α-Ketoglutarate dehydrogenase complex, Alzheimer’s disease, 346 Ketone body metabolism, oxidative phosphorylation, 51–52 KGDHC, see α-Ketoglutarate dehydrogenase complex Kreb’s cycle, see TCA cycle KSS, see Kearns–Sayre syndrome L Lactate, mitochondrial disease workup, 117–118 LCAD, see Long-chain acyl-CoA dehydrogenase LCFAs, see Long-chain fatty acids LCHAD, see Long-chain hydroxyacyl-CoA dehydrogenase Leber’s hereditary optic neuropathy cell models, 440– 441 characteristics, 97 genetics, 100–101 Leigh syndrome chromosome 2p16, 214 related encephalopathy, 112 related syndromes, 219–220 SURF-1, 209, 213–214 Leucine (UUR), point mutations, cell models, 436– 438

541

Lewy bodies, MPTP, 249 LHON, see Leber’s hereditary optic neuropathy Limb myopathy, 106–107 Liver amyotrophic lateral sclerosis, 420 Wilson’s disease therapy, 186–187 Long-chain acyl-CoA dehydrogenase, mitochondrial fatty acid oxidation, 50 Long-chain fatty acids, mitochondrial dysfunction, 264 –265 Long-chain hydroxyacyl-CoA dehydrogenase, mitochondrial fatty acid oxidation, 51 LSP, see L-strand promoter L-strand promoter, mitochondria transcription initiation, 12–13 Luft’s syndrome, 96 Lymphocytes, amyotrophic lateral sclerosis, 415 Lysine, point mutations, cell models, 438– 439 M Madelung syndrome, 111 Magnetic resonance imaging, Wilson’s disease, 183–184 Malonate, succinate dehydrogenase inhibition, 257–258 Manganese, mitochondrial dysfunction, 260–262 Maternally inherited cardiomyopathy, 232 Maternally inherited progressive external ophthalmoplegia, 231 MCAD, see Medium-chain acyl-CoA dehydrogenase deﬁciency Medium-chain acyl-CoA dehydrogenase deﬁciency clinical presentation, 494 – 495 diagnosis, 495 genetics, 496 mitochondrial fatty acid oxidation, 50 pathogenesis, 498– 499 treatment, 498 MELAS, see Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes Menadione, mitochondrial disorder treatment, 127

542

INDEX

Menkes’ Disease, 177–178 MERRF, see Myoclonic epilepsy with ragged-red ﬁbers N-Methyl-D-aspartate, Parkinson’s disease, 297–298 1-Methyl-4-phenylpyridinium ion, MPTP conferrence calcium homeostasis, 248–249 cellular damage mechanisms, 249–250 free radical generation, 248–249 mitochondrial energetic defects, 247–248 toxicity, 244 –247 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine calcium homeostasis, 248–249 cellular damage mechanisms, 249–250 free radical generation, 248–249 inﬂammatory processes, 250–251 intoxication, 244 Lewy bodies, 249 mitochondrial energetic defects, 247–248 Parkinson’s disease, 285–286, 288–289, 299, 301 toxicity, 244 –247 Mitochondria biological clock function, 519–520 cell models of rearrangements, 434 – 436 mitochondrion origins, 25–26 Mitochondrial disorders adPEO, 109–110 Alzheimer’s disease, 344 –345 anasthetic agents, 124 –125 ancillary investigations, 118 animal models, 179 ascorbate therapy, 127 biochemical overview, 112–113 biochemical studies, 121–122 carnitine treatment, 129 chloramphenicol treatment, 130 clinical features, 103–105, 117 coenzyme Q10 therapy, 125–126 coenzyme Q deﬁciency, 115 complex I deﬁciency, 113–114 complex II deﬁciency, 114 complex IV deﬁciency, 114 complex V deﬁciency, 115 counseling, 125 CPEO, 108 creatine treatment, 129–130 cytochrome b deﬁciency, 114

dichloroacetate therapy, 128–129 drugs, 124 –125 drug therapy, 124 –125 electron microscopy, 120–121 encephalomyopathies, 110–112 exercise therapy, 123 Friedreich’s ataxia, 159–161 gene therapy, 130–132 genetics, 98–102 histochemistry, 119–120 history, 94 –98 human disease, 180–181 Huntington’s disease, 324 –325 initial diagnostic approaches, 115–117 Kearns–Sayre syndrome, 108 lactate measurement, 117–118 menadione therapy, 127 muscle biopsy, 118–119 mutant and wild-type mtDNA segregation, 105 mutation signiﬁcance, 122 neurological investigations, 118 neuropathy, 110 Parkinson’s disease apoptosis, 299–301 excitotoxicity, 297–299 oxidative stress, 294 –297 protein aggregation, 301–302 pedigree studies, 98–102 phylloquinone therapy, 127 progressive limb myopathy without CPEO, 106–107 quinone therapy, 125–126 recurrent myoglobinuria, 107 riboﬂavin therapy, 127–128 steroid therapy, 128 succinate treatment, 129 thiamine therapy, 127–128 tissue energy requirements, 106 toxin-induced, see Toxin-induced mitochondrial dysfunction Mitochondrial DNA Alzheimer’s disease cybrid data, 366–370 cybrid studies, 371 genetic component theory, 359–361 genetic studies, 361–365 amyotrophic lateral sclerosis, 414 – 415, 418– 419 analysis, 122

INDEX

associated respiratory chain disease, 449– 452 cell models leucine (UUR) point mutations, 436– 438 lysine point mutations, 438– 439 model types, 432–434 mtDNA rearrangements, 434 – 436 protein-coding gene point mutations, 440– 441 therapeutic strategies, 447– 449 chloramphenicol-resistant, mouse model, 450– 452 COX subunit I mutations, 225–228 COX subunit II mutations, 228 COX subunit III mutations, 228–229 depletion syndromes, 221–223, 446– 447 D-loop region structure, 7 hereditary spastic paraplegia, 200–201 mitochondrial disorders, 122 mutant, replication, mitochondrial disorder therapy, 131–132 mutant and wild-type, segregation, 105 mutation, genetic background interaction, 105–106 mutation interactions, 105–106 overview, 3–5, 430– 431 oxidative damage, 520–521 oxidative damage accumulation, 522–525 Parkinson’s disease, 290–293 posttranscriptional modiﬁcations, 15 repair, 525–528 repair changes with age, 528–530 replication alternative mode, 9 DNA polymerase γ , 10–11 mitochondrial single-strand binding protein, 11 overview, 7–8 regulation, 11–12 respiratory chain disease, 431 structural overview, 5–7 transcription elongation and termination, 14–15 transcription initiation, 12–14 translation, COX deﬁciency mutations Kearns–Sayre syndrome, 232 maternally inherited myopathy and cardiomyopathy, 232

