Neurodegenerative Diseases and Metal Ions: Metal Ions in Life Sciences Vol 1

METAL IONS IN LIFE SCIENCES VOLUME 1

Neurodegenerative Diseases and Metal Ions

METAL IONS IN LIFE SCIENCES edited by

Astrid Sigel,1 Helmut Sigel,1 and Roland K. O. Sigel2 1

2

Department of Chemistry Inorganic Chemistry University of Basel Spitalstrasse 51 CH-4056 Basel, Switzerland Institute of Inorganic Chemistry University of Zürich Winterthurerstrasse 190 CH-8057 Zürich, Switzerland

VOLUME 1

Neurodegenerative Diseases and Metal Ions

Copyright © 2006

John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex PO19 8SQ, England Telephone

(+44) 1243 779777

Email (for orders and customer service enquiries): [email protected] Visit our Home Page on www.wiley.com All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning or otherwise, except under the terms of the Copyright, Designs and Patents Act 1988 or under the terms of a licence issued by the Copyright Licensing Agency Ltd, 90 Tottenham Court Road, London W1T 4LP, UK, without the permission in writing of the Publisher. Requests to the Publisher should be addressed to the Permissions Department, John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex PO19 8SQ, England, or emailed to [email protected], or faxed to (+44) 1243 770620. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The Publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the Publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. Other Wiley Editorial Offices John Wiley & Sons Inc., 111 River Street, Hoboken, NJ 07030, USA Jossey-Bass, 989 Market Street, San Francisco, CA 94103-1741, USA Wiley-VCH Verlag GmbH, Boschstr. 12, D-69469 Weinheim, Germany John Wiley & Sons Australia Ltd, 42 McDougall Street, Milton, Queensland 4064, Australia John Wiley & Sons (Asia) Pte Ltd, 2 Clementi Loop #02-01, Jin Xing Distripark, Singapore 129809 John Wiley & Sons Canada Ltd, 22 Worcester Road, Etobicoke, Ontario, Canada M9W 1L1 Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Library of Congress Cataloging in Publication Data Neurodegenerative diseases and metal ions / edited by Astrid Sigel, Helmut Sigel, and Roland K. O. Sigel. p. ; cm. — (Metal ions in life sciences ; v. 1) Includes bibliographical references and index. ISBN-13: 978-0-470-01488-2 (cloth : alk. paper) ISBN-10: 0-470-01488-1 (cloth : alk. paper) 1. Nervous system—Degeneration. 2. Nervous system—Diseases. 3. Metal ions—Health aspects. I. Sigel, Astrid. II. Sigel, Helmut. III. Sigel, Roland K. O. IV. Series. [DNLM: 1. Neurodegenerative Diseases—chemistry of. 2. Metals—adverse effects. 3. Metals— metabolism. 4. Prion Diseases—chemistry of. 5. Protein Folding. 6. Alzheimer’s Disease. 7. Parkinson’s Disease. 8. Creutzfeldt-Jakob Disease. WL 359 N49438 2006] RC365.N454 2006 616.8—dc22 2005032056 British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN-13 978-0-470-01488-2 (HB) ISBN-10 0-470-01488-1 (HB) Typeset in 10/12pt Times by Integra Software Services Pvt. Ltd, Pondicherry, India Printed and bound in Spain by Grafos S.A. Barcelona This book is printed on acid-free paper responsibly manufactured from sustainable forestry in which at least two trees are planted for each one used for paper production. The figure on the dustcover is part of Figure 6 of Chapter 3 by Henryk Kozlowski, Marek Luczkowski, Daniela Valensin, and Gianni Valensin

Historical Development and Perspectives of the Series Metal Ions in Life Sciences

It is an old wisdom that metals are indispensable for life. Indeed, several of them, like sodium, potassium, and calcium, are easily discovered in living matter. However, the role of metals and their impact on life remained largely hidden until inorganic chemistry and coordination chemistry experienced a pronounced revival in the 1950s. The experimental and theoretical tools created in this period and their application to biochemical problems led to the development of the field or discipline now known as Bioinorganic Chemistry, Inorganic Biochemistry, or more recently also often addressed as Biological Inorganic Chemistry. By 1970 Bioinorganic Chemistry was established and further promoted by the book series Metal Ions in Biological Systems founded in 1973 (edited by H.S., who was soon joined by A.S.) and published by Marcel Dekker, Inc., New York, for more than 30 years. After this company ceased to be a family endeavor and its acquisition by another company, we decided, after having edited 44 volumes of the MIBS series (the last two together with R.K.O.S.) to launch a new and broader-minded series to cover today’s needs in the Life Sciences. Therefore, the Sigels’ new series is entitled Metal Ions in Life Sciences and we are happy to join forces in this new endeavor with a most experienced publisher in the Sciences, John Wiley & Sons, Ltd, Chichester, UK. The development of Biological Inorganic Chemistry during the past 40 years was and still is driven by several factors; among these are: (i) the attempts to reveal the interplay between metal ions and peptides, nucleotides, hormones or vitamins, etc.; (ii) the efforts regarding the understanding of accumulation, transport, metabolism and toxicity of metal ions; (iii) the development and application of metal-based drugs; (iv) biomimetic syntheses with the aim to understand biological processes as well as to create efficient catalysts; (v) the determination of high-resolution structures of proteins, nucleic acids, and other biomolecules; (vi) the utilization of powerful spectroscopic tools allowing studies of structures and dynamics; and (vii), more recently, the widespread use of

Preface to Volume 1 Neurodegenerative Diseases and Metal Ions

Over the years substantial evidence has accumulated implicating that metal ions play a role in the pathophysiology and pathogenesis of neurodegenerative disorders. This is emphasized in Chapter 1, which sets the scene for this volume and provides an organizational frame for metal-related disorders, namely: (i) those caused by a defect in metal ion transport or homeostasis; (ii) those caused by toxicological exposure to metals; and (iii) those caused or associated with metalloprotein aggregation and/or misfolding. Indeed, misfolded proteins are implicated in a rapidly growing list of debilitating illnesses like Alzheimer’s, Parkinson’s, and Creutzfeldt–Jakob diseases. Therefore Chapter 2 deals with protein folding and misfolding; structures, energetics, and dynamics of transient species are considered in detail because their characterization is an essential step in understanding the benign and malignant pathways of protein folding. The three chapters following these general considerations are devoted to metal ion interactions, mainly of copper, with mammalian prion proteins and their fragments, to transmissible spongiform encephalopathies (Creutzfeldt–Jakob and related diseases) as well as to the amyloid precursor protein (Alzheimer’s disease). Chapters 6 and 7 consider in particular the role of iron in Parkinson’s and Huntington’s diseases, respectively, whereas Chapter 8 details the interrelation between copper–zinc superoxide dismutase and familial amyotrophic lateral sclerosis. The malfunctioning of copper transport in Wilson and Menkes diseases, where copper accumulates or is not absorbed, respectively, is dealt with in Chapter 9. The special role of iron in neurodegenerative diseases and the chemical interplay between catecholamines and metal ions are in the focus of Chapters 10 and 11, respectively; in this context Parkinson’s, Alzheimer’s and Huntington’s diseases are considered again, but from a different viewpoint, in addition to neurodegeneration with brain iron accumulation (NBIA, formerly Hallervorden– Spatz syndrome), neuroferritinopathy, aceruloplasminemia, Friedreich’s ataxia, and taupathies. The disruption of the homeostasis of metal ions can have devastating effects as is evident throughout the book. This also holds for the essential zinc; its

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PREFACE TO VOLUME 1

metalloneurochemistry, i.e. its physiology and pathology as well as probes and sensors to detect it, are covered extensively in Chapter 12. Next to metal ions like manganese, iron, copper, and zinc, which are essential but may also be toxic due to the creation of reactive oxygen species resulting in oxidative stress, there are other metal ions which are a priori neurotoxic. Among these is aluminum, and its role in neurodegenerative processes is reviewed in Chapter 13. Cadmium, lead, and mercury are important from the viewpoint of public health because they are released into the environment by human activities; their neurotoxicity is covered in Chapter 14. The terminating Chapter 15 summarizes in a general way the medicinal chemistry of metal-centered brain diseases and indicates other neurological disorders that may involve metal ions like polyneuropathy, multiple sclerosis, macular degeneration, progressive supranuclear palsy or the restless leg syndrome which are not otherwise covered in the book because knowledge is scarce. It is clear that there is an urgent need for developing novel drugs and classes of drugs that manipulate metal-centered neuropathology more precisely and elegantly than the presently (only partly) available chelation therapies. It is hoped that this volume stimulates research into this direction. Astrid Sigel Helmut Sigel Roland K. O. Sigel

Contents

HISTORICAL DEVELOPMENT AND PERSPECTIVES OF THE SERIES PREFACE TO VOLUME 1 CONTRIBUTORS TO VOLUME 1 TITLES OF VOLUMES 1–44 IN THE METAL IONS IN BIOLOGICAL SYSTEMS SERIES CONTENTS OF VOLUMES IN THE METAL IONS IN LIFE SCIENCES SERIES

1 THE ROLE OF METAL IONS IN NEUROLOGY. AN INTRODUCTION

v vii xv xix xxi

1

Dorothea Strozyk and Ashley I. Bush 1 Introductory Remarks 2 Comments on Metal Ions in Neurology Acknowledgments Abbreviations References

2 PROTEIN FOLDING, MISFOLDING, AND DISEASE

1 2 5 5 5

9

Jennifer C. Lee, Judy E. Kim, Ekaterina V. Pletneva, Jasmin Faraone-Mennella, Harry B. Gray, and Jay R. Winkler 1 2 3 4 5 6

Introduction Experimental Methods The Denatured State Protein Folding Dynamics -Synuclein and Parkinson’s Disease Conclusions and Outlook Acknowledgments

10 10 16 26 45 50 50

x

CONTENTS

Abbreviations and Definitions References

3 METAL ION BINDING PROPERTIES OF PROTEINS RELATED TO NEURODEGENERATION

51 51

61

Henryk Kozlowski, Marek Luczkowski, Daniela Valensin, and Gianni Valensin 1 Introduction 2 Cu2+ Interactions with Mammalian Prion Proteins and Their Fragments 3 Interactions of Metal Ions with the Amyloid Precursor Protein and Its Fragments 4 Concluding Remarks Acknowledgments Abbreviations References

4 METALLIC PRIONS: MINING THE CORE OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES

62 63 76 82 83 83 83

89

David R. Brown 1 Introduction 2 Historical Connections Between Copper and Transmissible Spongiform Encephalopathies 3 Copper Binding to Prion Protein 4 Copper Coordination by Prion Protein 5 Copper Uptake and Prion Protein Internalization 6 Prion Protein as an Antioxidant 7 Manganese Binding 8 Transmissible Spongiform Encephalopathies and Metals 9 Conclusions Abbreviations References

5 THE ROLE OF METAL IONS IN THE AMYLOID PRECURSOR PROTEIN AND IN ALZHEIMER’S DISEASE

89 92 93 95 97 101 104 107 109 109 110

115

Thomas A. Bayer and Gerd Multhaup 1 2 3 4

Introduction Amyloid Precursor Protein and Brain Copper Homeostasis Amyloid Precursor Protein and Cu,Zn-Superoxide Dismutase-1 General Conclusions Abbreviations References

115 117 119 120 121 122

CONTENTS

6 THE ROLE OF IRON IN THE PATHOGENESIS OF PARKINSON’S DISEASE

xi

125

Manfred Gerlach, Kay L. Double, Mario E. Götz, Moussa B. H. Youdim, and Peter Riederer 1 2 3 4 5

Introduction Iron in the Etiology of Parkinson’s Disease Sources of Increased Iron in Parkinson’s Disease Consequences of Iron Overload in Parkinson’s Disease General Conclusions Acknowledgments Abbreviations and Definitions References

7 IN VIVO ASSESSMENT OF IRON IN HUNTINGTON’S DISEASE AND OTHER AGE-RELATED NEURODEGENERATIVE BRAIN DISEASES

126 128 133 139 141 142 142 143

151

George Bartzokis, Po H. Lu, Todd A. Tishler, and Susan Perlman 1 Introduction. Puzzling Changes in Cell Numbers in Huntington’s Disease Brain 2 Human Brain Development and Disease Phenotypes 3 Oligodendrocytes and Iron in Brain Development and Degeneration 4 Transition Metal Metabolism and Proteinopathies 5 In Vivo Measurement of Brain Iron 6 Novel Treatment Considerations 7 Conclusions Acknowledgments Abbreviations References

159 165 166 168 168 169 169 170

8 COPPER-ZINC SUPEROXIDE DISMUTASE AND FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS

179

152 154

Lisa J. Whitson and P. John Hart 1 2 3 4 5 6 7

Introduction Molecular Mechanisms of fALS SOD1 Pathogenesis Structural Features of Human SOD1 ‘Wild-Type-like’ fALS Mutants ‘Metal Binding Region’ fALS Mutants Monomeric SOD1 and Pathogenesis Conclusions Acknowledgments

180 181 187 190 191 194 198 199

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CONTENTS

Abbreviations References

9 THE MALFUNCTIONING OF COPPER TRANSPORT IN WILSON AND MENKES DISEASES

199 199

207

Bibudhendra Sarkar 1 Introduction 2 Clinical and Biochemical Features of Copper Transport Disorders 3 Genes Identified in Copper Transport Disorders 4 Structure and Function of Copper-Transporting ATPases 5 Treatment of Copper Transport Disorders 6 Conclusions Acknowledgments Abbreviations References

10 IRON AND ITS ROLE IN NEURODEGENERATIVE DISEASES

208 210 212 213 217 221 221 221 221

227

Roberta J. Ward and Robert R. Crichton 1 Introduction 2 The Inorganic Chemistry of Iron and Its Role in Human Biology 3 Iron Metabolism 4 The Role of the ‘Labile Iron Pool’ in Free Radical Production 5 The Importance of Iron in Brain 6 The Involvement of Iron in Neurodegenerative Diseases 7 Experimental Approaches to Brain Iron Loading 8 Conclusions Acknowledgments Abbreviations References

11 THE CHEMICAL INTERPLAY BETWEEN CATECHOLAMINES AND METAL IONS IN NEUROLOGICAL DISEASES

228 229 230 238 239 244 265 270 270 270 272

281

Wolfgang Linert, Guy N. L. Jameson, Reginald F. Jameson, and Kurt A. Jellinger 1 2 3 4 5

General Introduction Neurodegenerative Diseases Relevant in Vitro Chemistry Iron and Parkinson’s Disease Relevant Manganese Chemistry

282 283 291 298 306

CONTENTS

6 Manganese and Manganosis 7 Other Metal Ions and Catecholamines 8 Summary of the Effect of Metal Ions on Autoxidation of Dopamine 9 Conclusions Acknowledgments Abbreviations and Definitions References

12 ZINC METALLONEUROCHEMISTRY: PHYSIOLOGY, PATHOLOGY, AND PROBES

xiii

306 308 311 312 313 314 315

321

Christopher J. Chang and Stephen J. Lippard 1 2 3 4 5 6

Introduction Zinc in the Brain Zinc Sensing for Neuroscience Applications Biomolecule Fluorescent Probes for Zinc Small-Molecule Fluorescent Probes for Zinc Concluding Remarks and Future Directions Acknowledgments Abbreviations References

322 323 328 329 339 359 360 360 361

13 THE ROLE OF ALUMINUM IN NEUROTOXIC AND NEURODEGENERATIVE PROCESSES

371

Tamás Kiss, Krisztina Gajda-Schrantz, and Paolo F. Zatta 1 2 3 4 5 6

Introduction Chemical Forms of Aluminum in Biological Systems Aluminum Loading in Humans Toxicology of Aluminum in Animals and Humans Aluminum and Alzheimer’s Disease General Conclusions Acknowledgments Abbreviations References

372 374 377 380 384 387 388 388 389

14 NEUROTOXICITY OF CADMIUM, LEAD, AND MERCURY

395

Hana R. Pohl, Henry G. Abadin, and John F. Risher 1 2 3 4 5

Introduction Cadmium Lead Mercury Conclusions

396 397 400 409 415

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CONTENTS

Abbreviations References

15 NEURODEGENERATIVE DISEASES AND METAL IONS. A CONCLUDING OVERVIEW

415 416

427

Dorothea Strozyk and Ashley I. Bush 1 2 3 4

Introduction The Medicinal Chemistry of Metal-centered Brain Disorders Other Neurological Disorders that May Involve Metals Conclusion and Outlook Acknowledgments Abbreviations and Definitions References

SUBJECT INDEX

428 428 430 432 433 433 434 437

Contributors

Numbers in parentheses indicate the pages on which the authors’ contributions begin. Henry G. Abadin Agency for Toxic Substances and Disease Registry (ATSDR), US Department of Health and Human Services, Division of Toxicology, Mailstop E-29, 1600 Clifton Road, Atlanta, GA 30333, USA (395) George Bartzokis (1) Department of Neurology, Alzheimer’s Disease Center, The David Geffen School of Medicine at UCLA, 710 Westwood Plaza, Los Angeles, CA 90095-1769, USA, , (2) Laboratory of Neuroimaging, Department of Neurology, Division of Brain Mapping, UCLA, Los Angeles, CA 90095, USA, (3) Greater Los Angeles VA Healthcare System, Department of Psychiatry, West Los Angeles, CA 90073, USA, and (4) Department of Psychiatry, Charles R. Drew University of Medicine and Science, Los Angeles, CA 90043, USA (151) Thomas A. Bayer Universität des Saarlandes, Klinik für Psychiatrie, Abteilung für Neurobiologie, D-66421 Homburg/Saar, Germany, (115) David R. Brown Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK, (89) Ashley I. Bush (1) Laboratory for Oxidation Biology, Genetics and Aging Research Unit, Harvard Medical School, Massachusetts General Hospital, Bldg 114, 16th Street, Charlestown, MA 02129, USA, , and (2) Oxidation Disorders Laboratory, Mental Health Research Institute of Victoria, and Department of Pathology, The University of Melbourne, 155 Oak Street, Parkville, Victoria 3052, Australia (1, 427) Christopher J. Chang Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA (321)

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CONTRIBUTORS

Robert R. Crichton Unité de Biochimie, Université Catholique de Louvain, Place Louis Pasteur 1, B-1348 Louvain-la-Neuve, Belgium, (227) Kay Double Prince of Wales Medical Research Institute, Barker St., Randwick, Sydney, NSW, 2031, Australia, (125) Jasmin Faraone-Mennella Beckman Institute, California Institute of Technology, Pasadena, CA 91125, USA (9) Krisztina Gajda-Schrantz Department of Inorganic and Analytical Chemistry, Bioinorganic Chemistry Research Group of the Hungarian Academy of Sciences, University of Szeged, P.O. Box 440, H-6701 Szeged, Hungary (371) Manfred Gerlach Department of Child and Adolescence Psychiatry and Psychotherapy, Clinical Neurochemistry, University of Würzburg, Füchsleinstrasse 15, D-97080 Würzburg, Germany, <[email protected]> (125) Mario E. Götz Department of Pharmacology, Universitätsklinikum Kiel, Hospitalstrasse 4, D-24105 Kiel, Germany, <[email protected]> (125) Harry B. Gray Beckman Institute, California Institute of Technology, Pasadena, CA 91125, USA, (9) P. John Hart Department of Biochemistry and the X-Ray Crystallography Core Laboratory, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA, (179) Guy N. L. Jameson Institute of Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9/163-AC, A-1060 Vienna, Austria, <[email protected]> (281) Reginald F. Jameson Emeritus Professor, University of Dundee, Scotland, UK, (281) Kurt Jellinger Institute of Clinical Neurobiology, Kenyongasse 18, A-1070 Vienna, Austria, (281) Judy E. Kim Beckman Institute, California Institute of Technology, Pasadena, CA 91125, USA (9)

CONTRIBUTORS

xvii

Tamás Kiss Department of Inorganic and Analytical Chemistry, Bioinorganic Chemistry Research Group of the Hungarian Academy of Sciences, University of Szeged, P.O. Box 440, H-6701 Szeged, Hungary, (371) Henryk Kozlowski Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie 14, 50-383 Wroclaw, Poland, (61) Jennifer C. Lee Beckman Institute, California Institute of Technology, Pasadena, CA 91125, USA, (9) Wolfgang Linert Institute of Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9/163-AC, A-1060 Vienna, Austria, <[email protected]> (281) Stephen J. Lippard Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA, (321) Po H. Lu (1) Department of Neurology, Alzheimer’s Disease Center, The David Geffen School of Medicine at UCLA, 710 Westwood Plaza, Los Angeles, CA 90095-1769, USA, and (2) Greater Los Angeles VA Healthcare System, Department of Psychiatry, West Los Angeles, CA 90073, USA (151) Marek Luczkowski Faculty of Chemistry, University of Wroclaw, F. JoliotCurie 14, 50-383 Wroclaw, Poland, <[email protected]> (61) Gerd Multhaup Institut für Chemie/Biochemie, Freie Universität Berlin, Thielallee 63, D-14195 Berlin, Germany, <[email protected]> (115) Susan Perlman Department of Neurology, Alzheimer’s Disease Center, the David Geffen School of Medicine at UCLA, 710 Westwood Plaza, Los Angeles, CA 90095-1769, USA (151) Ekaterina V. Pletneva Beckman Institute, California Institute of Technology, Pasadena, CA 91125, USA (9) Hana R. Pohl Agency for Toxic Substances and Disease Registry (ATSDR), US Department of Health and Human Services, Division of Toxicology, Mailstop F-32, 1600 Clifton Road, Atlanta, GA 30333, USA, (395) Peter Riederer Department of Psychiatry and Psychotherapy, Clinical Neurochemistry, National Parkinson Foundation Centers of Excellence for Neurodegenerative Diseases Research, University of Würzburg, Füchsleinstrasse 15, D-97080 Würzburg, Germany, (125)

xviii

CONTRIBUTORS

John F. Risher Agency for Toxic Substances and Disease Registry (ATSDR), US Department of Health and Human Services, Division of Toxicology, Mailstop E-29, 1600 Clifton Road, Atlanta, GA 30333, USA (395) Bibudhendra Sarkar (1) The Research Institute, The Hospital for Sick Children, Toronto, and (2) The Department of Biochemistry, University of Toronto, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada, (207) Dorothea Strozyk Department of Neurology, Albert Einstein College of Medicine, Kennedy Center, Suite 220, Pelham Parkway South, Bronx, NY 10461, USA, <[email protected]> (1, 427) Todd A. Tishler (1) Department of Neurology, Alzheimer’s Disease Center, The David Geffen School of Medicine at UCLA, 710 Westwood Plaza, Los Angeles, CA 90095-1769, USA, (2) Greater Los Angeles VA Healthcare System, Department of Psychiatry, West Los Angeles, CA 90073, and (3) Neuroscience Interdepartmental Graduate Program, The David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA (151) Daniela Valensin Department of Chemistry, University of Siena, Via Aldo Moro, I-53100 Siena, Italy (61) Gianni Valensin Department of Chemistry, University of Siena, Via Aldo Moro, I-53100 Siena, Italy, (61) Roberta J. Ward Unité de Biochimie, Université Catholique de Louvain, Place Louis Pasteur 1, B-1348 Louvain-la-Neuve, Belgium, <[email protected]> (227) Lisa J. Whitson Department of Biochemistry and the X-Ray Crystallography Core Laboratory, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA, <[email protected]> (179) Jay R. Winkler Beckman Institute, California Institute of Technology, Pasadena, CA 91125, USA, <[email protected]> (9) Moussa B. H. Youdim Eve Topf and National Parkinson Foundation Centers of Excellence for Neurodegenerative Diseases Research, and Department of Pharmacology, Technion-Faculty of Medicine, Efron St., PO Box 9697, Haifa 31096, Israel, (125) Paolo F. Zatta CNR Institute for Biomedical Technologies, Metalloproteins Unit, Department of Biology, University of Padova, Viale G. Colombo 3, I-35121 Padova, Italy, (371)

Titles of Volumes 1–44 in the Metal Ions in Biological Systems Series edited by the SIGELs and published by Dekker/Taylor & Francis Volume 1: Volume 2: Volume 3: Volume 4: Volume 5: Volume 6: Volume 7: Volume 8: Volume 9: Volume 10: Volume 11: Volume 12: Volume 13: Volume 14: Volume 15: Volume 16: Volume Volume Volume Volume Volume

17: 18: 19: 20: 21:

Volume 22: Volume 23: Volume 24: Volume 25: Volume 26:

Simple Complexes Mixed-Ligand Complexes High Molecular Complexes Metal Ions as Probes Reactivity of Coordination Compounds Biological Action of Metal Ions Iron in Model and Natural Compounds Nucleotides and Derivatives: Their Ligating Ambivalency Amino Acids and Derivatives as Ambivalent Ligands Carcinogenicity and Metal Ions Metal Complexes as Anticancer Agents Properties of Copper Copper Proteins Inorganic Drugs in Deficiency and Disease Zinc and Its Role in Biology and Nutrition Methods Involving Metal Ions and Complexes in Clinical Chemistry Calcium and Its Role in Biology Circulation of Metals in the Environment Antibiotics and Their Complexes Concepts on Metal Ion Toxicity Applications of Nuclear Magnetic Resonance to Paramagnetic Species ENDOR, EPR, and Electron Spin Echo for Probing Coordination Spheres Nickel and Its Role in Biology Aluminum and Its Role in Biology Interrelations Among Metal Ions, Enzymes, and Gene Expression Compendium on Magnesium and Its Role in Biology, Nutrition, and Physiology

xx

VOLUMES IN THE MIBS SERIES

Volume 27: Electron Transfer Reactions in Metalloproteins Volume 28: Degradation of Environmental Pollutants by Microorganisms and Their Metalloenzymes Volume 29: Biological Properties of Metal Alkyl Derivatives Volume 30: Metalloenzymes Involving Amino Acid-Residue and Related Radicals Volume 31: Vanadium and Its Role for Life Volume 32: Interactions of Metal Ions with Nucleotides, Nucleic Acids, and Their Constituents Volume 33: Probing Nucleic Acids by Metal Ion Complexes of Small Molecules Volume 34: Mercury and Its Effects on Environment and Biology Volume 35: Iron Transport and Storage in Microorganisms, Plants, and Animals Volume 36: Interrelations Between Free Radicals and Metal Ions in Life Processes Volume 37: Manganese and its Role in Biological Processes Volume 38: Probing of Proteins by Metal Ions and Their Low-Molecular-Weight Complexes Volume 39: Molybdenum and Tungsten. Their Roles in Biological Processes Volume 40: The Lanthanides and Their Interrelations with Biosystems Volume 41: Metal Ions and Their Complexes in Medication Volume 42: Metal Complexes in Tumor Diagnosis and as Anticancer Agents Volume 43: Biogeochemical Cycles of Elements Volume 44: Biogeochemistry, Availability, and Transport of Metals in the Environment

Contents of Volumes in the Metal Ions in Life Sciences Series edited by the SIGELs and published by John Wiley & Sons, Ltd, Chichester, UK Volume 1: Neurodegenerative Diseases and Metal Ions (this book) Volume 2: Nickel and Its Surprising Impact in Nature (tentative contents) 1. The Biogeochemistry of Nickel and Its Release into the Environment Tiina M. Nieminen, Liisa Ukonmaanaho, Nicole Rausch, and William Shotyk 2. Nickel in the Environment and Its Role in Plant Metabolism Hendrik Küpper and Peter M. H. Kroneck 3. Nickel Ion Complexes of Amino Acids and Peptides Teresa Kowalik-Jankowska, Henryk Kozlowski, Etelka Farkas, and Imre Sóvágó 4. Complex Formation of Nickel(II) with Nucleobases, Phosphates, Nucleotides, and Nucleic Acids Roland K. O. Sigel and Helmut Sigel 5. Synthetic Models for the Active Sites of Nickel-containing Enzymes Franc Meyer 6. Urease. Recent Insights in the Role of Nickel Stefano Ciurli 7. Nickel-Iron Hydrogenases Wolfgang Lubitz 8. Methyl-Coenzyme M Reductase and Its Nickel Corphin Cofactor F430 Rudolf K. Thauer 9. Acetyl-Coenzyme A Synthases and Nickel-containing Carbon Monoxide Dehydrogenases Paul A. Lindahl and David E. Graham 10. Nickel Superoxide Dismutase Peter A. Bryngelson and Michael J. Maroney 11. Biochemistry of the Nickel-dependent Glyoxalase I Enzymes Nicole Sukdeo, Elizabeth Daub, and John F. Honek

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CONTENTS OF FUTURE MILS VOLUMES

12. Nickel in Acireductone Dioxygenase Thomas C. Pochapsky 13. The Nickel-Regulated Peptidyl-Prolyl cis/trans Isomerase SlyD Frank Erdmann and Gunter Fischer 14. Chaperones of Nickel Metabolism Soledad Quiroz, Jong K. Kim, Scott Mulrooney, and Robert P. Hausinger 15. The Role of Nickel in Environmental Adaptation of the Gastric Pathogen Helicobacter pylori Florian D. Ernst, Arnoud H. M. van Vliet, Manfred Kist, Johannes G. Kusters, and Stefan Bereswill 16. The Effect of Nickel on Gene Expression Konstantin Salnikow and Kazimierz Kasprzak 17. Nickel Toxicity and Carcinogenesis Kazimierz Kasprzak and Konstantin Salnikow Subject Index Volume 3: The Ubiquitous Roles of Cytochrome P450 Proteins (tentative contents) 1. Diversity and Similarity of P450 Systems. An Introduction Stephen G. Sligar and Mary A. Schuler 2. Structural and Functional Mimics of Cytochromes P450 Wolf-D. Woggon 3. Structures of P450 Proteins and Their Molecular Phylogeny Thomas L. Poulos 4. Aquatic P450 Species Mark J. Snyder 5. The Electrochemistry of Cytochrome P450 Systems Alan M. Bond, Barry D. Fleming, and Lisandra L. Martin 6. Electron Transfer Reactions of Cytochrome P450 Stephen M. Contakes, Yen Hoang Le Nguyen, Andrew K. Udit, and Harry B. Gray 7. Cytochromes P450. Mechanistic Considerations Michael T. Green 8. Leakage in Cytochrome P450 Reactions in Relation to Protein Structural Properties Christiane Jung 9. Cytochromes P450. Structural Basis for Binding and Catalysis Ilme Schlichting 10. Beyond Heme-Thiolate Interactions. Roles of the Secondary Coordination Sphere in P450 Systems Yi Lu 11. Interactions of Cytochrome P450 with Nitrogen Monoxide and Related Ligands Andrew W. Munro

CONTENTS OF FUTURE MILS VOLUMES

xxiii

12. Cytochrome P450-Catalyzed Hydroxylations and Epoxidations Roshan Perera, S. Jin, Masanori Sono, and John H. Dawson 13. Cytochromes P450 and Steroid Hormones Michael R. Waterman and Rita Bernhard 14. Carbon–Carbon Bond Cleavage by P450 Systems James J. De Voss and Max J. Cryle 15. Drug Metabolism as Catalyzed by Cytochrome P450 Systems F. Peter Guengerich 16. Chemical Defence and Exploitation: Biotransformation of Xenobiotics by Cytochrome P450 Elizabeth M. J. Gillam 17. Design and Engineering of P450 Systems Stephen Bell, Nicola Hoskins, Christopher Whitehouse, and Luet L. Wong Subject Index Comments and suggestions with regard to contents, topics, and the like for future volumes of the series are welcome.

Met. Ions Life Sci. 1, 1–7 (2006)

1 The Role of Metal Ions in Neurology. An Introduction Dorothea Strozyk1 and Ashley I. Bush23 1

Department of Neurology, Albert Einstein College of Medicine, Kennedy Center, Suite 220, Pelham Parkway South, Bronx, NY 10461, USA <[email protected]> 2

3

Laboratory for Oxidation Biology, Genetics and Aging Research Unit, Harvard Medical School, Massachusetts General Hospital, Building 114, 16th Street, Charlestown, MA 02129, USA

Oxidation Disorders Laboratory, Mental Health Research Institute of Victoria, and Department of Pathology, The University of Melbourne, 155 Oak Street, Parkville, Victoria 3052, Australia

1 INTRODUCTORY REMARKS 2 COMMENTS ON METAL IONS IN NEUROLOGY ACKNOWLEDGMENTS ABBREVIATIONS REFERENCES

1 2 5 5 5

1 INTRODUCTORY REMARKS Substantial evidence has accumulated implicating metals in the pathophysiology and pathogenesis of neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease and amyotrophic lateral sclerosis. Interactions between redoxactive metal ions and proteins can lead to damage of critical biological systems and initiate a cascade of events leading to oxidative damage, neurodegeneration

Metal Ions in Life Sciences, Volume 1 © 2006 John Wiley & Sons, Ltd

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and cell death. Here we address the recent advances in understanding the molecular mechanisms of metal ion activity in the brain and nervous system, and we list neurological diseases that have been associated with metal ions and propose a conceptual model for organizing neurological disorders where metal ions are implicated.