543

maternally inherited PEO, 231 MELAS, 230–231 MERRF, 230 overview, 229 translation, transcripts, 15–17 translocase, 77–78 Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes cell models, 436, 438 characteristics, 111, 230–231 clinical features, 104 –105 Mitochondrial inner membrane proteins, HSP, 81–82 Mitochondrial neurogastrointestinal encephalomyopathy, 225 Mitochondrial preprotein import mammalian translocase, 77–78 Mohr–Tranebjaerg syndrome, 78–81 pathway overview, 59–61 targeting and sorting matrix-targeting signals, 62–64 multiple interal signals, 66 overview, 61–62 subcompartments, 64 –66 TIM22 complex carrier proteins, 74 –76 TIM22 complex overview, 73–74 TIM23 complex, 70–73 TIM23 import, 76–77 TOM complex, 66–69 Mitochondrial proteins nuclear mutations, Parkinson’s disease, 293–294 overview, 57–59 Mitochondrial RNA processing, 8 Mitochondrial single-strand binding protein, 11 Mitochondrial-targeted therapy, Friedreich’s ataxia, 165 Mitochondrial toxins, Parkinson’s disease, 288–289 Mitochondrial transcription factor mTERF, 14 –15 mtTFA, 13–14 Mitochondrial trifunctional protein, 51 Mitotic segregation, Alzheimer’s disease, 359 MND, see Motor neurone disease MNGIE, see Mitochondrial neurogastrointestinal encephalomyopathy

544

INDEX

Models amyotrophic lateral sclerosis, 420– 423 Friedreich’s ataxia, 152–154 Huntington’s disease, 326–328 mitochondrial dysfunction, 179 mtDNA disease therapeutic strategies, 447– 449 respiratory chain disease, 432– 441, 445– 447, 449– 457 Wilson’s disease, 178–179 Mohr–Tranebjaerg syndrome, mitochondrial preprotein import, 78–81 Motor neurone disease, 200 MPP+, see 1-Methyl-4-phenylpyridinium ion MPTP, see 1-Methyl-4-phenyl-1,2,3,6tetrahydropyridine MRP, see Mitochondrial RNA processing mtDNA, see Mitochondrial DNA MTP, see Mitochondrial trifunctional protein Multiple dehydrogenase deﬁciency, 502–503 Muscle amyotrophic lateral sclerosis, 418– 419 biopsy in mitochondrial disorders, 118–119 regeneration in mitochondrial disorder therapy, 131 Mutations Alzheimer’s disease mtDNA, 371 amyloid precursor protein, 394 –395 COX deﬁciency, 229–232 COX subunit I, 225–228 COX subunit II, 228 COX subunit III, 228–229 Friedreich’s ataxia, 150–152 Kearns–Sayre syndrome, 232 maternally inherited myopathy and cardiomyopathy, 232 maternally inherited PEO, 231 MELAS, 230–231 MERRF, 230 mitochondrial disorders, 122 mtDNA translation overview, 229 Parkinson’s disease, 293–294 presenilin, 395–397 primary carnitine deﬁciency, 482 Myoclonic epilepsy with ragged-red ﬁbers cell models, 438–439 characteristics, 230 COX deﬁciencies, 230 overview, 110

Myopathy benign infantile myopathy, 218–219 fatal infantile myopathy, 218 infantile myopathy, 111–112, 218–219 limb myopathy, 106–107 maternally inherited cardiomyopathy, 232 maternally inherited myopathy, 232 mitochondrial dysfunction, 264 –265 Myotoxic agents, mitochondrial dysfunction, 264–265 N NADH:ubiquinone oxidoreductase, see Complex I NARP, cell models, 441 Neonatal carnitine palmitoyltransferase II deﬁciency, 490 Neurodegenerative cascades, Alzheimer’s disease, 387–389 Neurodegenerative diseases hereditary spastic paraplegia, 81–82, 200–201 Mohr–Tranebjaerg syndrome, 78–81 Neurolathyrism, 253–254 Neuropathy characteristics, 110 Leber’s hereditary optic neuropathy, 97, 100–101, 440– 441 Nitric oxide, Parkinson’s disease, 297–299 Nitric oxide synthase, Parkinson’s disease, 297–299 3-Nitropropionic acid Huntington’s disease, 324 succinate dehydrogenase inhibition, 254–257 NMDA, see N-Methyl-D-aspartate NO, see Nitric oxide NOS, see Nitric oxide synthase 3-NP, see 3-Nitropropionic acid Nuclear DNA defects, animal models, 452– 455 Nuclear genes intergenomic communication, 444 respiratory chain complexes assembly proteins, 442– 444 cell models, 445– 447 indirect defects, 445 subunit defects, 442

INDEX

O OAT, see 3-Oxoacyl-CoA thiolase OL, see Origin of the L-strand replication Oligomycin-sensitivity-conferring protein, 43 Organotypic spinal cord cultures, ALS, 423 Origin of the H-strand replication, 7–8 Origin of the L-strand replication, 7–9 OSCP, see Oligomycin-sensitivity-conferring protein Outer mitochondrial membrane, translocation system, 66–69 OXA, mitochondrial preprotein import, 61 β-N- Oxalylamino-L-alanine, toxicity, 253 β-Oxidation, fatty acids, 48–51 Oxidative damage mitochondria, 520–521 mtDNA, 522–525 Oxidative phosphorylation complexes early research, 26–27 human mtDNA, 5–6 ketone body metabolism, 51–52 mitochondrial ETC ADP–ATP translocator, 44– 45 complex I, 29–31 complex II, 32–34 complex III, 34 –37 complex IV, 38– 41 complex V, 41– 44 overview, 27–28 mitochondrial fatty acid oxidation, 47–51 pyruvate dehydrogenase, 45– 46 TCA cycle, 46– 47 Oxidative stress Friedreich’s ataxia, 161–162 Huntington’s disease, 324 –325 Parkinson’s disease, 294 –297 3-Oxoacyl-CoA thiolase, β oxidation, 478– 479 OXPHOS, see Oxidative phosphorylation complexes Oxyradical production, Alzheimer’s disease, 390–391 P Parkinson’s disease apoptosis, 299–301 complex I deﬁciency, 286–287 excitotoxicity, 297–299

545

mitochondria and genetic etiologies, 289–290 mitochondrial protein nuclear mutations, 293–294 mitochondrial toxins, 288–289 MPTP, 244, 249, 285–286 mtDNA, 290–293 overview, 283–284 oxidative stress, 294 –297 protein aggregation, 301–302 rotenone, 251–253 PD, see Parkinson’s disease PDH, see Pyruvate dehydrogenase PDHC, see Pyruvate dehydrogenase complex Pedigree studies, mitochondrial disorders, 98–102 Penicillamine, Wilson’s disease therapy, 185 PEO, see Progressive external ophthalmoplegia Permeability transition pore, Parkinson’s disease, 300–301 Phylloquinone, mitochondrial disorder treatment, 127 Physical activities, Alzheimer’s disease, 399– 400 Platelets, amyotrophic lateral sclerosis, 415– 416 Point mutations cell models, protein-coding genes, 440– 441 leucine (UUR), cell models, 436– 438 lysine, cell models, 438– 439 transfer RNA, COX deﬁciency, 229–232 Polypeptides, complex II, 32–34 Preprotein import, see Mitochondrial preprotein import Presenilin, mutations, 395–397 Primary carnitine deﬁciency clinical presentation, 480– 481 diagnosis, 481– 482 genetics/mutation analysis, 482 pathogenesis, 483 treatment, 482– 483 Progressive external ophthalmoplegia cell models, 434– 435 characteristics, 95–98, 108–110, 223–224, 231 maternally inherited PEO, 231 progressive limb myopathy without CPEO, 106–107

546

INDEX

Progressive sclerosing poliodystrophy, 220 Protein-coding genes, point mutation cell models, 440– 441 Proteins aggregation in Parkinson’s disease, 301–302 amyloid precursor protein, 343–344, 394 –395 CREB-binding protein, 322 frataxin protein, 151–152 heat shock proteins, 370 iron–sulfur protein, complex III, 34–37 mitochondrial inner membrane proteins, HSP, 81–82 mitochondrial protein overview, 57–59 mitochondrial single-strand binding protein, 11 mitochondrial trifunctional protein, 51 nuclear genes in respiratory chain complexes, 442– 444 nuclear mutations, Parkinson’s disease, 293–294 oligomycin-sensitivity-conferring protein, 43 SURF-1 protein, 208 PTP, see Permeability transition pore Pyruvate dehydrogenase complex Alzheimer’s disease, 345 oxidative phosphorylation, 45– 46 Q Q cycle, complex III, 36–37 QPs-1, complex II, 32–34 QPs-3, complex II, 32–34 Quinones, mitochondrial disorder treatment, 125–126 R Rca1p, autosomal-recessive HSP, 194 –195 Reactive oxygen species, 520–521 Recurrent myoglobinuria, 107 Replicative segregation, Alzheimer’s disease, 359 Respiratory chain disease animal models, 449– 455 assembly proteins, 442– 444 cell model applications, 445– 446 indirect defects, 445

leucine (UUR) point mutations, 436– 438 lysine point mutations, 438– 439 mitochondrial defects, 431 models, 455– 457 mtDNA depletion syndrome, 446– 447 mtDNA rearrangements, 434 – 436 protein-coding gene point mutations, 440– 441 subunit defects, 442 Rhodobacter capsulatus, 456– 457 Rho zero cells, 432– 433 Riboﬂavin, mitochondrial disorder treatment, 127–128 RNA processing endoribonuclease, 8 RNase MRP, see RNA processing endoribonuclease ROS, see Reactive oxygen species Rotenone, toxicity, 251–253 S Saccharomyces cerevisiae cytochrome c oxidase biogenesis, 206–207 respiratory chain disease model, 456 SBMA, see Spinobulbar muscular atrophy SCA, see Spinocerebellar ataxias SCAD, see Short-chain acyl-CoA dehydrogenase deﬁciency SCHAD, see Short-chain hydroxyacyl-CoA dehydrogenase deﬁciency SCO1, cytochrome c oxidase, 214–215 SCO2, cytochrome c oxidase, 215–217 SDH, see Succinate dehydrogenase Short-chain acyl-CoA dehydrogenase deﬁciency genetics, 499–500 mitochondrial fatty acid oxidation, 50 pathogenesis, 500 treatment, 500 Short-chain hydroxyacyl-CoA dehydrogenase deﬁciency characteristics, 501–502 mitochondrial fatty acid oxidation, 51 Skin, amyotrophic lateral sclerosis, 416– 417 SMA, see Spinal muscular atrophy SOD1, see Superoxide dismutase Sodium azide, cytochrome c oxidase inhibition, 260 SPG5, autosomal-recessive HSP, 197 SPG7, autosomal-recessive HSP, 193–196

INDEX

SPG11, autosomal-recessive HSP, 197 SPG14, autosomal-recessive HSP, 197 Spinal cord, amyotrophic lateral sclerosis, 418 Spinal muscular atrophy, 418– 419 Spinobulbar muscular atrophy, 318–319 Spinocerebellar ataxias, 318–319 Steroids, mitochondrial disorder treatment, 128 Succinate, mitochondrial disorder treatment, 129 Succinate dehydrogenase, 255 Succinate ubiquinol oxidoreductase malonate, 257–258 3-nitropropionic acid, 254 –257 Superoxide dismutase amyotrophic lateral sclerosis, 412, 416– 417, 420– 421, 423 associated respiratory chain disease, 454– 455 SURF-1 protein cytochrome c oxidase biogenesis, 208 Leigh syndrome, 209, 213–214 T TCA cycle, oxidative phosphorylation, 46– 47 Terathiomolybadate, Wilson’s disease therapy, 186 1,2,3,4-Tetrahydroisoquinoline, Parkinson’s disease, 288–289 TFAM, see Transcription factor A Thiamine, mitochondrial disorder treatment, 127–128 TIM complex, see Translocase of the inner membrane complex TIQ, see 1,2,3,4-Tetrahydroisoquinoline Tissues β oxidation, 480 mitochondrial disorder energy requirements, 106 TOM complex, see Translocase of the outer mitochondrial membrane Toxicity cyanide, 258–259 dopamine in Huntington’s disease, 325–326 hydrogen sulﬁde, 259–260 malonate, 257–258

547

MPTP, 244 –247 β-N- oxalylamino-L-alanine, 253 rotenone, 251–253 sodium azide, 260 Toxin-induced mitochondrial dysfunction 3-acetylpyridine, 262–264 cytochrome c oxidase inhibition cyanide, 258–259 hydrogen sulﬁde, 259–260 sodium azide, 260 manganese, 260–262 myopathies, 264–265 myotoxic agents, 264 –265 NADH ubiquinine oxidoreductase inhibition MPTP/MPP+, 243–251 neurolathyrism, 253–254 rotenone, 251–253 regional and cellular speciﬁcity, 265–266 succinate ubiquinol oxidoreductase inhibition malonate, 257–258 3-nitropropionic acid, 254–257 Transcription Friedreich’s ataxia gene mutations, 150–151 mtDNA replication elongation and termination, 14 –15 mtDNA replication initiation, 12–14 Transcription factor A, mtDNA depletion syndromes, 222 Transfer RNA, point mutations, COX deﬁciency, 229–232 Transgenic mouse models amyotrophic lateral sclerosis, 420– 423 Friedreich’s ataxia, 152–154 Huntington’s disease, 326–328 Translocase, mitochondrial preprotein import, 70–73, 77–78 Translocase of the inner membrane complex carrier proteins, 74–76 inner mitochondrial membrane import, 76–77 mitochondrial preprotein import, 60–61, 70–73, 78–81 mitochondrial preprotein matrix-targeting signals, 64 overview, 73–74