2 COMMENTS ON METAL IONS IN NEUROLOGY The relevance of biological metals to neuroscience has burgeoned in this first decade of the 21st century. In 2000 we noted that the neurobiology of the heavier metal ions did not arouse much interest since they were not notably linked to major disease syndromes [1]. As we foresaw, this outlook currently is changing rapidly, with a growing number of excellent publications pointing the way to a seminal relationship between Fe, Cu, Mn, and Zn in the generation (or defence) of oxygen and protein radicals that mediate the major neurological diseases. However, there continues to be notable resistance among the mainstream of the neuroscience community to the appreciation of the importance of this emerging literature. This is probably because neuroscientists are not usually exposed to the basics of metallochemistry and oxidation chemistry during training, which emphasizes the value of cellular and molecular approaches, and because biochemical training has traditionally deemphasized the role of metal ions in metabolic reactions, which is why they have been pejoratively termed ‘trace metals’. This is a misnomer since the concentrations of Fe, Zn and Cu in the gray matter are in the same order of magnitude as Mg (0.1–0.5 mM) [2]. The brain utilizes metal ions for myriad biochemical reactions, and cortical neurons release ionically exchangeable copper [3] and zinc [4] during depolarization and neurotransmission. Abnormalities of metal ion biochemistry in neural tissue arise by two basic mechanisms: (i) protein aggregation mediated by metal ions, e.g., zinc induction of Alzheimer’s disease -amyloid A deposits [5]; and (ii) oxidative reactions catalyzed by redox-active metals. Redox-active metals (Cu, Fe, Mn) can generate radicals and reactive oxygen (ROS) and nitrogen species, by inappropriately accepting or donating electrons (a redox-active metal refers to a metal ion that can change its valence state under biological conditions). However, metal ions like Cd(II), Hg(II), Al(III), and Pb(II) can also affect redox reactions in indirect ways. Redox-active metal ions like Cu, Fe, and Mn are needed for essential biochemical activities and antioxidant defences such as cytochrome c oxidase and superoxide dismutase 1 (Cu), hemoglobin and cis-aconitase (Fe), and superoxide dismutase 2 (Mn). Because of the problem of inappropriate electron transfer from these metal ions, metalloproteins have highly structured active sites that are small and substrate-specific. Also, all cells have stringent chaperone mechanisms in place to prevent side reactions of these metals with incorrect substrates. This is probably why there is no free Cu or Fe in the cell, rather these metals are always chaperoned [6]. The blood–brain barrier is relatively Met. Ions Life Sci. 1, 1–7 (2006)

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impermeable to passive fluctuation of the major metal ions Zn, Cu, and Fe (e.g., due to prandial or environmental status). Therefore, the etiology of neurodegenerative disease is more likely to be due to homeostatic failure of the endogenous content of metals in the brain than due to environmental or nutritional exposure. However, this important issue has not really been settled with empirical evidence. The brain has the highest metabolic rate of any tissue and has an obligate requirement for aerobic metabolism. This, combined with endogenously high concentrations of Cu and Fe, increase the vulnerability of brain gray matter to radical attack when metal chaperoning is less than perfect. Redox-active metals become available for ROS and radical generation when they either escape their chaperones (usually because the concentration of metal is too high to be completely chaperoned), or because of an accumulation of a damaged metalloprotein that inappropriately permits reaction of the metal with oxygen (Figure 1). Some –

O2 Reducing agents

e–

O2 H2O2 x–1

M

x

M

Mx–1

. OH Metalloprotein aggregates Lipid peroxidation Protein oxidation Protein aggregation DNA/RNA adducts

Figure 1. Reactive oxygen species generation by redox-active metals as the basis for ‘oxidation disorders’. The vast majority of biochemical radicals and ROS arise from the redox chemistry of metals. Dissolved molecular oxygen O2  is liable to react with redox active metals (Mx , representing Cu or Fe ions most commonly, but also occasionally Cd, Hg, and Pb which may indirectly participate in redox reactions). Cu and Fe ions in their reduced state Mx−1  will reduce O2 to superoxide O− 2 , which is then dismutated or disproportionated to H2 O2 , which is freely permeable across lipid membranes, and, if not cleared by scavenging mechanisms (e.g., catalase or glutathione peroxidase) can generate the highly reactive hydroxyl radical OH•  upon reaction with encountered reduced metal Mx−1 . The hydroxyl radical reacts within nanometers and generates a variety of oxidative damage adducts that typify the chemical damage observed in neurodegenerative disorders like Alzheimer’s disease and Parkinson’s disease. In these conditions, the reduced metal ions (especially Cu+ ) are generated by the aggregating protein (e.g., A in Alzheimer’s disease). Biological reducing agents (e.g., cholesterol) are recruited by the A  Cu complex, fostering the catalytic generation of H2 O2 [32]. The catalytic generation of H2 O2 in this manner has been considered to be possible in other neurodegenerative diseases characterized by protein aggregation such as Creutzfeldt–Jakob disease (PrP), Parkinson’s disease (-synuclein), and amyotrophic lateral sclerosis (CuZnSOD). Met. Ions Life Sci. 1, 1–7 (2006)

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metal ions like Hg(II), Cd(II), Pb(II), and Al(III) do not subserve physiological purposes, and accumulate in neurons where the high metabolic rate and oxygen requirement of the tissue make toxic exposure to these metals likely to exhibit a neurological phenotype. We propose an organizational scheme for neurological disease where metal ions are implicated (Table 1). In the last five years the category of neurological Table 1. Organizational framework for the involvement of metals in neurological disease. The disorders listed are confined to instances where there is a prominent or primary neurological phenotype, and does not address secondary syndromes (e.g., due to mineral deficiencies). Metal

Phenotype

1. Genetic disorders of metal metabolism Copper Wilson’s disease Copper Menkes’ disease Iron Neurodegeneration with brain iron accumulation (NBIA, formerly Hallervorden–Spatz disease) Iron Friedreich’s ataxia Iron, Copper Aceruloplasminemia 2. Toxicological Aluminum Cadmium Lead Mercury Manganese

exposure to metal ?Alzheimer’s disease Various Various Various Parkinsonism

Protein

References

Cu7B ATPase Cu7A ATPase Pantothenate kinase 2

[9], Chapter 9 [10,11], Chapter 9 [12,13], Chapter 10

Frataxin Ceruloplasmin

[14], Chapter 10 [15], Chapter 10

? ? Glutathione ? Glutathione ? Glutathione Cis-aconitase

[16], Chapter 13 Chapter 14 Chapter 14 Chapter 14 [17], Chapters 6, 11

3. Protein aggregation disorders involving metals in pathogenesis Zinc, copper Alzheimer’s disease -amyloid [5,18], Chapters Iron, copper Parkinson’s disease -synuclein [19–22], Chapters Copper, zinc Amyotrophic Superoxide [23–25], lateral sclerosis dismutase 1 Chapters Copper, zinc, Transmissible PrP [26–28], manganese spongiform Chapters encephalopathy [29,30] Zinc Drusen, Sorsby’s Tissue inhibitor of fundus dystrophy matrix metalloproteinase-3 (TIMP3) Copper Cataracts -B-crystallin [31]

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disorders of protein aggregation where metals are implicated has grown considerably. A accumulation by zinc in Alzheimer’s disease was the prototypic member of this disorder category [5]. This pathogenic mechanism received much support from recent studies showing that the accumulation of cerebral amyloid in the brains of APP transgenic mice (a model for AD) is markedly reduced by genetic ablation of ZnT3 [7,8], the gene for the zinc transporter responsible for concentrating zinc into synaptic vesicles. While metal-mediated aggregation and toxicity of protein aggregation targets in AD and the other protein aggregation diseases (e.g., prion protein (PrP) in Creutzfeldt–Jakob disease (CJD)) remains controversial, the concepts have increased adherents, and many more neurochemical studies are being published in support of the generic biochemical pathway described in Figure 1. While this model is useful, it will remain controversial until drugs are developed that target these reactions successfully in clinical trials. These drugs are currently being developed and tested, and will be discussed in the terminating chapter of this volume.

ACKNOWLEDGMENTS Supported by funding from the Australian Reseach Council, the National Health and Medical Research Council and the Alzheimer’s Association.

ABBREVIATIONS AD APP A CJD NBIA PrP ROS

Alzheimer’s disease amyloid precursor protein -amyloid Creutzfeldt–Jakob disease neurodegeneration with brain iron accumulation prion protein reactive oxygen species

REFERENCES 1. 2. 3. 4. 5.

A. I. Bush, Curr. Opin. Chem. Biol., 4, 184–191 (2000). A. I. Bush, Trends Neurosci., 26, 207–214 (2003). M. L. Schlief, A. M. Craig, and J. D. Gitlin, J. Neurosci., 25, 239–246 (2005). G. A. Howell, M. G. Welch, and C. J. Frederickson, Nature, 308, 736–738 (1984). A. I. Bush, W. H. Pettingell, G. Multhaup, M. d. Paradis, J. P. Vonsattel, J. F. Gusella, K. Beyreuther, C. L. Masters, and R. E. Tanzi, Science, 265, 1464–1467 (1994). Met. Ions Life Sci. 1, 1–7 (2006)

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6. T. D. Rae, P. J. Schmidt, R. A. Pufahl, V. C. Culotta, and T. V. O’Halloran, Science, 284, 805–808 (1999). 7. J.-Y. Lee, T. B. Cole, R. D. Palmiter, S. W. Suh, and J.-Y. Koh, Proc. Natl. Acad. Sci. USA, 99, 7705–7710 (2002). 8. A. L. Friedlich, J. Y. Lee, T. van Groen, R. A. Cherny, I. Volitakis, T. B. Cole, R. D. Palmiter, J. Y. Koh, and A. I. Bush, J. Neurosci., 24, 3453–3459 (2004). 9. R. E. Tanzi, K. Petrukhin, I. Chernov, J. L. Pellequer, W. Wasco, B. Ross, D. M. Romano, E. Parano, L. Pavone, L. M. Brzustowicz, M. Devoto, J. Peppercorn, A. I. Bush, I. Sternlieb, M. Pirastu, J. F. Gusella, O. Evgrafov, G. K. Penchaszadeh, B. Honig, I. S. Edelman, M. B. Soares, I. H. Scheinberg, and T. C. Gilliam, Nat. Genet., 5, 344–350 (1993). 10. J. F. Mercer, J. Livingston, B. Hall, J. A. Paynter, C. Begy, S. Chandrasekharappa, P. Lockhart, A. Grimes, M. Bhave, D. Siemieniak, and T. W. Glover, Nat. Genet., 3, 20–25 (1993). 11. L. Ambrosini and J. F. Mercer, Hum. Mol. Genet., 8, 1547–1555 (1999). 12. B. Zhou, S. K. Westaway, B. Levinson, M. A. Johnson, J. Gitschier, and S. J. Hayflick, Nat. Genet., 28, 345–349 (2001). 13. P. T. Kotzbauer, A. C. Truax, J. Q. Trojanowski, and V. M. Lee, J. Neurosci., 25, 689–698 (2005). 14. D. C. Radisky, M. C. Babcock, and J. Kaplan, J. Biol. Chem., 274, 4497–4499 (1999). 15. Z. L. Harris, Y. Takahashi, H. Miyajima, M. Serizawa, R. T. MacGillivray, and J. D. Gitlin, Proc. Natl. Acad. Sci. USA, 92, 2539–2543 (1995). 16. T. P. Flaten, Brain Res. Bull., 55, 187–196 (2001). 17. W. Zheng, S. Ren, and J. H. Graziano, Brain Res., 799, 334–342 (1998). 18. C. S. Atwood, R. D. Moir, X. Huang, N. M. E. Bacarra, R. C. Scarpa, D. M. Romano, M. A. Hartshorn, R. E. Tanzi, and A. I. Bush, J. Biol. Chem., 273, 12817–12826 (1998). 19. R. Riederer, E. Sofic, W. D. Rausch, B. Schmidt, G. P. Reynolds, K. Jellinger, and M. B. Youdim, J. Neurochem., 52, 515–520 (1989). 20. N. Ostrerova-Golts, L. Petrucelli, J. Hardy, J. M. Lee, M. Farer, and B. Wolozin, J. Neurosci., 20, 6048–6054 (2000). 21. D. Kaur, F. Yantiri, J. Kumar, J. O. Mo, S. Rajagopalan, V. Viswanath, R. Boonplueang, R. Jacobs, L. Yang, M. F. Beal, D. DiMonte, I. Volitakis, L. Ellerby, R. A. Cherny, A. I. Bush, and J. K. Andersen, Neuron, 37, 923–933 (2003). 22. S. R. Paik, H. J. Shin, J. H. Lee, C. S. Chang, and J. Kim, Biochem. J., 340, 821–828 (1999). 23. A. G. Estevez, J. P. Crow, J. B. Sampson, C. Reiter, Y. Zhuang, G. J. Richardson, M. M. Tarpey, L. Barbeito, and J. S. Beckman, Science, 286, 2498–2500 (1999). 24. M. Kiaei, A. I. Bush, B. M. Morrison, J. H. Morrison, R. A. Cherny, I. Volitakis, M. F. Beal, and J. W. Gordon, J. Neurosci., 24, 7945–7950 (2004). 25. S. M. Lynch, S. A. Boswell, and W. Colon, Biochemistry, 43, 16525–16531 (2004). 26. D. R. Brown, K. Qin, J. W. Herms, A. Madlung, J. Manson, R. Strome, P. E. Fraser, T. Kruck, A. von Bohlen, W. Schulz-Schaeffer, A. Giese, D. Westaway, and H. Kretzschmar, Nature, 390, 684–687 (1997). Met. Ions Life Sci. 1, 1–7 (2006)

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27. K. Nishina, S. Jenks, and S. Supattapone, J. Biol. Chem., 279, 40788–40794 (2004). 28. D. R. Brown, F. Hafiz, L. L. Glassmith, B. S. Wong, I. M. Jones, C. Clive, and S. J. Haswell, Embo J., 19, 1180–1186 (2000). 29. B. H. Weber, G. Vogt, R. C. Pruett, H. Stohr, and U. Felbor, Nat. Genet., 8, 352–356 (1994). 30. S. T. Leu, S. Batni, M. J. Radeke, L. V. Johnson, D. H. Anderson, and D. O. Clegg, Exp. Eye Res., 74, 141–154 (2002). 31. L. E. Goldstein, M. Hartshorn, M. C. Leopold, X. Huang, C. S. Atwood, A. J. Saunders, J. Lim, K. Faget, R. C. Scarpa, L. T. Chylack, E. F. Bowden, R. E. Tanzi, and A. I. Bush, Biochemistry, 39, 7266–7275 (2000). 32. C. Opazo, X. Huang, R. Cherny, R. Moir, A. Roher, A. White, R. Cappai, C. Masters, R. Tanzi, N. Inestrosa, and A. Bush, J. Biol. Chem., 277, 40302–40308 (2002).

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2 Protein Folding, Misfolding, and Disease Jennifer C. Lee, Judy E. Kim, Ekaterina V. Pletneva, Jasmin Faraone-Mennella, Harry B. Gray, and Jay R. Winkler Beckman Institute, California Institute of Technology, Pasadena, CA 91125, USA <[email protected]>

1 INTRODUCTION 2 EXPERIMENTAL METHODS 2.1 Photochemical Electron Transfer Triggers 2.2 Electron Transfer Kinetics 2.3 Fluorescence Energy Transfer Kinetics 3 THE DENATURED STATE 3.1 Hydration and Solvent Accessibility of Zinc-substituted Cytochrome c 3.2 Unfolded Cytochrome c 3.3 Cytochrome c Molten Globule 4 PROTEIN FOLDING DYNAMICS 4.1 Protein Folding Speed Limit: Intrachain Diffusion Dynamics 4.2 Cytochrome c Folding Landscapes 4.2.1 Zinc-substituted Cytochrome c 4.2.2 AEDANS-labeled Cytochrome c 4.2.3 Cobalt-substituted Cytochrome c 4.3 Four-helix Bundles 4.3.1 Cytochrome b562 4.3.2 Cytochrome c 4.3.3 Cytochrome c556 and c-b562

Metal Ions in Life Sciences, Volume 1 © 2006 John Wiley & Sons, Ltd

Edited by Astrid Sigel, Helmut Sigel and Roland K. O. Sigel

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1 INTRODUCTION The production of functional proteins requires that polypeptides find a unique conformation in a vast space of incorrect folds [1]. The consequences of failure are severe; misfolded proteins are implicated in a rapidly growing list of debilitating neurodegenerative illnesses that includes Alzheimer’s, Parkinson’s as well as Creutzfeldt–Jakob diseases [2]. To ensure that proteins reach their native states and avoid potentially toxic aggregates, cells have evolved a complex machinery of molecular chaperones that assists the folding of nascent polypeptides and rescues proteins from stressinduced denaturation [3]. Partially folded polypeptide structures are key intermediates in both the proper assembly of proteins, and in the formation of harmful misfolded structures [4–8]. Characterizing the structures, energetics, and dynamics of these transient species is an essential step in understanding their benign and malignant pathways.

2 EXPERIMENTAL METHODS Elucidation of key events in protein folding calls for investigations on time scales that range from picoseconds to minutes (Figure 1). The fastest nuclear motions in proteins, rotations about single bonds, occur on the picosecond time scale [9]. Short segments of helical structure can be formed in nanoseconds [10–12], whereas the large scale, collective motions associated with the development of tertiary structure fall in the microsecond to millisecond range [13–15]. Misfolded structures or traps are frequently encountered in folding processes; escape from these traps (e.g., proline isomerization) can take seconds or even minutes [16,17]. Formation of partially folded structures, the so-called ‘burst’ intermediates observed during a stopped-flow mixing deadtime, has been the most challenging to study because it occurs on a submillisecond time scale [13,14,18–21]. Identification and characterization of these compact structures are critical in Met. Ions Life Sci. 1, 9–60 (2006)

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DYNAMICS unfolded protein side-chain rotations

10 –12

helix formation

10 –10

10 –8

fluorescence anisotropy

hydrophobic collapse intrachain diffusion

10 –6

10 –4

molten globule ligand substitution

10 –2

folded protein proline isomerization

10 0

10 2 seconds

ultrafast mixing laser T-jump

stopped-flow T-jump photochemistry

METHODS Figure 1. Time regimes and experimental methods employed for the study of protein folding dynamics.

understanding the role of hydrophobic collapse during protein folding. Development of rapid folding triggers (e.g., ultrafast mixers and photochemical methods) is necessary for studies on these fast time scales [10,22–25]. Understanding the key events in folding and identifying any partially folded intermediates are major goals of theoretical [5,18,26–29] and experimental [20,22,30–34] work. Without detailed maps of folding energy landscapes, it is uncertain at what stage the conformational heterogeneity of the unfolded ensemble is lost. This issue is difficult to resolve experimentally; folding probes must report not just the average progress of the unfolded ensemble toward the folded state, but on the ensemble heterogeneity as well. In our research program, we have utilized electron transfer (ET) [35–46] and fluorescence energy transfer (FET) [47–52] techniques to trigger and probe the folding of redox-active proteins.

2.1 Photochemical Electron Transfer Triggers The basic requirement for experimental investigations of protein folding dynamics is some means of triggering the folding process. We have developed a new method for initiating protein folding using ET chemistry [35]. ET triggered folding is based on the observation that the reduction potentials of redox cofactors in the hydrophobic interiors of folded proteins often are shifted dramatically Met. Ions Life Sci. 1, 9–60 (2006)

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ΔG°ox Uox

Fox

E°U

EF°

Ured

ΔG°red

Fred

U

Pox

e–

Pred

F [Denaturant]

Figure 2. Thermodynamic cycle illustrating the relationship between the reduction potentials of redox cofactors (where F = folded; U = unfolded; ox = oxidized; red = reduced) and the free energy of folding (upper). Representative denaturation titration curves for oxidized Pox  and reduced Pred  forms of a redox-active protein with Ef > 0 eV. Under these conditions, rapid electron injection into Pox will initiate a folding reaction (lower).

from their aqueous solution values [53]. A simple thermodynamic cycle can be drawn connecting an oxidized Pox  and reduced Pred  protein in both folded and unfolded states (Figure 2). If the active-site reduction potentials are different for the folded and unfolded proteins Ef = EF − EU , then the folding free energies of the oxidized and reduced proteins will differ by a comparable amount Gf = −nFEf  [35,54]. The shift in the reduction potential reflects the differential stabilities of the oxidized and reduced folded proteins. If the difference between the reduction potentials is sufficiently large and their folding free energies depend similarly on denaturant concentration, it is possible to find denaturant conditions where one oxidation state of the protein is fully folded while the other is unfolded (Figure 2). Most heme proteins have Ef > 0; thus, Met. Ions Life Sci. 1, 9–60 (2006)

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Pred

Pox

pMDMA

pMDMA•+

2+

+

*Ru(bpy)3

Ru(bpy)3

hν λex = 480 nm

Pox

2+

Ru(bpy)3

Pred

2+

*Ru(bpy)3

+

Ru(bpy)3 10–7

10–5

10–3

10–1

101 seconds

– NAD• + eaq

NADH

hν

hν

λex = 355 nm

– NAD• + eaq

Pox

Pred NAD +

H+

Figure 3. Reversible (upper) and irreversible (lower) reaction schemes for photochemical ET triggered protein folding (Pox and Pred are oxidized and reduced proteins). Time regimes for different photochemical sensitizers also are illustrated.

in the denaturant concentration region that lies between the two unfolding transitions, electron injection into the oxidized protein will lead to the formation of the reduced folded state. One very attractive feature of this technique is that the initiation of the folding reaction is only limited by the ET time. There is a vast array of photoactive complexes that can rapidly inject and remove electrons from proteins on time scales as short as a few hundred nanosec2+ ∗  onds. Electronically excited Rubpy2+ 3  Rubpy3  bpy = 22 -bipyridine is 3+/∗2+  a powerful reductant E Ru  = −085 V vs NHE and its 600-ns decay time makes it an excellent reagent for triggering folding reactions on the Met. Ions Life Sci. 1, 9–60 (2006)

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microsecond time scale [35]. Furthermore, the millisecond-time-scale reoxidation of the reduced protein by Rubpy3+ 3 regenerates the initial species and permits extensive signal averaging. However, the folding observation window is limited to the 1–1000 s range (Figure 3) and often the yield of electron injection into an unfolded oxidized protein is low. Reductive quenching with p-methoxy-N,N dimethylaniline (pMDMA) provides an alternative photoreductant, Rubpy+ 3, that permits a tenfold increase in injection yield with only a modest sacrifice in injection time scale ∼10 s (Figure 3) [41]. Complete refolding of a protein can require tens to hundreds of milliseconds. Consequently, irreversible photochemical ET reagents are required to study the entire range of folding dynamics. We have found that NADH is an effective irreversible photochemical sensitizer for injecting electrons into unfolded proteins [37,40,42,46]. Two-photon, 355-nm excitation of this reagent generates two powerful reductants, a solvated electron and NAD• [55]; both reductants reduce unfolded heme proteins ∼100 M in about 100 s. The combination of ∗ Rubpy2+ 3 and NADH permits investigations of 1 s to >1 s folding events of heme proteins (Figure 3).

2.2 Electron Transfer Kinetics The reactivity of a polypeptide can be an extremely sensitive indicator of structural heterogeneity. With a carefully selected probe reaction, a bimodal distribution of protein conformations would exhibit biphasic kinetics, whereas a

Q

Q Q

Q

Q

Q Q

Q

Q

τfold < 1 s

Q

Q Q

Q

Q

Q

Q Q

Q

Q

Q Q

Figure 4. Differences in hydration and solvent accessibility of the heme cofactor in unfolded and folded cytochrome c (quenchers (Q) are represented by gray circles, denaturants are represented by green circles, and water molecules are represented by blue circles); in the unfolded polypeptide the heme cofactor is more solvent-exposed and hydrated, whereas in the native protein, the heme is embedded in the protein interior protected from quenchers. Met. Ions Life Sci. 1, 9–60 (2006)

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single-mode distribution would react in a single phase. The key requirement is that the probe reaction be fast compared to the time scale of folding. ET reactions could be excellent folding probes because rates at high driving forces are determined by the distance and medium separating the two redox partners [56]. Buried redox centers in proteins often exchange electrons rather slowly with reagents in solution. Unfolding will greatly increase the accessibility of a redox cofactor and can lead to much faster ET [43,45] (Figure 4). Compact intermediates might be expected to exhibit ET rates somewhere between those of the folded and unfolded molecules.

2.3 Fluorescence Energy Transfer Kinetics A heterogeneous ensemble of unfolded proteins should exhibit a broad, nearly Gaussian distribution of end-to-end distances, consistent with a large number of degenerate conformations and, hence, a high configurational entropy [57]. Proteins in their native states, by contrast, have definite three-dimensional orientations of their peptide backbones and, to a certain extent, the side chains as well. Most spectroscopic probes of protein folding report on average properties of the ensemble and provide little insight into its heterogeneity. Furthermore, in order to unravel the mechanism of protein folding, an effective probe must not only provide information on the populations of unfolded, intermediate, and folded forms, but also should report on the structural heterogeneity of these conformers during the folding process. FET measurements are particularly sensitive to the structural heterogeneity in polypeptides. Dipole–dipole energy-transfer rates kEnT  depend on the inverse sixth power of the distance r between a fluorescent donor (D) and energy acceptor (A) (Eq. (1)) [58]. A donor–acceptor (DA) pair is characterized by a Förster critical length ro , which is determined by , the orientation factor, n, the index of refraction, D , quantum yield of the donor, and J , overlap integral between donor emission and acceptor absorption spectra (Eq. (2)). Under typical conditions, FET rates can be measured for DA distances in the range 03ro ≤ r ≤ 15ro . Depending on the chromophores used, DA separations as long as 100 Å can be evaluated [59,60]. Energy transfer partners incorporated into a single biomolecule will furnish site-specific conformational detail. kEnT = ko

 r 6 o

r ro ∝  2 n−4 D J1/6

(1) (2)

The distribution of distances between DA-labeled residues in a polypeptide can be extracted from an analysis of FET kinetics [61]. An ensemble of unfolded Met. Ions Life Sci. 1, 9–60 (2006)

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U

P(k)

P(r)

I(t)

F distance

rates

time

Figure 5. Schematic representation of the relationship between polypeptide conformations and fluorescence decay kinetics. An ensemble of unfolded proteins found near the top of a funnel-shaped landscape (left) will have a broad distribution of distances Pr between fluorescence energy transfer donors and acceptors. The distance distribution function can be transformed to a distribution over fluorescence decay rates k, producing a slowly decaying fluorescence intensity profile It. An ensemble of folded proteins (bottom of the funnel) will have a narrow distance distribution, and faster excited-state decay. In FET kinetics measurements, the fluorescence intensity profiles are transformed into distance distributions.

polypeptides should have a broad DA distribution Pr (Figure 5), with a mean distance that increases with the number of residues between D and A. The many different distances in this ensemble produce a distribution of fluorescence decay rates Pk and a highly nonexponential decay profile It (Figure 5). By contrast, a folded protein will have a narrow Pr range, a smaller value of r, and faster, albeit still nonexponential decays. By monitoring FET kinetics during the entire course of protein folding, we are able to map DA distance distributions from the unfolded ensemble to the native state [48].

3 THE DENATURED STATE Denatured proteins contain rapidly exchanging polypeptide conformers. In early studies by Tanford and coworkers, several proteins at high concentrations of guanidine hydrochloride (GuHCl) were found to exhibit the hydrodynamic properties of random coils [62]. More recent studies also report random-coil behavior for some proteins and long peptides [63–65]. However, there are many unfolded proteins that retain substantial amounts of internal structure, even at high concentrations of denaturant [66]. In order to gain a detailed picture of the unfolded state, we have employed ET [43,45] and FET [49,50] kinetics measurements Met. Ions Life Sci. 1, 9–60 (2006)

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to elucidate the conformational heterogeneity within the cytochrome c (cyt c) polypeptide ensemble.

3.1 Hydration and Solvent Accessibility of Zinc-substituted Cytochrome c Solvent-mediated interactions often play key roles in protein folding reactions. Both folded and unfolded structures are associated with water molecules [67,68], and chemical denaturant activity is attributed to changes in water hydrogen bonding networks as well as direct solvation of peptide bonds and hydrophobic residues (Figure 4) [69,70]. Importantly, water and protein interactions are crucial for native structures [71], protein–protein recognition [72], and folding pathways [73–76]. Computational studies have suggested that the expulsion of water from the hydrophobic protein cavity could be an important step during folding. Molecular dynamics simulations reveal that rapidly-formed, collapsed structures trap a significant number of water molecules inside the protein [74]. Similarly, a theoretical model incorporating solvent effects found that the last step in protein folding involves ‘squeezing out’ water molecules from a partially hydrated polypeptide core [73]. While these computational studies have aimed to elucidate the role of water in protein structure and folding, very few experiments have probed the dynamics of solvation during a folding reaction. Cofactor solvation in folded and unfolded protein states has been investigated using Zn(II)-substituted cyt c, which is structurally similar with comparable folding free energy to the native protein (Fe-cyt c) [43,77,78]. The key advantage of the Zn-protein is the availability of a long-lived  ∼ 15 ms, powerfully reducing E = −08 V vs NHE triplet excited state ∗ Zn-cyt c [79,80]. Because the deactivation of ∗ Zn-cyt c is strongly dependent on outer sphere solvation, high frequency O–H vibrations facilitate nonradiative decay. Significant deuterium isotope effects on decay rates have been reported because O–D stretches are less efficient acceptor modes [81,82]. Another channel for triplet state deactivation involves electron transfer to an external redox reagent, such as RuNH3 3+ [80], producing Zn-cyt c•+ and RuNH3 2+ (Figure 6). Since 6 6 the degree to which there is an isotope effect or efficient triplet decay in the presence of quenchers directly reflects the extent of hydration and solvent accessibility of the Zn-porphyrin (Figure 4), each of these photophysical properties reports on the heme solvent environment of various conformational states of cyt c [43,45]. The cofactor in folded Zn-cyt c is partially hydrated, as indicated by the modest, yet reproducible, isotope effect kH2 O /kD2 O of 1.2 (Figure 7) [45]. This isotope effect is smaller than that observed for the fully exposed Zn-porphyrin in microperoxidase-8 (Zn-MP8), a heme octapeptide derived from enzymatic Met. Ions Life Sci. 1, 9–60 (2006)

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Ru3+ *Zn-cyt c Ru2+

Zn-cyt c•+

hν

Ru2+ Zn-cyt c Ru3+

Figure 6. Reaction scheme for bimolecular electron transfer quenching of triplet Zn-cytochrome c by RuNH3 3+ 6 .

NaPi H2O

D2O

Absorbance

5 M GuHCl H2O

D2O

9 M Urea

H2O

10–5

10–4

D2O

10–3 seconds

10–2

Figure 7. Transient absorption decay kinetics of triplet Zn-cyt c in protonated and deuterated solutions. Buffers were 20 mM phosphate buffer (top) with 5 M GuHCl (middle) or 9 M urea (bottom); solutions were pH = pD = 74. Met. Ions Life Sci. 1, 9–60 (2006)

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cleavage of cyt c [83] kH2 O /kD2 O ∼ 15, and the triplet lifetime of the folded protein  ∼ 10 ms is much longer than that for Zn-MP8  ∼ 300 s [45]. The difference in lifetimes arises because the folded protein restricts solvent access to the cofactor (Figure 4). The observed isotope effect may be explained by the presence of water molecules in the Zn-porphyrin binding pocket of the folded protein [84] combined with partial cofactor exposure to the bulk solvent. The observation that numerous H2 O molecules occupy conserved positions in a variety of cytochromes c indicates that these solvent molecules are critical for protein function [84]. Hydration of the cofactor in Zn-cyt c increases dramatically upon protein unfolding (Figure 4). The lifetime of the porphyrin triplet changes by nearly an order of magnitude, from ∼10 ms (folded) to ∼13 ms (unfolded) (Figure 7) [43,45]. This change in lifetime cannot be attributed solely to the change in heme–solvent vibronic coupling; our studies with Zn-MP8 indicate that a change in cosolvent only affects triplet lifetimes by a factor of ∼3 [45]. Instead, the decreased lifetime reflects enhanced Zn-porphyrin solvation, and this interpretation is further supported by the larger isotope effect for unfolded kH2 O /kD2 O = 14 relative to folded kH2 O /kD2 O = 12 protein [45]. Furthermore, by comparison to Zn-MP8 (kH2 O = 7000 s−1 in GuHCl), the triplet state decays nearly 10 times more slowly, suggesting that the cofactor is only partially exposed in the unfolded polypeptide (kH2 O = 810 s−1 in GuHCl) [45]. The presence of this shielding supports the notion that unfolded proteins are not random polymers, but instead may adopt partial structure even under fully denaturing conditions [66,85–88]. An unfolded structure may feature hydrophobic clustering [86], native-like topology [85], or in the case of heme proteins, well-defined heme ligation geometry [87]. The existence of such partially structured unfolded states with locally buried regions also can be inferred from the results of bimolecular ET quenching experiments with RuNH3 3+ 6 (Figure 6). The reported quenching rate constant measured under native conditions is 14 × 107 M−1 s−1 [80]. Under denaturing conditions  GuHCl > 3 M, the quenching reaction is 100 times faster 14 × 109 M−1 s−1 , GuHCl = 35 M than that measured in the absence of GuHCl [43]. The quenching rate is very high, owing to the greater accessibility of the Zn-porphyrin in the unfolded protein. The ∗ Zn-cyt c ET kinetics are generally consistent with a two-state unfolding process; and the unfolding isotherm generated from ET kinetics exhibits a transition midpoint at 2.8(1) M GuHCl, in good agreement with those obtained from far-UV CD and heme absorption measurements. A molecular description of the mechanism of chemical denaturation is a goal of much current research [70,89–91]. For Zn-cyt c, the free energies of unfolding are comparable in urea Gf = −31 kJ/mol and GuHCl Gf = −35 kJ/mol. In high urea and GuHCl concentrations Zn-cyt c exhibits similar triplet lifetimes (800 s−1 in GuHCl; 700 s−1 in urea) and isotope effects (kH2 O /kD2 O = 14 Met. Ions Life Sci. 1, 9–60 (2006)