548 Translocase of the outer mitochondrial membrane matrix-targeting signals, 62–64 mitochondrial preprotein import, 66–69 overview, 59–61 Trientene, Wilson’s disease therapy, 185 Trifunctional protein deﬁciencies clincial presentation and diagnosis, 500–501 genetics, 501 treatment, 501 U Ubiquinol:cytochrome c reductase, see Complex III deﬁciency V Very long-chain acyl-CoA dehydrogenase deﬁciency clinical presentation, 493 diagnosis, 493 genetics, 493– 494 mitochondrial fatty acid oxidation, 50 pathogenesis, 494 treatment, 494 Vitamin B1, mitochondrial disorder treatment, 127–128 Vitamin B2, mitochondrial disorder treatment, 127–128 Vitamin E, Friedreich’s ataxia therapy, 164

INDEX

Vitamin K1, mitochondrial disorder treatment, 127 Vitamin K3, mitochondrial disorder treatment, 127 VLCAD, see Very long-chain acyl-CoA dehydrogenase deﬁciency W Wilson’s disease animal models, 179 chelating therapy, 185–186 copper absorption inhibition, 186–187 copper transport role, transport overview, 176–177 cranial magnetic resonance imaging, 183–184 diagnosis, 183 experimental models, 178–179 mitochondrial dysfunction, 180–181 phenotype variation, 182–183 X X-linked hereditary spastic paraplegia, 199 Y Yeast, Friedreich’s ataxia model, 152 Yme1p, autosomal-recessive HSP, 195 Z Zinc, Wilson’s disease therapy, 186

CONTENTS OF RECENT VOLUMES

Volume 37 Section I: Selectionist Ideas and Neurobiology Selectionist and Instructionist Ideas in Neuroscience Olaf Sporns Population Thinking and Neuronal Selection: Metaphors or Concepts? Ernst Mayr Selection and the Origin of Information Manfred Eigen Section II: Development and Neuronal Populations Morphoregulatory Molecules and Selectional Dynamics during Development Kathryn L. Crossin Exploration and Selection in the Early Acquisition of Skill Esther Thelen and Daniela Corbetta Population Activity in the Control of Movement Apostolos P. Georgopoulos Section III: Functional Segregation and Integration in the Brain Reentry and the Problem of Cortical Integration Giulio Tononi Coherence as an Organizing Principle of Cortical Functions Wolf Singer

Memory and Forgetting: Long-Term and Gradual Changes in Memory Storage Larry R. Squire Implicit Knowledge: New Perspectives on Unconscious Processes Daniel L. Schacter Section V: Psychophysics, Psychoanalysis, and Neuropsychology Phantom Limbs, Neglect Syndromes, Repressed Memories, and Freudian Psychology V. S. Ramachandran Neural Darwinism and a Conceptual Crisis in Psychoanalysis Arnold H. Modell A New Vision of the Mind Oliver Sacks INDEX

Volume 38 Regulation of GABAA Receptor Function and Gene Expression in the Central Nervous System A. Leslie Morrow Genetics and the Organization of the Basal Ganglia Robert Hitzemann, Yeang Olan, Stephen Kanes, Katherine Dains, and Barbara Hitzemann

Section IV: Memory and Models

Structure and Pharmacology of Vertebrate GABAA Receptor Subtypes Paul J. Whiting, Ruth M. McKernan, and Keith A. Wafford

Selection versus Instruction: Use of Computer Models to Compare Brain Theories George N. Reeke, Jr.

Neurotransmitter Transporters: Molecular Biology, Function, and Regulation Beth Borowsky and Beth J. Hoffman

Temporal Mechanisms in Perception Ernst P¨oppel

549

550

CONTENTS OF RECENT VOLUMES

Presynaptic Excitability Meyer B. Jackson

Volume 40

Monoamine Neurotransmitters in Invertebrates and Vertebrates: An Examination of the Diverse Enzymatic Pathways Utilized to Synthesize and Inactivate Biogenic Amines B. D. Sloley and A. V. Juorio

Mechanisms of Nerve Cell Death: Apoptosis or Necrosis after Cerebral Ischemia R. M. E. Chalmers-Redman, A. D. Fraser, W. Y. H. Ju, J. Wadia, N. A. Tatton, and W. G. Tatton

Neurotransmitter Systems in Schizophrenia Gavin P. Reynolds

Changes in Ionic Fluxes during Cerebral Ischemia Tibor Kristian and Bo K. Siesjo

Physiology of Bergmann Glial Cells Thomas M¨uller and Helmut Kettenmann INDEX

Techniques for Examining Neuroprotective Drugs in Vitro A. Richard Green and Alan J. Cross

Volume 39

Techniques for Examining Neuroprotective Drugs in Vivo Mark P. Goldberg, Uta Strasser, and Laura L. Dugan

Modulation of Amino Acid-Gated Ion Channels by Protein Phosphorylation Stephen J. Moss and Trevor G. Smart

Calcium Antagonists: Their Role in Neuroprotection A. Jacqueline Hunter

Use-Dependent Regulation of GABAA Receptors Eugene M. Barnes, Jr.