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in GuHCl and urea) [45], suggesting nearly identical solvent exposure of the Zn-porphyrin. In contrast to fully denatured protein, the nature of the compact species depends on denaturant. Bimolecular quenching of the triplet Zn-porphyrin in the compact state in urea is ∼7 times slower than in GuHCl (59 × 107 M−1 s−1 in 2.7 M GuHCl; 86×106 M−1 s−1 in 6.7 M urea), and this difference likely reflects greater protection of the porphyrin group from the aqueous urea solvent [45] as well as electrostatic effects, since it has been shown that triplet Zn-cyt c is sensitive to ionic strength and quencher charge [92,93]. However, the bimolecular quenching rates observed here are similar to those reported for folded and molten globule states of Zn-cyt c [93], supporting our interpretation that the rate differences are attributable to variations in the equilibrium GuHCl- and urea-induced compact structures. It is possible that these partially unfolded species are similar to the intermediates associated with the burst phase of cyt c folding [31,43,48,77,94]. The compact structure likely represents this partially unfolded intermediate as well, and the finding that the structure of this species is denaturant-dependent supports the notion of nonnative heme environments for this compact, partially unfolded species. The observation of more collapsed nonnative structures in urea may reflect the relative strengths of these two widely used denaturants. There is general consensus that chemical denaturants act through a combination of direct binding to the peptide backbone and side chains, and by altering the hydrogen bonding network of water in a structure-making or structure-breaking manner, thereby diminishing the hydrophobic effect [69,70]. It is well established that the transfer free energy for the peptide group from water to aqueous solutions of urea or GuHCl is favorable, with GuHCl being a better solvent for the peptide backbone than urea [95]. In contrast, side chains show wide variability in transfer free energy to denaturant solutions; however, for a given denaturant concentration, GuHCl is more effective in its ability to solvate both hydrophobic and hydrophilic residues [96,97]. Studies on charge effects suggest, not surprisingly, that denaturation by urea is more sensitive to protein charge and ionic strength than GuHCl [90,91,98,99]. Collectively, these reports indicate that GuHCl is more effective than urea in its ability to disrupt and solvate hydrophobic pockets of folded proteins, in accord with the finding that the Zn-porphyrin in the compact state of Zn-cyt c is more exposed to solvent in GuHCl than in urea. It is clear that Zn-porphyrin triplet lifetimes are sensitive indicators of hemepocket hydration. The finding that folded and unfolded species have different isotope effects and bimolecular quenching rates indicates that changes in hydration can be monitored during a folding reaction (Figure 4). Important issues such as the time scales for water expulsion, the nature of dehydrated and hydrated intermediates, and even the effect of denaturant on folding pathways can be addressed by means of triplet lifetime measurements. Met. Ions Life Sci. 1, 9–60 (2006)

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3.2 Unfolded Cytochrome c FET kinetics measurements have been employed to characterize the conformational heterogeneity in unfolded yeast cyt c [49,50]. Six different Cys variants were engineered by site-directed mutagenesis [50] and labeled with the thiol-reactive fluorophore 5-((((2-iodoacetyl)-amino)-ethyl)-amino)naphthalene-1-sulfonic acid (AEDANS) (Figure 8, inset). FET kinetics were measured in each of the six DA proteins (K4C, H39C, D50C, E66C, L85C, K99C) (Figure 8) with AEDANS and heme ro = 39 Å serving as the fluorescent donor and energy acceptor, respectively [49,50]. The AEDANS fluorescence decay kinetics are nonexponential for all variants, revealing multiple conformations of the polypeptide segments between D and A in the unfolded protein. To test whether the heterogeneous unfolded ensemble of cyt c can be described as a randomly configured polypeptide, the data were fit to the simplest polymer model, a freely jointed chain. This model, which produces a distance distribution with a single adjustable parameter, the mean-squared end-to-end distance [100], does not provide good fits to the FET kinetics. A less restrictive empirical

Cys

H N HN

O

4 –

99

85

SO3

66 H18 39

M80

50

Figure 8. Structure of yeast iso-1 cytochrome c (PDB 1YCC) showing the AEDANS labeling positions (K4C, H39C, D50C, E66C, L85C, K99C). Inset: molecular structure of the AEDANS fluorophore. Met. Ions Life Sci. 1, 9–60 (2006)

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model assuming a Gaussian distribution of DA distances characteristic of random coils [64] also poorly approximates the experimental data. Moreover, the extracted distributions exhibit peculiar trends; these distributions do not show the linear dependence of the mean and width of the distribution on the number of residues separating DA that is expected for a random coil [100]. Given the poor quality of the fits and the peculiar trends in the distance distributions, it is apparent that the range of DA distances in unfolded cyt c is not adequately described by a single Gaussian distribution. In order to characterize the ensemble heterogeneity, the distance distributions (P(r)) were extracted directly from the AEDANS fluorescence decays (Figure 9). A maximum-entropy (ME) analysis was chosen as the least biased approach [101] and gave consistently better fits than the random coil distributions. ME fits to the FET kinetics data for folded variants yield DA distances that are consistent with C −Fe distances determined from the crystal structure [102]

AEDANS(C4)

AEDANS(C39)

AEDANS(C50)

0 M GuHCl

0 M GuHCl

2.8 M GuHCl

2.7 M GuHCl

2.7 M GuHCl

5.6 M GuHCl

5.4 M GuHCl

4.4 M GuHCl

P (r )

0 M GuHCl

10 20 30 40 50 ≥60 10 20 30 40 50 ≥60

P (r )

AEDANS(C66)

AEDANS(C85)

10 20 30 40 50 ≥60 AEDANS(C99)

0 M GuHCl

0 M GuHCl

0 M GuHCl

2.8 M GuHCl

2.9 M GuHCl

2.9 M GuHCl

5.9 M GuHCl

5.7 M GuHCl

5.8 M GuHCl

10 20 30 40 50 ≥60 10 20 30 40 50 ≥60 10 20 30 40 50 ≥60 distance, Å

Figure 9. Distributions of DA distances, P(r), from maximum-entropy analyses of the AEDANS-labeled cyt c fluorescence decay kinetics in the presence of GuHCl. Met. Ions Life Sci. 1, 9–60 (2006)

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with a 6–8 Å increase due to the linker (Figure 8, inset). Unfolding with GuHCl affects differently the distance distributions of the six AEDANS-labeled variants (Figure 9). For AEDANS(C4)-cyt c, P(r) broadens somewhat and moves to slightly longer distances (Figure 9). This labeling site remains close to the heme, even in the unfolded protein. The broadening of the P(r) distribution likely reflects the transition to a less ordered state. For the other variants, addition of GuHCl leads to more extended polypeptide structures (Figure 9). Interestingly, even at very high GuHCl concentrations where the protein would be expected to be completely unfolded, a significant fraction of the polypeptide ensemble retains short DA distances. At pH 7, His26, His33, and His39 can bind to the heme in the denatured state [103,104], as well as the N-terminus, replacing the native Met80 ligand (Figure 8) [105]. Heme misligation retards cyt c refolding and formation of the native Met80-iron bond is rate-limiting. Although heme misligation produces some compact states in the N-terminal sites, AEDANS(C4) and AEDANS(C39),

P (r )

AEDANS(C4)

AEDANS(C39)

2.8 M GuHCl

2.8 M GuHCl

+ 0.15 M Im

+ 0.15 M Im

10

20

30

40

50

≥60 10

20

P (r )

AEDANS(C50)

30

2.8 M GuHCl

+ 0.15 M Im

+ 0.15 M Im

20

30

40

50

50

≥60

AEDANS(C66)

2.8 M GuHCl

10

40

≥60 10

20

30

40

50

≥60

distance, Å

Figure 10. Effects of added imidazole ([Im]) on DA distances distributions (P(r)) in AEDANS-labeled variants (C4, C39, C50, C66) in 28±01 M GuHCl at pH 7. Met. Ions Life Sci. 1, 9–60 (2006)

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it is not the only source of structural complexity in the unfolded protein (Figure 10). Interestingly, the polypeptide regions that show large populations of compact structures are located in the middle of the protein sequence (C39, C50, C66) and their proximity to the heme (linked to the polypeptide via Cys14 and Cys17 [102]) is likely caused by their interactions with this prosthetic group. The existence of residual structure in denatured proteins is well established [66,85,86,106–114]. Under mildly denaturing conditions, cyt c is a compact molten globule with a covalently bound heme [47,115,116], but the apoprotein is unstructured. Even noncovalent binding of heme to the apoprotein produces a compact structure [117]. In addition, studies of synthetic heme– peptide complexes provide compelling evidence for noncovalent bonding interactions between hydrophobic amino acid residues and hemes [118,119]. There are several hydrophobic residues in the vicinity of labeling sites 39, 50, and 66, and these hydrophobic residues may interact preferentially with the heme in unfolded cyt c. Refolding studies of iso-1 cyt c spin-labeled at positions 39, 50, and 66 revealed very large burst phase components [120], thereby indicating that interactions of this region with the heme promote hydrophobic core formation during the folding process.

3.3 Cytochrome c Molten Globule Under mild denaturing conditions, many proteins adopt a nonnative state characterized by compactness, a native-like secondary structure, but the absence of rigid tertiary structure [20,21,115,116]. It has been proposed that this molten globule state is a common intermediate that occurs early in the folding pathways of all globular proteins [21]. Recently, it has been suggested that in some cases molten globules correspond to late-folding intermediates [121]. Cytochrome c was the first protein in which a molten globule state was detected at low pH in the presence of high salt concentration [122]. This conformational transition from acid-unfolded protein to the globular state is mediated by anion binding [115] and depends strongly on the size of the added anion [123]. In addition, polyols [124] and some alcohols [125,126] can produce the molten globule state of cyt c at low pH. The Saccaromyces cerevisiae iso-1 cytochrome c molten globule has been probed using FET kinetics [47]: AEDANS-modified Cys102 fluoresces intensely when the protein is unfolded, but is strongly quenched by energy transfer to the heme in compact conformations. As expected, the molten globule has very similar helix content to the native state, whereas the acid-denatured protein exhibits no secondary structure. CD spectroscopy confirms the absence of tertiary structure for both the acid-denatured protein and the molten globule  SO2− 4 > 07 M [115]. Far-UV CD spectra show that the AEDANS(C102)-cyt c molten Met. Ions Life Sci. 1, 9–60 (2006)

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globule thermal unfolding transition at pH 2 is cooperative [47], in excellent agreement with related work on the unmodified molten globule [127]. DA distance distributions in AEDANS(C102)-cyt c have been obtained under conditions favoring the molten globule [47]. At salt concentrations of 100 mM or lower, the polypeptide ensemble is highly heterogeneous: 50% of the polypeptides are in extended conformations r > 35 Å; the remaining 50% are in compact conformations r < 35 Å (Figure 11). As the salt concentration is increased further, the fraction of polypeptides in extended conformations decreases in favor of compact structures, but both populations remain heterogeneous. At high salt concentrations ≥700 mM, all of the polypeptides are compact with DA distances between 25 and 30 Å (Figure 11). The structural homogeneity of folded proteins can be disrupted by a variety of chemical and physical perturbations. FET kinetics measurements on AEDANS(C102)-cyt c reveal a large degree of such heterogeneity in the aciddenatured protein. High salt concentrations convert the observed complex mixture of conformations into an ensemble of compact r < 35 Å polypeptides with a mean DA separation quite close to that of the native protein 25 Å. This molten globule is somewhat more compact and far more homogeneous than the ensemble of polypeptides in the burst intermediate formed during

50 mM Na2SO4

330 mM Na2SO4

P (r )

450 mM Na2SO4

700 mM Na2SO4

folded

20

30

40

50

≥60

distance, Å

Figure 11. Structural features of the cytochrome c molten globule. Na2 SO4 -induced changes in the distribution of AEDANS(C102)–heme distances (P(r)) at pH ∼2 22  C. Met. Ions Life Sci. 1, 9–60 (2006)

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cyt c folding [48,94,128–130]. A clear transition between the acid-denatured and molten globule forms of cyt c is not apparent from most ensemble-averaged spectroscopic probes (UV-vis absorption, CD, fluorescence intensity). FET kinetics, however, provide definitive evidence for the formation of a uniformly compact molten globule at salt concentrations greater than 700 mM. It remains to be determined if polyols, alcohols, and other molten-globule stabilizing agents [131–133] are as effective as anions in shifting the collapsed/extended equilibrium to a position in which compact states are dominant.

4 PROTEIN FOLDING DYNAMICS Proteins often fold quite rapidly (< 1 s) because the energy bias on a funnelshaped landscape steers unfolded peptides toward native conformations [5,134]. However, proteins do not fold by randomly searching a large number of nearly degenerate configurations (Levinthal’s paradox) [135]; instead, an ensemble of unfolded molecules must traverse a complicated energy landscape to reach a thermodynamically stable structure [1,5,29,134]. The configurational entropy of a polypeptide, represented by the lateral dimension on a folding energy landscape, is minimized at the native conformation. The high energy portion of the conformational landscape represents a heterogeneous ensemble of unfolded polypeptides; partially folded conformations appear as local minima on this energy surface and misfolded structures can be in deep energy wells that are separated from the native minimum (Figure 12). In order to map this complex energy landscape, it is necessary to probe structural features of the polypeptide ensemble as it evolves to the native state.

4.1 Protein Folding Speed Limit: Intrachain Diffusion Dynamics The formation of tertiary contacts during protein folding is recognized as a pivotal step in the formation of native structure. It is clear that proteins can fold no faster than the rate at which native tertiary contacts are made. The time scales of tertiary contact formation in peptides and proteins have been investigated both experimentally [41,136–143] and theoretically [144–149]. Statistical mechanical models suggest that the probability and rate of forming transient tertiary contacts in random polymers are determined largely by two factors: the coefficient for intrachain diffusion and the size of the resultant loop [144–146]. The most probable loop formed in a random polypeptide is estimated to be comprised of 10 residues [146]. Experimental investigations of tertiary contact formation in proteins and polymers have been aimed at determining the upper limit for protein folding Met. Ions Life Sci. 1, 9–60 (2006)

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Energy

PROTEIN FOLDING, MISFOLDING, AND DISEASE

I1

I2

I3

N

M Conformation Figure 12. Idealized protein folding energy landscape. The configurational coordinate of the unfolded ensemble is represented by the lateral dimension. Unfolded polypeptides must traverse a complicated landscape to reach the native protein (N); partially folded intermediates (I) provide kinetics complexity in the folding process and misfolded (M) structures can trap proteins in deep energy wells.

rates [136–138]. The 40- s timescale for methionine (Met65 and Met80) binding to the ferroheme in denatured cytochrome c suggested a ∼106 s−1 folding speed limit [136]. Subsequent investigations of ET quenching of triplet excited tryptophan by cysteine in synthetic peptides led to a value closer to 107 s−1 [139]. This larger value is in line with estimates based on triplet energy transfer in dye-labeled peptides [143]. These experimental determinations of polymer contact times exploit measurements of the rates of reactions with very small intrinsic barriers. ET reactions are well suited to investigations of this type because the barriers can be minimized by tuning the reaction driving force [150–154]. In prior studies, we measured ET kinetics and identified likely tunneling pathways in Zn-cyt c labeled at moiety [80,155–157]. The dominant surface His residues with a RuNH3 3+ 5 electronic coupling pathway between the redox centers in RuNH3 5 (His33)Zn-cyt c (Ru(His33)-Zn-cyt c) has 15 covalent bonds and one H-bond between Met. Ions Life Sci. 1, 9–60 (2006)

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the carbonyl oxygen of Pro30 and the proton on the N atom of His18 [156]. Chemical denaturants will disrupt this pathway, leaving only the 15-residue covalent link between His33 and the Zn-coordinated His18 residue. Electron tunneling along this 45-bond covalent pathway is extremely unfavorable [44,157]. ET from the Zn-porphyrin triplet ∗ ZnP to the RuHis333+ complex in the denatured protein, then, will likely be an adiabatic process within an encounter complex formed by intrachain diffusion to bring the two redox complexes into van der Waals contact. In the presence of denaturant, then, ∗ ZnP → RuHis333+ ET should be a diffusion-limited process and the time scale for the reaction will set the speed limit for formation of a ∼15-residue loop in an unfolded polypeptide. In the native protein, the ∗ ZnP → RuHis333+ ET rate constant is 75 × 105 s−1 [80]. Denaturation of Ru(His33)-Zn-cyt c produces species in which intramolecular ET is faster than the bond-mediated tunneling process in the folded molecule [44]. Unfolded Ru(His33)-Zn-cyt c ET kinetics are dominated by a component (83%) with a 250-ns lifetime. Our measured 250-ns time constant for formation of a ∼15-residue loop  GuHCl = 54 M in Ru(His33)-Zn-cyt c demonstrates that tertiary contact formation can occur very rapidly in denatured cyt c [44]. If tertiary contact formation is diffusion limited, then the statistical mechanical model predicts that the most probable loop in denatured cyt c will form in ∼65 ns. This value agrees well with measurements of end-to-end contact formation in small n = 3–19 peptides [136,139]. Taken together, these measurements suggest that a 10-residue polypeptide loop will form in ∼100 ns, which sets the speed limit for protein folding near 107 s−1 .

4.2 Cytochrome c Folding Landscapes 4.2.1 Zinc-substituted Cytochrome c The folding energy landscape of cytochrome c has been investigated by exploiting the widely different ET reactivities of buried and exposed zinc-substituted hemes [43]. Zn-cyt c is structurally homologous to the iron protein, which has been studied in great detail [94,129,136,158]. A nonnative axial ligand (His26 or His33) replaces Met80 at neutral pH in denatured equine Fe-cyt c [121]; the rate-limiting folding step in this case is correction of heme misligation. Owing to weaker binding and faster substitution at the sixth coordination site, replacement of Fe with Zn will eliminate axial ligand traps during refolding [159]. Addition of GuHCl to solutions of Zn-cyt c produces a blue shift in the Soret absorption band [43], giving a species with a spectrum similar to that of Zn(II)substituted myoglobin [160]. GuHCl unfolding curves generated from UV-vis absorption and far-UV CD spectra show that the stability of folded Zn-cyt c is comparable to that of the Fe(III) form [36,161]. In contrast to Fe-cyt c, the zinc Met. Ions Life Sci. 1, 9–60 (2006)

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center in the unfolded protein is not coordinated by any peptide side chain other than the native His18 [159]. Changes in Zn-cyt c Soret absorption after stopped-flow dilution of denaturant were examined: the transient absorption kinetics are exponential functions and the observed rate constants depend linearly on denaturant concentrations. The extrapolated time constant for refolding in the absence of denaturant is about a millisecond. The Zn-cyt c folding rate is about 10 times higher than that of the Fe(III) protein at comparable driving forces [94,129], consistent with the absence of heme misligation. More complex Zn-cyt c folding becomes apparent when the process is probed with transient absorption measurements of ∗ Zn-cyt c/RuNH3 3+ 6 ET kinetics. As the polypeptide chain folds around the porphyrin, the ∗ Zn-cyt c ET rate decreases from its value in the unfolded protein ∼7 × 106 s−1  RuNH3 3+ 6  = 3–5 mM to that characteristic of folded molecules ∼25 × 105 s−1 . Biphasic ∗ Zn-cyt c decay kinetics are observed 1 ms after GuHCl dilution: two-thirds of the excited Zn-porphyrins decay in a fast phase 68 × 106 s−1  attributable to unfolded protein; the remaining third exhibits a rate constant 41 × 105 s−1  closer to that expected for folded molecules. ET kinetics measured at longer folding times remain biphasic, with the amplitude of the faster component decreasing in favor of an increase in the amplitude of the slow component (Figure 13 upper panel). Both ∗ Zn − cyt c decay rates decrease by about a factor of 2 1 ms 68 × 106  41×105 s−1  50 ms 32×106  24×105 s−1  as the folding reaction proceeds (Figure 13, upper panel). This variation in quenching efficiency reflects a gradual collapse of polypeptide structures during folding, revealing the conversion of compact, nonnative conformations to a fully folded protein. The amplitudes of the two ∗ Zn-cyt c decay phases vary exponentially with folding time and the rate constant is in reasonable agreement with that measured by Soret absorption spectroscopy (Figure 13, lower panel). It is noteworthy that, at the earliest measured folding times (1 ms), there are significant amplitudes in both the fast and slow ∗ Zn-cyt c ET phases. This is substantially more than would be expected on the basis of the stopped-flow dead-time ∼1 ms and the observed rate constants extracted from the signal amplitudes. Measurements of ∗ Zn-cyt c kinetics at different GuHCl concentrations consistently extrapolate back to a burst-phase ensemble [94,128,129,162] with a 2:1 ratio of fast and slow ET components (Figure 13, lower panel): these results demonstrate that the burst ensemble is heterogeneous; molecules in one-third of the protein population have compact structures, and ones in the remaining fraction have exposed Znporphyrins. It is apparent that there is underlying complexity in Zn-cyt c folding. The fraction of the burst ensemble with slow ∗ Zn-cyt c decay kinetics could be fully folded protein or an ensemble of compact nonnative structures. The former possibility would be an example of fast-track folding [163–165], where about a third of the unfolded Zn-cyt c molecules adopt conformations that can Met. Ions Life Sci. 1, 9–60 (2006)

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P (k)

Δ + 3 ms Δ + 5 ms Δ + 10 ms Δ + 20 ms Δ + 50 ms

5.0

5.5

6.0

6.5

7.0

log (k) 1.0

F

Amplitude

0.8

0.6

0.4

U 0.2

0

0

10

20

30 time, ms

40

50

60

Figure 13. Zn-cyt c folding probed by ET kinetics. Upper panel: Bimolecular quenching rate constants and relative amplitudes from biexponential fits of ∗ Zn-cyt c during folding ( Zn-cyt c = 10 M RuNH3 3+ 6  = 4 mM GuHClfinal = 146 M pH 7  is the stopped-flow mixer dead-time ∼1 ms). Lower panel: Amplitudes of fast and slow ∗ Zn-cyt c decay constants as functions of time after the initiation of protein folding. The amplitudes vary exponentially kfold = 33 s−1  with folding time and extrapolate to a ∼2:1 fast:slow ratio at time zero  GuHClfinal = 146 M.

refold very quickly. The remaining protein molecules have relatively exposed porphryin groups, and fold on a substantially longer time scale. Alternatively, the fast and slow ∗ Zn-cyt c decay components formed immediately after dilution of [GuHCl] may reflect a shift in the equilibrium between unfolded and compact nonnative structures. Although it is often assumed that native solvent conditions will strongly favor compact structures, the ∗ Zn-cyt c ET kinetics clearly indicate that two-thirds of the molecules in the burst intermediates have highly Met. Ions Life Sci. 1, 9–60 (2006)

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exposed porphyrins. Ultimately, the entire protein ensemble folds because, at low [GuHCl], the native structure is much more stable than unfolded conformations.

4.2.2 AEDANS-labeled Cytochrome c In our laboratory, Lyubovitsky examined DA distance distributions during the folding of AEDANS-labeled S. cerevisiae iso-1 cytochrome c [48]. It was shown in prior work that the folding kinetics of iso-1 cyt c are comparable to those of the more extensively studied equine protein [36,94,166–169]. In the folded protein at neutral pH, an imidazole nitrogen (His18) and a thioether sulfur (Met80) axially ligate the heme iron (Figure 8). The nitrogenous base of an amino acid side chain (pH 7: His26, His33, His39) replaces Met80 in the unfolded protein [121]. This misligation retards refolding, because ligand exchange is required for the peptide to adopt its native conformation [40,170]. The AEDANS fluorescence intensity, a measure of the ensemble-averaged extent of folding in AEDANS(C102)-cyt c, exhibits a biphasic kinetics profile when the refolding is triggered by stopped-flow dilution of GuHCl. The major fraction (90%) of the decrease in fluorescence occurs within 2 s of mixing; the remaining 10% of the signal change proceeds in the 2–10 s time interval [48]. The integrated AEDANS fluorescence only provides an indication of the extent of folding and reveals nothing about the polypeptide ensemble heterogeneity. Measurements of I(t) at various times during folding (Figure 14) provide snapshots of DA distance distributions, P(r). Immediately after the folding is triggered (1 ms), 40% of the protein ensemble has collapsed, producing a population with

AEDANS fluorescence

1 ms 40 ms 380 ms 760 ms 1.8 s 4.8 s 10 s 16 s

10

20 τAE

30 40 ns

DANS ,

50

,s τ fold

Figure 14. AEDANS fluorescence decay kinetics measured at the indicated times after initiation of AEDANS(C102)-cyt c refolding. Met. Ions Life Sci. 1, 9–60 (2006)

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1 ms

10 ms

P (r )

40 ms

380 ms

760 ms

16 s

20

30

40

≥50

distance, Å

Figure 15. Evolution of the distributions of DA distances (P(r)) during the refolding of AEDANS(C102)-cyt c  GuHClfinal = 013 M pH 7.

an average DA distance of ∼27 Å (Figure 15). Surprisingly, 60% of the protein remains in extended conformations with DA distances greater than 40 Å. Within 10 ms, the P(r) distribution develops a component at r = 25 Å, a value comparable to that of the folded protein. By 50 ms, the 25-Å component is larger than the 27-Å population, and after 400 ms, the latter fraction has nearly disappeared (Figure 15). Concomitant with the evolution of the collapsed ensemble, there is a decrease in the population of extended conformations. The formation of correctly folded cyt c is limited by heme axial ligand substitution processes at neutral pH [170]. Displacement of misligated His groups in denatured cyt c by imidazole dramatically accelerates refolding [30]. NMR investigations of the imidazole adduct of equine cyt c reveal only modest disruption of the protein structure in the vicinity of the Met80 residue [171]. Measurements of FET kinetics during AEDANS(C102)-cyt c refolding in the presence of imidazole at room temperature indicate that the native DA distance distribution is formed in less than 20 ms. At lower temperature (1  C), the evolution of DA distance distributions is remarkably similar to that in the absence of imidazole. The key difference is the overall time scale of refolding; formation of folded Met. Ions Life Sci. 1, 9–60 (2006)

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protein is largely complete within 200 ms. Roughly equal populations of extended r > 50 Å and collapsed r = 32 Å structures are observed immediately after folding is triggered. The 32-Å distribution evolves into a 25-Å native distribution in ∼200 ms. The FET kinetics measured during AEDANS(C102)-cyt c folding indicate that dilution of denaturant to concentrations favoring native protein conformations does not produce a complete collapse of the polypeptide ensemble. Within the deadtime of stopped-flow measurements, only half of the protein population has formed compact structures. The compact ensemble (C) is characterized by a mean AEDANS-heme separation of ∼27 Å. This distance is greater than that of the native protein, indicating that the collapsed species are not fully folded. As the population of proteins with the native fold (N) increases, the extended (E) and compact (C) populations disappear on comparable time scales. It is surprising that such a large fraction of the protein ensemble remains in an extended conformation after denaturant dilution. These extended conformations are not a consequence of His misligation in the unfolded protein; high concentrations of imidazole displace the His ligands and speed refolding, yet both E and C fractions are observed at 1  C. The accelerated cyt c refolding in the presence of imidazole also demonstrates that E does not arise from kinetically trapped conformations containing proline isomers or incorrect topomers; there is no obvious mechanism by which added imidazole could eliminate such traps. Indeed, the parallel disappearance of E and C, rapidly in the presence of imidazole, slowly when His residues misligate the heme, strongly suggests that the two populations are in rapid equilibrium [129,169]. The time-resolved DA distance distributions extracted from FET measurements lead to an idealized folding landscape for DNS(C102)-cyt c (Figure 16). The lateral dimension of the landscape is the deviation of the DA distance from its value in the folded protein R − RF  RF ∼ 25 Å; and the vertical axis reflects the polypeptide free energy. The cross-section of this landscape (Figure 16, right panel) illustrates two possible fates for a polypeptide that was in an extended conformation r > 40 Å prior to the initiation of folding. Denaturant dilution shifts the E  C equilibrium to produce comparable populations of extended and compact polypeptides undergoing rapid exchange ∼100 s. Collapsed conformations (C) with favorable arrangements of the polypeptide backbone can transform into the native structure (N) at pH 7 by surmounting an energy barrier corresponding to a heme axial-ligand substitution process (Figure 16, right panel, left path). Rapid collapse of a polypeptide is likely to produce conformations that cannot evolve into the native fold (C→N) because of topological frustration [164] (Figure 16, right panel, right path). This model implies that the population of collapsed polypeptides is heterogeneous and that only a fraction of the collapsed conformers is competent to transform into N [164,172]. Met. Ions Life Sci. 1, 9–60 (2006)

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34

Fe-cyt c + imidazole E

C

C′

E

N N R-Rnative

Figure 16. Idealized representations of the AEDANS-labeled cytochrome c folding landscape. The lateral dimension in both plots is the deviation from the native DA distance ∼25 Å. The three-dimensional folding landscape (left panel) reveals a flat region on the periphery for extended and compact polypeptides that surrounds the global energy minimum (blue) of the native (N) fold. Some of the compact structures are separated from the native fold by low energy barriers (yellow). The remainder have high barriers (red) to native state formation. Polypeptides that fall into these minima must rearrange to extended conformations that can collapse into productive compact structures. The twodimensional cross-section of the landscape (right panel) shows nearly degenerate, shallow energy minima corresponding to extended (E) and collapsed C C  conformations. The collapsed structures on the left side (C) of the global energy minimum can surmount a ligand substitution barrier (blue curve) which can be lowered by the addition of imidazole (red curve) to reach the native (N) structure. Collapsed peptides on the right side C  face a high barrier to formation of the native fold; this population must exchange with other collapsed structures with lower barriers to folding.

For topologically frustrated compact conformations, the only route to the native state involves formation of an extended polypeptide that can recollapse to a more favorable structure. This mechanism is illustrated by the three-dimensional contour plot of the AEDANS(C102)-cyt c folding landscape (Figure 16, left panel). The native fold is represented by the central free-energy minimum. Owing to the near degeneracy of collapsed and extended polypeptides, the landscape consists of a relatively flat outer rim surrounding a narrow funnel. Rapid interchange among extended conformations via intrachain diffusion proceeds on the outer rim of the landscape [136,139,173]. These extended polypeptides frequently fall into collapsed conformations toward the interior of the landscape; some of these (4 of the 12 collapsed conformers shown on the idealized surface (Figure 16, left panel)) can form the native structure; the others must extend and try again. Met. Ions Life Sci. 1, 9–60 (2006)

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Collapsed and extended polypeptides in rapid equilibrium at the top of the funnel must wait for a ligand substitution event to open the way to conversion to the native structure at the bottom. Addition of imidazole lowers the ligand substitution barrier and speeds the transformation of collapsed polypeptides into the native form. The picture that emerges is one in which extended polypeptide conformations play a pivotal role in AEDANS(C102)-cyt c refolding. Time-resolved FET measurements reveal that, at the onset of folding, fully half of the polypeptide ensemble is found in extended conformations reminiscent of the denatured protein [128,174]. The near degeneracy and rapid equilibration of collapsed and extended populations would enable AEDANS(C102)-cyt c to escape from topologically frustrated compact structures that cannot produce the native fold because of extremely high energy barriers [164]. If collapsed intermediates were substantially more stable than extended geometries, formation of extended structures would occur infrequently, exacerbating the problem of topological frustration [164]. Instead, AEDANS(C102)-cyt c can collapse and extend many times as it searches for compact structures that have low-barrier routes to the native conformation [172]. Since collapsed cyt c intermediates are not substantially more stable than the fully denatured protein, the likelihood of a native structure rearranging to a partially folded species is substantially lower than would have been the case if collapsed conformations were found in deeper wells on the folding landscape. If this is a common protein folding characteristic, it may be an important means of avoiding the partially folded intermediates that can aggregate into the misfolded structures that characterize a variety of disease states.

4.2.3 Cobalt-substituted Cytochrome c Years of experimental work on the energetics and dynamics of selfassembly of cytochrome c have failed to resolve whether unfolded polypeptides undergo global collapse to compact conformations upon dilution or laser triggering to solution conditions that strongly favor the native structure [30,35,94,129,130,158,166,169]. Indeed, recent kinetics experiments suggest that comparable populations of compact and extended polypeptides are formed rapidly <1 ms, and these two populations disappear in parallel as the protein folds [43,48]. To test this new energy landscape model rigorously, Tezcan isolated the structures that comprise the burst-phase by replacing the native iron center with cobalt(III) [175]: owing to very high ligand substitution barriers, the final step of cobalt(III)-cytochrome c (Co-cyt c) folding is well separated in time from all early events; and, in this kinetically stabilized system, the early folding intermediates can be examined carefully over a period of hours by standard physical methods [52]. Met. Ions Life Sci. 1, 9–60 (2006)

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The folding free energies Gf  of equine, tuna, and yeast Co-cyt c (9.9, 9.8, and 6.1 kcal/mol, respectively) determined by monitoring changes in farUV CD and Soret absorption spectra in GuHCl solutions are comparable to those of the corresponding Fe(III)-protein (9.0, 9.4, and 4.6 kcal/mol) [36,161]. The folding thermodynamics accord with the crystal structure of tuna Co-cyt c (15-Å resolution), which confirms that replacing Fe(III) with Co(III) does not disrupt the native protein fold (022-Å RMS C displacements in Fe(III)- and Co(III)-proteins) [176,177]. Both equine and tuna Co-cyt c refold several orders of magnitude more slowly than the corresponding native protein; the observed kinetics are biphasic, owing to the presence of misligated unfolded protein populations [175] with different activation energies for ligand substitution. In the equine protein, His26 and His33 are the likely axial ligands; and there is evidence that Lys side chains coordinate to Co(III) in the unfolded tuna protein, which lacks His33 [175]. Analysis of the equine Co-cyt c folding kinetics suggests that His33 binds more tightly to the metal center than His26 in the unfolded protein [175]. The refolding phases associated with His26- and His33-misligated proteins exhibit comparable amplitudes after relatively short incubation periods in denaturant solution, indicating that the binding rates for the two histidines are similar [175]. However, with longer incubation time, the dissociation rate for His26 is 3 times greater than that for His33, a finding that is in excellent agreement with the 3- to 4-fold faster folding rate for the His26-misligated protein. It is apparent from these results that folding is limited by His dissociation from Co(III); and that the weaker binding of His26 to the metal center (relative to His33) is due to unfavorable steric interactions and the lower entropy associated with formation of a smaller loop [121]. Pulsed H/D exchange NMR measurements have revealed cyt c folding intermediates in which the C- and N-terminal helices are docked [30,178]. As expected, then, there is a large decrease in the far-UV CD amplitude (40% of total amplitude) immediately after diluting equine Co-cyt c/GuHCl solutions (Figure 17). The helical intermediate persists at low temperature for several hours without significant change. The CD spectrum of the Co-cyt c folding intermediate is similar to that reported recently for its Fe-protein analogue [174]. Unfolded Co-cyt c displays a 1 H NMR spectrum characteristic of a random coil: the resonances are sharp and not dispersed. The upfield signals attributable to Met80 side chain protons [179] in the spectrum of the folded protein are absent, consistent with His-misligation. Shortly after the initiation of folding, when helical intermediates are fully populated, resonances in the NMR spectrum are broadened and overlapped, but remain negligibly dispersed. Such a spectrum indicates conformational exchange times comparable to the chemical shift time scale ∼ ms. Thus, the NMR experiments confirm that folding intermediates containing elements of secondary structure are in rapid equilibrium with unstructured conformations. Met. Ions Life Sci. 1, 9–60 (2006)

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0

CD signal

–10

–20

–30

–40

–50

210

220

230

240

250

nm

Figure 17. Far-UV CD spectra of equine Co-cyt c species observed during refolding in 0.4 M GuHCl at 7 C: unfolded (circle), ∼ 1, 10, 55 min, and folded (triangle).