Sodium and Potassium Channel Modulators: Their Role in Neuroprotection Tihomir P. Obrenovich

Synaptic Transmission and Modulation in the Neostriatum David M. Lovinger and Elizabeth Tyler

NMDA Antagonists: Their Role in Neuroprotection Danial L. Small

The Cytoskeleton and Neurotransmitter Receptors Valerie J. Whatley and R. Adron Harris

Development of the NMDA Ion-Channel Blocker, Aptiganel Hydrochloride, as a Neuroprotective Agent for Acute CNS Injury Robert N. McBurney

Endogenous Opioid Regulation of Hippocampal Function Michele L. Simmons and Charles Chavkin Molecular Neurobiology of the Cannabinoid Receptor Mary E. Abood and Billy R. Martin Genetic Models in the Study of Anesthetic Drug Action Victoria J. Simpson and Thomas E. Johnson Neurochemical Bases of Locomotion and Ethanol Stimulant Effects Tamara J. Phillips and Elaine H. Shen Effects of Ethanol on Ion Channels Fulton T. Crews, A. Leslie Morrow, Hugh Criswell, and George Breese INDEX

The Pharmacology of AMPA Antagonists and Their Role in Neuroprotection Rammy Gill and David Lodge GABA and Neuroprotection Patrick D. Lyden Adenosine and Neuroprotection Bertil B. Fredholm Interleukins and Cerebral Ischemia Nancy J. Rothwell, Sarah A. Loddick, and Paul Stroemer Nitrone-Based Free Radical Traps as Neuroprotective Agents in Cerebral Ischemia and Other Pathologies Kenneth Hensley, John M. Carney, Charles A. Stewart, Tahera Tabatabaie, Quentin Pye, and Robert A. Floyd

CONTENTS OF RECENT VOLUMES

Neurotoxic and Neuroprotective Roles of Nitric Oxide in Cerebral Ischemia Turgay Dalkara and Michael A. Moskowitz A Review of Earlier Clinical Studies on Neuroprotective Agents and Current Approaches Nils-Gunnar Wahlgren

551

Sensory and Cognitive Functions Lawrence M. Parsons and Peter T. Fox Skill Learning Julien Doyon Section V: Clinical and Neuropsychological Observations

INDEX

Executive Function and Motor Skill Learning Mark Hallett and Jordon Grafman

Volume 41

Verbal Fluency and Agrammatism Marco Molinari, Maria G. Leggio, and Maria C. Silveri

Section I: Historical Overview Rediscovery of an Early Concept Jeremy D. Schmahmann

Classical Conditioning Diana S. Woodruff-Pak

Section II: Anatomic Substrates

Early Infantile Autism Margaret L. Bauman, Pauline A. Filipek, and Thomas L. Kemper

The Cerebrocerebellar System Jeremy D. Schmahmann and Deepak N. Pandya Cerebellar Output Channels Frank A. Middleton and Peter L. Strick Cerebellar-Hypothalamic Axis: Basic Circuits and Clinical Observations Duane E. Haines, Espen Dietrichs, Gregory A. Mihailoff, and E. Frank McDonald Section III. Physiological Observations Amelioration of Aggression: Response to Selective Cerebellar Lesions in the Rhesus Monkey Aaron J. Berman Autonomic and Vasomotor Regulation Donald J. Reis and Eugene V. Golanov Associative Learning Richard F. Thompson, Shaowen Bao, Lu Chen, Benjamin D. Cipriano, Jeffrey S. Grethe, Jeansok J. Kim, Judith K. Thompson, Jo Anne Tracy, Martha S. Weninger, and David J. Krupa Visuospatial Abilities Robert Lalonde Spatial Event Processing Marco Molinari, Laura Petrosini, and Liliana G. Grammaldo Section IV: Functional Neuroimaging Studies Linguistic Processing Julie A. Fiez and Marcus E. Raichle

Olivopontocerebellar Atrophy and Friedreich’s Ataxia: Neuropsychological Consequences of Bilateral versus Unilateral Cerebellar Lesions Th´er`ese Botez-Marquard and Mihai I. Botez Posterior Fossa Syndrome Ian F. Pollack Cerebellar Cognitive Affective Syndrome Jeremy D. Schmahmann and Janet C. Sherman Inherited Cerebellar Diseases Claus W. Wallesch and Claudius Bartels Neuropsychological Abnormalities in Cerebellar Syndromes—Fact or Fiction? Irene Daum and Hermann Ackermann Section VI: Theoretical Considerations Cerebellar Microcomplexes Masao Ito Control of Sensory Data Acquisition James M. Bower Neural Representations of Moving Systems Michael Paulin How Fibers Subserve Computing Capabilities: Similarities between Brains and Machines Henrietta C. Leiner and Alan L. Leiner

552

CONTENTS OF RECENT VOLUMES

Cerebellar Timing Systems Richard Ivry

Volume 43

Attention Coordination and Anticipatory Control Natacha A. Akshoomoff, Eric Courchesne, and Jeanne Townsend

Early Development of the Drosophila Neuromuscular Junction: A Model for Studying Neuronal Networks in Development Akira Chiba

Context-Response Linkage W. Thomas Thach

Development of Larval Body Wall Muscles Michael Bate, Matthias Landgraf, and Mar Ruiz G´omez Bate

Duality of Cerebellar Motor and Cognitive Functions James R. Bloedel and Vlastislav Bracha Section VII: Future Directions

Development of Electrical Properties and Synaptic Transmission at the Embryonic Neuromuscular Junction Kendal S. Broadie

Therapeutic and Research Implications Jeremy D. Schmahmann

Ultrastructural Correlates of Neuromuscular Junction Development Mary B. Rheuben, Motojiro Yoshihara, and Yoshiaki Kidokoro

Volume 42

Assembly and Maturation of the Drosophila Larval Neuromuscular Junction L. Sian Gramates and Vivian Budnik

Alzheimer Disease Mark A. Smith Neurobiology of Stroke W. Dalton Dietrich Free Radicals, Calcium, and the Synaptic Plasticity-Cell Death Continuum: Emerging Roles of the Trascription Factor NFκB Mark P. Mattson AP-I Transcription Factors: Short- and LongTerm Modulators of Gene Expression in the Brain Keith Pennypacker Ion Channels in Epilepsy Istvan Mody Posttranslational Regulation of Ionotropic Glutamate Receptors and Synaptic Plasticity Xiaoning Bi, Steve Standley, and Michel Baudry Heritable Mutations in the Glycine, GABAA , and Nicotinic Acetylcholine Receptors Provide New Insights into the Ligand-Gated Ion Channel Receptor Superfamily Behnaz Vafa and Peter R. Schoﬁeld INDEX

Second Messenger Systems Underlying Plasticity at the Neuromuscular Junction Frances Hannan and Yi Zhong Mechanisms of Neurotransmitter Release J. Troy Littleton, Leo Pallanck, and Barry Ganetzky Vesicle Recycling at the Drosophila Neuromuscular Junction Daniel T. Stimson and Mani Ramaswami Ionic Currents in Larval Muscles of Drosophila Satpal Singh and Chun-Fang Wu Development of the Adult Neuromuscular System Joyce J. Fernandes and Haig Keshishian Controlling the Motor Neuron James R. Trimarchi, Ping Jin, and Rodney K. Murphey

Volume 44 Human Ego-Motion Perception A. V. van den Berg Optic Flow and Eye Movements M. Lappe and K.-P. Hoffman