FET kinetics provide nanosecond-time-scale snapshots of DA distance distributions in rapidly equilibrating populations of folding intermediates [48]. FET kinetics from AEDANS at Cys102 to the Co(III)-porphyrin were monitored during yeast Co-cyt c folding. Similar to the iron protein [48], AEDANS(C102) fluoresces intensely when unfolded by GuHCl; and it is quenched by the Co-porphyrin in the folded structure. As expected, the folded protein has a narrow DA distance distribution centered at ∼27 Å and, notably, the unfolding process is accompanied by a broadening of Pr with appearance of extended conformations with r > 50 Å. A fraction of the AEDANS(C102)-Co-cyt c population collapses shortly after triggering folding by manual dilution of denaturant (Figure 18) [52]. Interestingly, however, most of the molecules are still in extended conformations hours after initiating the refolding reaction; and, on an even longer time scale, both extended and compact conformations disappear as native structure forms. The time required to reach Pr of the Co-cyt c is >18 h. Comparison of the FET kinetics for Co-cyt c with those obtained for Fe-cyt c [48] is especially revealing. Immediately after triggering AEDANS(C102)Fe-cyt c folding ∼1 ms, the measurements reveal a bimodal distribution of AEDANS-Fe(III) distances: half of the protein molecules adopt compact Met. Ions Life Sci. 1, 9–60 (2006)

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unfolded

2 min

P (r )

3 h 40 min

12 h

17 h 45 min

folded

20

30

40

≥50

distance, Å

Figure 18. Evolution of the distributions of DA distances Pr during the refolding of AEDANS(C102)-Co-cyt c  GuHClfinal < 05 M pH 7.

structures and the remainder are in extended conformations. As folding progresses, the compact and extended populations decrease in parallel and are replaced by folded protein. It is apparent, then, that similarly structured intermediates are populated in both the Fe(III) and Co(III) proteins. The key difference between the two is the refolding time scale: Fe-cyt c evolves to the native structure in a few hundred milliseconds, whereas folding the Co(III)-protein requires several hours. The combined spectroscopic probes of the burst-phase folding ensemble in Cocyt c shed new light on the nature of polypeptide structures that form immediately after dilution of denaturant solutions. CD spectra demonstrate that there are elements in the ensemble with some secondary structure. Since the FET kinetics reveal populations of both compact and extended structures, it is likely that the compact structures give rise to the CD signal. The absence of dispersion in the NMR spectra demonstrates that the compact and extended structures equilibrate on a submillisecond time scale. The Co-cyt c folding data also are consistent with time-resolved small-angle X-ray scattering measurements that show that the Fe-cyt c ensemble has a bimodal pair distribution function a few milliseconds after the initiation of folding [128]. Met. Ions Life Sci. 1, 9–60 (2006)

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The possibility that global hydrophobic collapse is not an obligatory step in protein folding has been examined in computational experiments [18]. Relative stabilities of collapsed and extended conformations were suggested to correlate with the properties of the primary sequences and overall stability of the folded protein: more hydrophobic and less optimized sequences favored formation of collapsed intermediates, whereas extended structures evolved directly to the native fold in less hydrophobic and strongly optimized sequences [18]. The sequence and stability of cyt c appear to place it between these two limits: in this case, extended and collapsed structures are degenerate; and nearly all of the folding free energy is released when the compact conformations convert to the native fold. The relative instability of collapsed nonnative structures not only prevents formation of misfolded structures during the self-assembly process, but also reduces the probability that the native protein will transiently adopt an incorrect conformation.

4.3 Four-helix Bundles Whether topologically similar proteins necessarily have similar folding pathways is an open question [180]. It has become clear that amino acid sequences with little or no homology can assume nearly identical three-dimensional structures. Theoretical models suggest [181–183], and most experimental studies confirm [184–187], that a helical bundle is a fast folding structural motif. The presence of heme cofactors, however, can introduce new features into the helical bundle energy landscape that can greatly alter refolding pathways.

4.3.1 Cytochrome b562 ET triggering has been employed to study the folding of cytochrome b562 (Figure 19) [37,41]. Although the porphyrin is not covalently attached to the protein, the heme iron is ligated axially by the side chains of Met7 and His102. As expected for a heme protein with a 0.18-V reduction potential, titrations with GuHCl confirm that reduced cytochrome b562 is more stable toward unfolding than the oxidized protein [37,41,54,188]. Unfolding experiments probed using CD and Soret-band absorbance gave identical results, consistent with a two-state process. In contrast to the bis-His ligation of unfolded cyt c, absorption spectra of the unfolded cyt b562 indicate that the heme iron is high-spin in both oxidation states. Oxidized cyt b562 is fully denatured at 2 M GuHCl, whereas reduced cyt b562 does not unfold below 6 M GuHCl. Electron injection into unfolded, oxidized cyt b562 produces a significant amount of folded, reduced protein at GuHCl concentrations between 2 and 3 M [37,41]. The ferrocytochrome b562 folding kinetics can be described by a dominant kinetics phase with a first-order rate constant of 800 ± 200 s−1 at a Met. Ions Life Sci. 1, 9–60 (2006)

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Figure 19. In cyt b562 (grey), the heme iron is axially ligated to His102 and Met7, whereas in cyt c (black), the heme has only one axial ligand (His117). Backbone atoms of four -helices of cyt c (PDB 1A7V) are superimposed on the corresponding atoms in cyt b562 (PDB 256B), with a calculated RMS deviation of 16 Å.

driving force of ∼25 kJ/mol. At a similar driving force, ferrocytochrome c folds much more slowly 10 s−1  [40]. The absence of nonnative His ligands is certainly one explanation for the faster folding of cyt b562 ; but even at reduced pH, the rate of Met80 binding to the ferroheme in cyt c (16(5) s−1 ) [40] is far slower than in cyt b562 .

4.3.2 Cytochrome c Although folded reduced cyt b562 was observed within milliseconds after reduction of the unfolded oxidized protein, no more than half of the reduced protein successfully developed native structure [41]. Rapid heme dissociation from the polypeptide on the millisecond time scale limited the yield of the folding reaction. The heme-loss step selects fast-folding conformations from the unfolded ensemble; if there are slow-folding components, they cannot be detected. Under these circumstances, the kinetics reflect heme-dissociation dynamics rather than folding. Cytochrome c from the photosynthetic bacterium Rhodopseudomonas palustris is a monomeric, soluble, 125-residue, four-helix-bundle heme protein. Met. Ions Life Sci. 1, 9–60 (2006)

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Importantly, the porphyrin is bound to the polypeptide with two thioether links near the C terminus (Cys113 and Cys116) [189–191]. Although sharing just 19% sequence identity and 40% similarity [192], cyt c and b562 have quite similar folds (Figure 19) [193,194]. Cyt c has a high-spin, five-coordinate heme that is axially ligated by His117, and a reduction potential of 100 mV [195–200]. Rapid electron injection ∼100 s into unfolded cyt c in the 2.0–2.9-M GuHCl concentration range following laser excitation of NADH [40,55] initiates the folding of the reduced protein [42]. Under these conditions, heme reduction is slower than binding of the nonnative sixth ligand. Highly heterogeneous kinetics were observed in studies where the progress of the folding reaction was monitored by heme absorption from hundreds of microseconds to seconds after photoexcitation. A small fraction ∼20% of the population forms a high-spin heme species in about a millisecond. Complete formation of the fully folded ensemble requires several seconds. Rate constants for Fe(II)-cyt c span a range from 103 to 10−1 s−1 revealing fast- ∼103 s−1  17%, intermediate- (∼102 to 101 s−1  24%), and slow-folding ∼10−1 s−1  43% components in the protein ensemble. The transient difference spectrum recorded 100 s after electron injection is characteristic of a mixture of five-coordinate, high-spin and six-coordinate, low-spin ferrohemes, suggesting that the slower folding populations are misligated. It is reasonable to expect that topologically homologous proteins would fold at similar rates, yet cytochromes b562 and c display quite disparate folding kinetics. Apparently, there are factors beyond structural topology that must be considered in order to explain the folding kinetics of these four-helix bundles. Three key features of cytochromes b562 and c are likely to contribute to the differences in folding kinetics: covalent attachment of the porphyrin to the polypeptide; nonnative heme-ligand traps; and folding driving force. The rapid dissociation of the heme from unfolded cyt b562 limits the time scale for observation of folding kinetics. Since the porphyrin is covalently attached in Fe-cyt c , the folding kinetics cover a far wider time range and are considerably more complex. A fraction of the unfolded cyt c ensemble refolds rapidly, but several seconds are required for the entire sample to fold. It is interesting to note that the fast-folding fraction of Fe(II)-cyt c is roughly comparable to the yield of folded Fe(II)-cyt b562 . It is possible, then, that if heme dissociation could be inhibited, Fe(II)-cyt b562 would display slower and more complex folding kinetics. The refolding of Fe(II)-cyt c can be described qualitatively by a kinetics partitioning mechanism [164,201]. At the instant that folding is initiated, a fraction of the denatured proteins rapidly folds to the native structure. Folding of the remaining polypeptides is slowed by transient trapping in local minima on the folding energy landscape. Escape from these misfolded structures is an activated process that retards formation of the native structure. The partition factor that determines the balance between fast- and slow-folding populations depends on the primary sequence as well as the refolding conditions [163,164,181,201–203]. Met. Ions Life Sci. 1, 9–60 (2006)

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Simulated folding kinetics of model three-helix-bundle proteins [163,181] point to a central role for the folding free-energy gap in determining the partitioning between fast- and slow-track folding. The heterogeneous folding kinetics of cyt c are strikingly similar to the simulated folding kinetics of a small-gap three-helix-bundle [181]. Nonnative methionine ligands in unfolded Fe(II)-cyt c contribute to the heterogeneity of the folding kinetics. In addition to the fast-folding population, there are intermediate and slow-folding components. Iron–sulfur bond dissociation is not rate limiting because ferroheme–methionine ligand exchange is faster than either folding phase [136]. In the presence of CO, the nonnative Met ligands are displaced from the ferroheme and folding is dominated by a slow phase with a time constant of ∼350 ms. Similarly, Fe(III)-cyt c folding is not complicated by heme-ligand binding and a single 1-s phase predominates [42]. These observations suggest that, in the absence of misligation, cyt c requires 500–1000 ms to adopt a folded structure. The presence of an intermediate ∼102 s−1  folding phase in Fe(II)-cyt c implies that nonnative methionine ligation can, in some instances, facilitate refolding. Nonnative ligand binding clearly can perturb heme protein folding; it is likely that noncovalent, nonnative heme–polypeptide contacts represent additional sources of frustration.

4.3.3 Cytochrome c556 and c-b562 Although cyt b562 and c have nearly identical three-dimensional structures (Figure 19), they have very low sequence identity (15%) and exhibit quite disparate folding kinetics [41,42,51,71,204]. Fe(II)-cyt b562 folds in less than a millisecond, while Fe(II)-cyt c folding is quite heterogeneous, spanning time scales from milliseconds to seconds (Figure 20). Clearly, topology alone does not dictate these refolding rates. In order to circumvent complications arising from heme dissociation, a variant of E. coli cyt b562 (cyt c-b562 ) in which two thioether linkages bind the porphyrin to the polypeptide chain in the fashion of a c-type cytochrome was expressed [191,205,206]. Cytochrome c556 from R. palustris [207], a protein with the same four-helix-bundle fold as cyt b562 and cyt c [194,208–210], also has been investigated. In cyt c556 and c-b562 , the heme group is covalently attached to the polypeptide chain through two C-terminal thioether linkages and the iron center is axially ligated by Met12 and His121 (cyt c556 ) or Met 7 and His102 (cyt c-b562 ) [210]. GuHCl titrations monitored by absorbance, CD, and Trp fluorescence spectra reveal cooperative unfolding transitions and, as expected on the basis of their high reduction potentials, the oxidized forms are less stable than the reduced proteins [35,39]. It is interesting to note that the introduction of two thioether linkages to the heme in cyt c-b562 leads to a substantial stabilization of the folded protein [46]. Met. Ions Life Sci. 1, 9–60 (2006)

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7 6 Fe(II)-cyt c-b562

log (k)

5

Fe(II)-cyt c556

4 Fe(II)-cyt b562

3 2 1 0

Fe(II)-cyt c' 0

1

2

3 4 [GuHCl], M

5

6

Figure 20. Folding rates of four-helix-bundle heme proteins as a function of [GuHCl] (the vertical bar reflects the range of observed rate constants in cyt c folding).

At suitable denaturant concentrations cyt c556  15–25 M cyt c-b562 , 38–48 M, electron injection into the denatured oxidized protein will initiate folding of the reduced form [35]. Both Rubpy2+ 3 and NADH phototrigger ferrocytochrome c556 and c-b562 refolding [46]. In experiments with ∗ Rubpy2+ 3 as reductant, the observed transient absorption kinetics are adequately described by a biexponential function. The higher rate is independent of [GuHCl] and corresponds to decay of ∗ Rubpy2+ 3 with parallel microsecond reduction of the oxidized protein. The rate constant for the slower phase varies with [GuHCl]; this reaction channel represents early events in the folding of the protein around the heme cyt c556  k ∼ 5 × 104 s−1  cyt c-b562  k ∼ 1 × 105 s−1  (Figure 20) [46]. The transient spectra measured at the end of the slower phase  ∼ 100 s are consistent with the formation of reduced folded protein. Biexponential kinetics also are observed when NADH is used as photoreductant, with rate constants that are attributable to the reduction of the protein − by eaq 4–5 × 104 s−1  and NAD• 9 × 103 s−1 . The relative signal amplitudes observed following excitation of samples at low and high [GuHCl] suggest that folded reduced protein is formed 300 s after excitation [46]. No additional changes in absorption were detected on time scales as long as several seconds; the formation of folded reduced protein appears to be limited by the rate of reduction by NAD• . Moreover, steady-state UV-visible and CD spectra recorded after laser excitation of NADH-containing samples confirm that the photochemically reduced proteins adopt native folds. Met. Ions Life Sci. 1, 9–60 (2006)