CONTENTS OF RECENT VOLUMES

The Role of MST Neurons during Ocular Tracking in 3D Space K. Kawano, U. Inoue, A. Takemura, Y. Kodaka, and F. A. Miles Visual Navigation in Flying Insects M. V. Srinivasan and S.-W. Zhang Neuronal Matched Filters for Optic Flow Processing in Flying Insects H. G. Krapp A Common Frame of Reference for the Analysis of Optic Flow and Vestibular Information B. J. Frost and D. R. W. Wylie Optic Flow and the Visual Guidance of Locomotion in the Cat H. Sherk and G. A. Fowler Stages of Self-Motion Processing in Primate Posterior Parietal Cortex F. Bremmer, J.-R. Duhamel, S. B. Hamed, and W. Graf Optic Flow Analysis for Self-Movement Perception C. J. Duffy Neural Mechanisms for Self-Motion Perception in Area MST R. A. Andersen, K. V. Shenoy, J. A. Crowell, and D. C. Bradley Computational Mechanisms for Optic Flow Analysis in Primate Cortex M. Lappe Human Cortical Areas Underlying the Perception of Optic Flow: Brain Imaging Studies M. W. Greenlee What Neurological Patients Tell Us about the Use of Optic Flow L. M. Vaina and S. K. Rushton INDEX

Volume 45 Mechanisms of Brain Plasticity: From Normal Brain Function to Pathology Philip. A. Schwartzkroin

553

Brain Development and Generation of Brain Pathologies Gregory L. Holmes and Bridget McCabe Maturation of Channels and Receptors: Consequences for Excitability David F. Owens and Arnold R. Kriegstein Neuronal Activity and the Establishment of Normal and Epileptic Circuits during Brain Development John W. Swann, Karen L. Smith, and Chong L. Lee The Effects of Seizures of the Hippocampus of the Immature Brain Ellen F. Sperber and Solomon L. Moshe Abnormal Development and Catastrophic Epilepsies: The Clinical Picture and Relation to Neuroimaging Harry T. Chugani and Diane C. Chugani Cortical Reorganization and Seizure Generation in Dysplastic Cortex G. Avanzini, R. Preaﬁco, S. Franceschetti, G. Sancini, G. Battaglia, and V. Scaioli Rasmussen’s Syndrome with Particular Reference to Cerebral Plasticity: A Tribute to Frank Morrell Fredrick Andermann and Yvonne Hart Structural Reorganization of Hippocampal Networks Caused by Seizure Activity Daniel H. Lowenstein Epilepsy-Associated Plasticity in gammaAmniobutyric Acid Receptor Expression, Function and Inhibitory Synaptic Properties Douglas A. Coulter Synaptic Plasticity and Secondary Epileptogenesis Timothy J. Teyler, Steven L. Morgan, Rebecca N. Russell, and Brian L. Woodside Synaptic Plasticity in Epileptogenesis: Cellular Mechanisms Underlying Long-Lasting Synaptic Modiﬁcations that Require New Gene Expression Oswald Steward, Christopher S. Wallace, and Paul F. Worley Cellular Correlates of Behavior Emma R. Wood, Paul A. Dudchenko, and Howard Eichenbaum

554

CONTENTS OF RECENT VOLUMES

Mechanisms of Neuronal Conditioning David A. T. King, David J. Krupa, Michael R. Foy, and Richard F. Thompson Plasticity in the Aging Central Nervous System C. A. Barnes Secondary Epileptogenesis, Kindling, and Intractable Epilepsy: A Reappraisal from the Perspective of Neuronal Plasticity Thomas P. Sutula Kindling and the Mirror Focus Dan C. McIntyre and Michael O. Poulter Partial Kindling and Behavioral Pathologies Robert E. Adamec The Mirror Focus and Secondary Epileptogenesis B. J. Wilder Hippocampal Lesions in Epilepsy: A Historical Review Robert Naquet

Biosynthesis of Neurosteroids and Regulation of Their Synthesis Synthia H. Mellon and Hubert Vaudry

Clinical Evidence for Secondary Epileptogensis Hans O. Luders Epilepsy as a Progressive (or Nonprogressive “Benign”) Disorder John A. Wada Pathophysiological Aspects of LandauKleffner Syndrome: From the Active Epileptic Phase to Recovery Marie-Noelle Metz-Lutz, Pierre Maquet, Annd De Saint Martin, Gabrielle Rudolf, Norma Wioland, Edouard Hirsch and Chriatian Marescaux Local Pathways of Seizure Propagation in Neocortex Barry W. Connors, David J. Pinto, and Albert E. Telefeian Multiple Subpial Transection: A Clinical Assessment C. E. Polkey

Neurosteroid Modulation of Recombinant and Synaptic GABAA Receptors Jeremy J. Lambert, Sarah C. Harney, Delia Belelli, and John A. Peters

The Legacy of Frank Morrell Jerome Engel, Jr.

Neurosteroid 7-Hydroxylation Products in the Brain Robert Morfin and Luboslav Starka ´ Neurosteroid Analysis Ahmed A. Alomary, Robert L. Fitzgerald, and Robert H. Purdy Role of the Peripheral-Type Benzodiazepine Receptor in Adrenal and Brain Steroidogenesis Rachel C. Brown and Vassilios Papadopoulos Formation and Effects of Neuroactive Steroids in the Central and Peripheral Nervous System Roberto Cosimo Melcangi, Valerio Magnaghi, Mariarita Galbiati, and Luciano Martini

GABAA -Receptor Plasticity during LongTerm Exposure to and Withdrawal from Progesterone Giovanni Biggio, Paolo Follesa, Enrico Sanna, Robert H. Purdy, and Alessandra Concas Stress and Neuroactive Steroids Maria Luisa Barbaccia, Mariangela Serra, Robert H. Purdy, and Giovanni Biggio Neurosteroids in Learning and Memory Processes Monique Vall´ee, Willy Mayo, George F. Koob, and Michel Le Moal Neurosteroids and Behavior Sharon R. Engel and Kathleen A. Grant

Volume 46

Ethanol and Neurosteroid Interactions in the Brain A. Leslie Morrow, Margaret J. VanDoren, Rebekah Fleming, and Shannon Penland

Neurosteroids: Beginning of the Story Etienne E. Baulieu, P. Robel, and M. Schumacher

Preclinical Development of Neurosteroids as Neuroprotective Agents for the Treatment of Neurodegenerative Diseases Paul A. Lapchak and Dalia M. Araujo

CONTENTS OF RECENT VOLUMES

Clinical Implications of Circulating Neurosteroids Andrea R. Genazzani, Patrizia Monteleone, Massimo Stomati, Francesca Bernardi, Luigi Cobellis, Elena Casarosa, Michele Luisi, Stefano Luisi, and Felice Petraglia Neuroactive Steroids and Central Nervous System Disorders Mingde Wang, Torbj¨orn B¨ackstr¨om, Inger Sundstr¨om, G¨oran Wahlstr¨om, Tommy Olsson, Di Zhu, Inga-Maj Johansson, Inger Bj¨orn, and Marie Bixo Neuroactive Steroids in Neuropsychopharmacology Rainer Rupprecht and Florian Holsboer Current Perspectives on the Role of Neurosteroids in PMS and Depression Lisa D. Griffin, Susan C. Conrad, and Synthia H. Mellon INDEX

Volume 47 Introduction: Studying Gene Expression in Neural Tissues by in Situ Hybridization W. Wisden and B. J. Morris Part I: In Situ Hybridization with Radiolabelled Oligonucleotides In Situ Hybridization with Oligonucleotide Probes Wl. Wisden and B. J. Morris

555

Processing Human Brain Tissue for in Situ Hybridization with Radiolabelled Oligonucleotides Louise F. B. Nicholson In Situ Hybridization of Astrocytes and Neurons Cultured in Vitro L. A. Arizza-McNaughton, C. De Felipe, and S. P. Hunt In Situ Hybridization on Organotypic Slice Cultures A. Gerﬁn-Moser and H. Monyer Quantitative Analysis of in Situ Hybridization Histochemistry Andrew L. Gundlach and Ross D. O’Shea Part II: Nonradioactive in Situ hybridization Nonradioactive in Situ Hybridization Using Alkaline Phosphatase-Labelled Oligonucleotides S. J. Augood, E. M. McGowan, B. R. Finsen, B. Heppelmann, and P. C. Emson Combining Nonradioactive in Situ Hybridization with Immunohistological and Anatomical Techniques Petra Wahle Nonradioactive in Situ Hybridization: Simpliﬁed Procedures for Use in Whole Mounts of Mouse and Chick Embryos Linda Ariza-McNaughton and Robb Krumlauf INDEX

Cryostat Sectioning of Brains Victoria Revilla and Alison Jones

Volume 48

Processing Rodent Embryonic and Early Postnatal Tissue for in Situ Hybridization with Radiolabelled Oligonucleotides David J. Laurie, Petra C. U. Schrotz, Hannah Monyer, and Ulla Amtmann

Assembly and Intracellular Trafﬁcking of GABAA Receptors Eugene Barnes

Processing of Retinal Tissue for in Situ Hybridization Frank M¨uller

Subcellular Localization and Regulation of GABAA Receptors and Associated Proteins Bernhard Luscher ¨ and Jean-Marc Fritschy D1 Dopamine Receptors Richard Mailman

Processing the Spinal Cord for in Situ Hybridization with Radiolablled Oligonucleotides A. Berthele and T. R. T¨olle

Molecular Modeling of Ligand-Gated Ion Channels: Progress and Challenges Ed Bertaccini and James R. Trudel

556

CONTENTS OF RECENT VOLUMES

Alzheimer’s Disease: Its Diagnosis and Pathogenesis Jillian J. Kril and Glenda M. Halliday DNA Arrays and Functional Genomics in Neurobiology Christelle Thibault, Long Wang, Li Zhang, and Michael F. Miles INDEX

Volume 49 What Is West Syndrome? Olivier Dulac, Christine Soufﬂet, Catherine Chiron, and Anna Kaminski The Relationship between encephalopathy and Abnormal Neuronal Activity in the Developing Brain Frances E. Jensen Hypotheses from Functional Neuroimaging Studies Csaba Juh´asz, Harry T. Chugani, Ouo Muzik, and Diane C. Chugani Infantile Spasms: Unique Sydrome or General Age-Dependent Manifestation of a Diffuse Encephalopathy? M. A. Koehn and M. Duchowny

The Treatment of Infantile Spasms: An Evidence-Based Approach Mark Mackay, Shelly Weiss, and O. Carter Snead III ACTH Treatment of Infantile Spasms: Mechanisms of Its Effects in Modulation of Neuronal Excitability K. L. Brunson, S. Avishai-Eliner, and T. Z. Baram Neurosteroids and Infantile Spasms: The Deoxycorticosterone Hypothesis Michael A. Rogawski and Doodipala S. Reddy Are there Speciﬁc Anatomical and/or Transmitter Systems (Cortical or Subcortical) That Should Be Targeted? Phillip C. Jobe Medical versus Surgical Treatment: Which Treatment When W. Donald Shields Developmental Outcome with and without Successful Intervention Rochelle Caplan, Prabha Siddarth, Gary Mathern, Harry Vinters, Susan Curtiss, Jennifer Levitt, Robert Asarnow, and W. Donald Shields Infantile Spasms versus Myoclonus: Is There a Connection? Michael R. Pranzatelli

Histopathology of Brain Tissue from Patients with Infantile Spasms Harry V. Vinters

Tuberous Sclerosis as an Underlying Basis for Infantile Spasm Raymond S. Yeung

Generators of Ictal and Interictal Electroencephalograms Associated with Infantile Spasms: Intracellular Studies of Cortical and Thalamic Neurons M. Steriade and I. Timofeev

Brain Malformation, Epilepsy, and Infantile Spasms M. Elizabeth Ross

Cortical and Subcortical Generators of Normal and Abnormal Rhythmicity David A. McCormick Role of Subcortical Structures in the Pathogenesis of Infantile Spasms: What Are Possible Subcortical Mediators? F. A. Lado and S. L. Mosh´e What Must We Know to Develop Better Therapies? Jean Aicardi

Brain Maturational Aspects Relevant to Pathophysiology of Infantile Spasms G. Auanzini, F. Panzica, and S. Franceschetti Gene Expression Analysis as a Strategy to Understand the Molecular Pathogenesis of Infantile Spasms Peter B. Crino Infantile Spasms: Criteria for an Animal Model Carl E. Stafstrom and Gregory L. Holmes INDEX

CONTENTS OF RECENT VOLUMES

Volume 50 Part I: Primary Mechanisms How Does Glucose Generate Oxidative Stress In Peripheral Nerve? Irina G. Obrosova Glycation in Diabetic Neuropathy: Characteristics, Consequences, Causes, and Therapeutic Options Paul J. Thornalley Part II: Secondary Changes

Nerve Growth Factor for the Treatment of Diabetic Neuropathy: What Went Wrong, What Went Right, and What Does the Future Hold? Stuart C. Apfel Angiotensin-Converting Enzyme Inhibitors: Are there Credible Mechanisms for Beneﬁcial Effects in Diabetic Neuropathy? Rayaz A. Malik and David R. Tomlinson Clinical Trials for Drugs Against Diabetic Neuropathy: Can We Combine Scientiﬁc Needs With Clinical Practicalities? Dan Ziegler and Dieter Luft

Protein Kinase C Changes in Diabetes: Is the Concept Relevant to Neuropathy? Joseph Eichberg

INDEX

Are Mitogen-Activated Protein Kinases Glucose Transducers for Diabetic Neuropathies? Tertia D. Purves and David R. Tomlinson

Volume 51

Neuroﬁlaments in Diabetic Neuropathy Paul Fernyhough and Robert E. Schmidt Apoptosis in Diabetic Neuropathy Aviva Tolkovsky Nerve and Ganglion Blood Flow in Diabetes: An Appraisal Douglas W. Zochodne Part III: Manifestations Potential Mechanisms of Neuropathic Pain in Diabetes Nigel A. Calcutt Electrophysiologic Measures of Diabetic Neuropathy: Mechanism and Meaning Joseph C. Arezzo and Elena Zotova Neuropathology and Pathogenesis of Diabetic Autonomic Neuropathy Robert E. Schmidt Role of the Schwann Cell in Diabetic Neuropathy Luke Eckersley

557

Energy Metabolism in the Brain Leif Hertz and Gerald A. Dienel The Cerebral Glucose-Fatty Acid Cycle: Evolutionary Roots, Regulation, and (Patho) physiological Importance Kurt Heininger Expression, Regulation, and Functional Role of Glucose Transporters (GLUTs) in Brain Donard S. Dwyer, Susan J. Vannucci, and Ian A. Simpson Insulin-Like Growth Factor-1 Promotes Neuronal Glucose Utilization During Brain Development and Repair Processes Carolyn A. Bondy and Clara M. Cheng CNS Sensing and Regulation of Peripheral Glucose Levels Barry E. Levin, Ambrose A. Dunn-Meynell, and Vanessa H. Routh

Part IV: Potential Treatment

Glucose Transporter Protein Syndromes Darryl C. De Vivo, Dong Wang, Juan M. Pascual, and Yuan Yuan Ho

Polyol Pathway and Diabetic Peripheral Neuropathy Peter J. Oates

Glucose, Stress, and Hippocampal Neuronal Vulnerability Lawrence P. Reagan

558 Glucose/Mitochondria Conditions John P. Blass

CONTENTS OF RECENT VOLUMES

in

Neurological

Energy Utilization in the Ischemic/Reperfused Brain John W. Phillis and Michael H. O’Regan Diabetes Mellitus and the Central Nervous System Anthony L. McCall Diabetes, the Brain, and Behavior: Is There a Biological Mechanism Underlying the Association between Diabetes and Depression? A. M. Jacobson, J. A. Samson, K. Weinger, and C. M. Ryan Schizophrenia and Diabetes David C. Henderson and Elissa R. Ettinger Psychoactive Drugs Affect Glucose Transport and the Regulation of Glucose Metabolism Donard S. Dwyer, Timothy D. Ardizzone, and Ronald J. Bradley INDEX

Neural Control of Salivary S-IgA Secretion Gordon B. Proctor and Guy H. Carpenter Stress and Secretory Immunity Jos A. Bosch, Christopher Ring, Eco J. C. de Geus, Enno C. I. Veerman, and Arie V. Nieuw Amerongen Cytokines and Depression Angela Clow Immunity and Schizophrenia: Autoimmunity, Cytokines, and Immune Responses Fiona Gaughran Cerebral Lateralization and the Immune System Pierre J. Neveu Behavioral Conditioning of the Immune System Frank Hucklebridge Psychological and Neuroendocrine Correlates of Disease Progression Julie M. Turner-Cobb The Role of Psychological Intervention in Modulating Aspects of Immune Function in Relation to Health and Well-Being J. H. Gruzelier

Volume 52 Neuroimmune Relationships in Perspective Frank Hucklebridge and Angela Clow Sympathetic Nervous System Interaction with the Immune System Virginia M. Sanders and Adam P. Kohm Mechanisms by Which Cytokines Signal the Brain Adrian J. Dunn Neuropeptides: Modulators of Responses in Health and Disease David S. Jessop

Systemic Stress-Induced Th2 Shift and Its Clinical Implications Ilia J. Elenkov

Immune

Brain–immune Interactions in Sleep Lisa Marshall and Jan Born Neuroendocrinology of Autoimmunity Michael Harbuz

INDEX

Volume 53 Section I: Mitochondrial Structure and Function Mitochondrial DNA Structure and Function Carlos T. Moraes, Sarika Srivastava, Ilias Kirkinezos, Jose Oca-Cossio, Corina vanWaveren, Markus Woischnick, and Francisca Diaz Oxidative Phosphorylation: Structure, Function, and Intermediary Metabolism Simon J. R. Heales, Matthew E. Gegg , and John B. Clark

559

CONTENTS OF RECENT VOLUMES

Import of Mitochondrial Proteins Matthias F. Bauer, Sabine Hofmann, and Walter Neupert

Huntington’s Disease: The Unfolds? ˚ Peters´en and Patrik Brundin Asa

Section II: Primary Respiratory Chain Disorders

Mitochondria in Alzheimer’s Disease Russell H. Swerdlow and Stephen J. Kish

Mitochondrial Disorders of the Nervous System: Clinical, Biochemical, and Molecular Genetic Features Dominic Thyagarajan and Edward Byrne

Contributions of Mitochondrial Alterations, Resulting from Bad Genes and a Hostile Environment, to the Pathogenesis of Alzheimer’s Disease Mark P. Mattson

Section III: Secondary Respiratory Chain Disorders Friedreich’s Ataxia J. M. Cooper and J. L. Bradley

Mystery

Mitochondria and Amyotrophic Lateral Sclerosis Richard W. Orrell and Anthony H. V. Schapira Section VI: Models of Mitochondrial Disease

Wilson Disease C. A. Davie and A. H. V. Schapira

Models of Mitochondrial Disease Danae Liolitsa and Michael G. Hanna

Hereditary Spastic Paraplegia Christopher J. McDermott and Pamela J. Shaw

Section VII: Defects of β-Oxidation Including Carnitine Deﬁciency

Cytochrome c Oxidase Deﬁciency Giacomo P. Comi, Sandra Strazzer, Sara Galbiati, and Nereo Bresolin

Defects of β-Oxidation Including Carnitine Deﬁciency K. Bartlett and M. Pourfarzam

Section IV: Toxin-Induced Mitochondrial Dysfunction

Section VIII: Mitochondrial Involvement in Aging

Toxin-Induced Mitochondrial Dysfunction Susan E. Browne and M. Flint Beal

The Mitochondrial Theory of Aging: Involvement of Mitochondrial DNA Damage and Repair Nadja C. de Souza-Pinto and Vilhelm A. Bohr

Section V: Neurodegenerative Disorders Parkinson’s Disease L. V. P. Korlipara and A. H. V. Schapira

INDEX

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