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The transient absorption data suggest that ferrocytochrome c556 and c-b562 refolding rates are faster than 104 s−1 [46]. UV-visible spectra provide information about the immediate environment of the heme cofactor, but do not directly report on the conformation of the polypeptide. In order to gain more insight into the submillisecond cyt c556 and c-b562 folding dynamics, ET refolding experiments were repeated in the presence of CO. The deeply buried iron centers of native c-b562 and c556 are six-coordinate and therefore not available for CO binding. Under denaturing conditions, the ferroheme remains low-spin, likely due to Met ligation in the sixth coordination site. Nevertheless, CO will replace Met as an Fe(II) ligand in unfolded c-b562 and c556 . Kinetics of CO rebinding to the heme were examined after photodissociation from denatured Fe(II)(CO)cyt c-b562 . Under 1 atm of CO, the rate constant for CO rebinding to the Fe(II)-heme is approximately 65 s−1 [46]. If the polypeptide wraps around the heme rapidly >102 s−1 , little CO should bind to the heme. On the other hand, if folding is slower than ∼102 s−1 , substantial CO binding is expected. At 7 M GuHCl, where formation of native, reduced c-b562 is disfavored, there is a major change in absorbance consistent with CO binding after reduction of the oxidized protein. In contrast, very little CO binding is apparent under conditions favoring formation of the folded Fe(II)-protein. ET triggered experiments reveal that early events in Fe(II)-cyt c556 and cyt c-b562 refolding involve formation of a low-spin heme and some degree of heme encapsulation by the polypeptide. A lower limit to the time required for the heme to ligate a Met residue can be estimated from studies of tertiary contact dynamics in unfolded proteins and peptides [44,137,138,143]. Met ligation to the heme in Fe(II)-cyt c556 will produce polypeptide loops comprised of 96, 97, and 104 residues; and loop sizes of 39 and 90 residues in Fe(II)-cyt c-b562 are possible. Energy transfer quenching studies in synthetic polypeptides suggest that tertiary contact rate constants for 90–100 residue loops are 106−107 s−1 [137]. These values compare with the rates of 105−106 s−1 for formation of native heme absorption spectra in ET triggered Fe(II)-cyt c556 and c-b562 refolding experiments. Denaturant dependences observed for Fe(II)-cyt c556 and c-b562 refolding suggest that the early kinetics phases involve more than intrachain diffusion leading to Met-Fe ligation. Rapid Met-Fe ligation could facilitate refolding of Fe(II)-cyt c556 and c-b562 , because formation of one or more strong native tertiary contacts will substantially reduce the size of the conformational space available to the polypeptide [211]. Constraining the folding energy landscape in this manner could lead to a substantial reduction in the time required to find the native structure [212]. Following reduction of the unfolded oxidized proteins, CO will bind to the ferroheme under solution conditions where formation of native structure is disfavored. The observed rate constant for CO binding to denatured Fe(II)-cyt c556 and c-b562 is about 10 times smaller than the corresponding rate found for Fe(II)-cyt c [42], and about 20 times smaller than the rate for Fe(II)-cyt c [213]. Met. Ions Life Sci. 1, 9–60 (2006)

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The smaller CO binding rate constant for cyt c556 and c-b562 may be a consequence of stronger Met binding to the reduced denatured proteins. Indeed, the similarity in the absorption spectra of folded and denatured reduced proteins suggests that Met is completely bound in the denatured forms. The Soret absorption of denatured Fe(II)-cyt c is less intense than those of denatured Fe(II)-cyt c556 and c-b562 , possibly owing to less complete Met binding. The finding that CO does not bind to the Fe(II)-cyt c556 and c-b562 hemes under GuHCl conditions favoring folding points to a rapid encapsulation of the heme by the polypeptide. The spectroscopic data do not reveal whether the polypeptide has developed secondary and tertiary structure by this time. Ultrafast mixing measurements on a F65W mutant of apo-cyt b562 , which adopts a 3-helix-bundle fold [71,214], are consistent with a refolding rate constant of 2600 s−1 in the absence of denaturant [204]. This value represents a reasonable upper limit to the folding rate for the holoprotein and is in line with the lower limit indicated by our CO ligation measurements. The refolding of cyt c556 and c-b562 clearly begins from an extensively denatured state. This contrasts with recent results on cyt b562 [204] where, as we had suggested earlier [41], heme dissociation preselects fast-folding members of the denatured ensemble. The only events revealed by changes in heme absorption spectra in Fe(II)-cyt c556 and c-b562 occur on very early microsecond time scales and involve Met-iron ligation processes. The absence of separate kinetics phases attributable to heme encapsulation by the polypeptide, and the observation that the heme is protected from CO binding, confirm that a substantial degree of refolding occurs on submillisecond time scales. Formation of one persistent native contact in the early stages of Fe(II)-cyt c556 and Fe(II)-cyt c-b562 refolding puts each polypeptide on a fast track to its native structure [212].

5 -SYNUCLEIN AND PARKINSON’S DISEASE Parkinson’s disease (PD) is the most common age-related neurodegenerative movement disorder [215]. The primary symptoms of PD are caused by the loss of dopaminergic neurons in the substantia nigra region of the brain stem [216]. A diagnostic hallmark of PD is the presence in the cerebral cortex of intracellular inclusions (Lewy bodies and neurites) [217,218], but the role of these proteinacious materials in the pathogenic process has not been established. -Synuclein (-syn), an abundant 140-residue neuronal protein of unknown function [219], is the primary component of the fibrillar inclusions. Autosomal dominant earlyonset PD has been linked to two point mutations (A53T and A30P) in the gene encoding -syn [220,221]. Introduction of human wild-type, A30P, or A53T -syn into transgenic animal models produces age-dependent motor dysfunction, neuronal deposits of fibrillar protein, and loss of dopaminergic neurons, consistent with suggestions that PD may arise from these protein aggregates [222,223]. Met. Ions Life Sci. 1, 9–60 (2006)

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In vitro fibrillogenesis experiments also have shown that the two mutants aggregate faster than the wild-type protein [224,225], consistent with the proposal that the disease is caused by protein aggregation. Fibrillar -syn, however, may be a symptom rather than a cause of the disease. Recent reports suggest that the formation of soluble oligomeric intermediates is accelerated in the A30P mutant [226]. Although there are contradictory results, the A30P mutant has been reported to bind more weakly to lipid vesicles than the wild-type protein [227–230]. These observations point to a role for prefibrillar species in PD [226,231] and, further, that -syn interaction with synaptic-vesicle membranes may be involved in the pathogenic mechanism [232,233]. Indeed, it has been proposed that fibrillar aggregates are a byproduct of neuronal death and that the formation of Lewy bodies could be a protective mechanism to sequester neurotoxic species [234–237]. -Synuclein is localized in the cytosol and presynaptic terminals of neurons, with some fractions associated with synaptic-vesicle membranes [238–241]. Although its function has not been determined definitively, suggestions include a role in neuronal plasticity and synaptogenesis [238,242] as well as a protein folding chaperone [243]. The amino-acid sequence includes seven imperfect repeats in the N-terminal portion that are similar to an 11 residue repeating motif in exchangeable apolipoproteins [238]. This similarity suggests that the protein may be capable of reversibly binding to the surfaces of lipid membranes [228,229,232,244]. A distinguishing characteristic of the amino-acid sequence is the highly acidic C-terminal region. In vitro, -syn has been characterized as a monomeric, intrinsically unstructured (natively unfolded) protein [245]. Small-angle X-ray scattering studies indicate that the protein has a radius of gyration Rg ∼ 40 Å that is larger than expected for a folded globular 15 Å polypeptide [246]. However, in the presence of acidic phospholipid vesicles, the protein undergoes a conformational change, forming some -helical structure observable by CD [244] and NMR [247–249], as well as by EPR [250,251]. Interestingly, in contrast to the monomeric protein, larger oligomeric intermediates form -sheet structures that cause membrane leakage in vitro [233,252], providing further support for the hypothesis that prefibrillar structures may be cytotoxic. Large environmentally induced conformational changes of this type are particularly interesting. Toxic conformers of otherwise benign proteins have been invoked as pathogens in a number of neurodegenerative diseases [253,254]. We have employed FET kinetics to probe the structure and dynamics of pseudo-wild-type and disease-related (A30P) -synucleins [255]. A fluorescent amino acid (tryptophan) and a chemically modified tyrosine (3-nitrotyrosine, YNO2 ) were chosen as DA; this pair has a ro of 26 Å [256]. Tryptophan fluorescence decay and energy transfer kinetics were used to characterize the conformational heterogeneity of the protein under a variety of solution conditions. Met. Ions Life Sci. 1, 9–60 (2006)

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Trp residues were incorporated at the sites of three different aromatic amino acids (F4, Y39, F94) in -syn; in buffer all three variants exhibited emission properties characteristic of water-exposed indole side chains. In the presence of SDS micelles, Trp4 and Trp39 emission maxima exhibited a pronounced blueshift indicative of a more hydrophobic micellar environment that is consistent with previous NMR studies [247–249]. Time-resolved fluorescence polarization measurements also revealed an increase in the microenvironment viscosity and rigidity of the polypeptide structure in the presence of SDS micelles. In order to probe the equilibrium state of aggregation in solution, Trp fluorescence decay kinetics were examined at different -syn concentrations. For Trp39 and Trp94, the observed kinetics were independent of protein concentration, whereas Trp4 fluorescence decay kinetics exhibited a modest dependence on [-syn], suggesting some type of interprotein interaction at protein concentrations above 20 M. To explore possible N-terminal interpeptide interactions, FET kinetics of mixtures of D-only and A-only proteins (F4W and Y39NO2 ; Y39W and Y39NO2  were measured; however, there was no evidence for interprotein contacts in the 3–15 M concentration range. It is clear that the state of protein aggregation within the low micromolar concentration range is constant and that, most likely, only monomeric species are present. Moreover, Trp fluorescence decay provides no evidence to suggest that the A30P mutation induces a change in the aggregation state of the protein at concentrations below 30 M. Since -syn has been characterized as a natively unfolded protein, FET kinetics were fit to DA distance distributions for freely jointed polymer chains [100]. Although this model does not capture all the structural features, the results provide an approximate description of the conformational heterogeneity in terms of a well-defined Gaussian chain model (Figure 21). As expected, the mean DA distances scale with the number of residues separating D and A. Also, from this model an effective length parameter (l ) can be extracted which is related to the stiffness of the chain [100]. For a freely jointed polypeptide, the length of the chain segment is generally taken to be the length of a residue of 38 Å. Stiffer polypeptide chains will have larger values of l . At physiological pH, five of the DA pairs have chain segments in the 13–16 Å range, corresponding in each case to 3–4 amino-acid residues. Under acidic conditions, where -syn aggregation is accelerated [257], the chain segments in the N-terminal region of the protein lengthen slightly, whereas the C-terminal DA pair exhibits a dramatic decrease in chain segment length (∼2 amino-acid residues) at pH 4.4. Apparently, the neutralization of the negative charges in the C-terminus produces a relatively flexible polymer that behaves like a Gaussian coil. Upon addition of SDS, CD spectra indicate that our mutant -synucleins adopt ∼65% helical secondary structure (CD, 222 nm). However, the fits do not reveal a consistent trend in segment lengths in SDS. The greatest effect appears as a substantial increase in segment length (∼5 residues) in the C-terminal region of the protein. Met. Ions Life Sci. 1, 9–60 (2006)

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Y19-W39

W4-Y39

Y74-W94

W94-Y136

W4-Y136

P (r )

W4-Y19

0

20

40

60

80 0 20 distance, Å

40

60

80

Figure 21. Tryptophan to 3-nitrotyrosine distance distributions for freely jointed polymer chain model (dotted line) and DA distributions directly extracted from FET kinetics (solid line) of -synuclein under physiological pH conditions.

Fits to the FET kinetics data reveal that the Gaussian chain distance distribution is just a rough approximation to the conformational heterogeneity of -syn. DA distances also were extracted directly from the Trp fluorescence decay kinetics using a linear least squares procedure without recourse to a specific polymer model [48]. This fitting procedure produces the narrowest distance distributions required to fit the data. In all cases, the LLS method gave better fits to the data than the Gaussian chain model, but the general trends in the fitting results were comparable. The distance distributions projected from Trp decay kinetics in pH 7.4 solutions reveal both compact and highly extended conformations. For proteins with the DA pair separated by 15 and 20 residues, regardless of location in the sequence, the protein ensemble includes short 15 Å ≤10%, intermediate ∼20 Å ∼45%, and extended (≥ 30 Å; ∼45%) polypeptides (Figure 21). As expected, increasing the number of residues between DA pairs shifts the majority of the population to distances beyond >40 Å. In the presence of SDS, average DA distances decrease, particularly for the Y19-W39 and W4-Y39 pairs. Based on chemical shifts in C NMR spectra, residues 18–31 are expected to have the highest degree of -helical structure [247]. As found in the Gaussian chain fits, the distance between the W94-Y136 pair increases significantly in SDS, presumably owing to repulsive electrostatic interactions with the negative micelle surface. The most striking feature of the -syn structures in the presence of SDS micelles is the lack of tertiary contacts; there is no evidence for globular structure despite the development of Met. Ions Life Sci. 1, 9–60 (2006)

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-helical secondary structure. Although -syn has substantial helical content in the presence of micelles, the FET data reveal that the protein remains highly disordered and largely extended. Acidic conditions do not change the general character of the six DA distance distributions, but there is a slight increase in the amplitudes of the shorter distance populations. Our results are consistent with small angle X-ray scattering studies that indicate the average radius of gyration shrinks from 40 at pH 7 to 30 Å at pH 3.0 [246]. The greatest structural change induced by acidic conditions is the contraction in the C-terminal region. The correlation with accelerated aggregation under acidic conditions [246] raises the possibility that the high charge and extended conformation in the C-terminus serve to inhibit aggregation. When the acidic side chains become neutralized, reduced electrostatic repulsion induces shortening of the C-terminus and overall collapse of the polypeptide chain. Notably, C-terminally truncated -syn (1–110) aggregates faster than the full-length protein [258,259]. FET kinetics of two W/YNO2  pairs in the vicinity of one of these PD related mutations (A30P) were examined as functions of solution conditions. Interestingly, the presence of this mutation leads to an increase in the average DA distance under all solution conditions except acidic pH, where shorter distances are observed for some of the polypeptide chains. With the Pro30 mutation, both pairs lose populations with DA distances < 20 Å. The expansion of the polypeptide in the vicinity of the Pro30 mutation may be attributable to the increased stiffness of the polypeptide backbone (segment length ∼5 residues) and to the helix-disrupting property of Pro. From C chemical shift analysis, the slight bias (10%) for residual helical conformation in the N-terminal region is abolished in the A30P protein in solution [260]. The finding that single mutations in -syn are linked to familial early-onset forms of PD points to a central role for the protein in the etiology of the disease. A crucial question is whether a conformationally altered (misfolded) protein is directly involved in the pathogenic mechanism. Our analysis of the FET kinetics emphasizes that -syn is a highly disordered polymer at pH 7.4, pH 4.4, and in the presence of micelles. On average, the polypeptide is more extended than expected for a freely jointed polymer, and under some conditions the protein is substantially less flexible than a random coil. Nevertheless, it is likely that the protein is highly dynamic and that conformers interchange on very short ∼ s time scales. We find that modifications of solution conditions and amino-acid sequence do not produce unique conformational changes; rather, these perturbations result in subtle redistributions of the structures comprising the protein ensemble. It has been suggested that the death of dopaminergic neurons in Parkinson’s disease is the ultimate result of a cascade of events involving inhibition of mitochondrial complex I, -syn aggregation, and proteosome dysfunction [253,254]. The role of -syn in this complex disease progression is likely to involve its Met. Ions Life Sci. 1, 9–60 (2006)

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interactions with other biomolecules (e.g., vesicles, enzymes, chaperones, proteosomes) [253,254]. FET kinetics measurements have provided unique insights into the conformational heterogeneity of -syn that are not apparent from other spectroscopic measurements. The greatest power of this approach may lie in its ability to determine how different -syn subpopulations interact with neuronal compounds and structures that are implicated in the pathogenic process.

6 CONCLUSIONS AND OUTLOOK Elucidating the structures and properties of disordered biopolymers is an extremely challenging endeavor that urgently needs the development of new experimental probes and new modes of analysis. The usual structuredetermination tools (X-ray crystallography, NMR spectroscopy) provide little insight into the properties of dynamic and heterogeneous polypeptide ensembles. In one amyloid-forming polypeptide, -synuclein, we have shown that analysis of fluorescence decay kinetics can provide probability distributions of DA distances. Measurements of the FET and ET kinetics of other amyloidogenic peptides and proteins will allow us to take nanosecond snapshots of structures as well as determine the conformational dynamics of soluble prefibrillar aggregates. These powerful photophysical methods also will be used to investigate the interactions of -synuclein and Alzheimer’s A peptides with metal ions and other ionic and molecular species. Of special interest will be the characterization of transient intermediates generated during fibril formation. An extremely important outcome associated with the development of new probes for -synuclein and A structure and dynamics lies in the area of intervention. If we can use time-resolved spectroscopy to identify critical differences between wild-type and malignant proteins, then we also can use these laser methods to screen for chemical agents that disrupt pathogenic processes. Developing a detailed understanding of the conformational properties of -synuclein and A is a key step in the rational design of new therapeutic agents for Parkinson’s and Alzheimer’s diseases.

ACKNOWLEDGMENTS Our research is supported by the National Institutes of Health (GM068461 to JRW; DK19038 to HBG; Ruth L. Kirschstein National Research Service Award to JEK), the Department of Energy (DE-FG02-02ER15359 to JRW), the Arnold and Mabel Beckman Foundation (Beckman Senior Research Fellowship to JCL), Parkinson’s Disease Foundation (to JRW), National Parkinson Foundation (to JRW), and the Ellison Medical Foundation (Senior Scholar Award in Aging to HBG). Met. Ions Life Sci. 1, 9–60 (2006)

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ABBREVIATIONS AND DEFINITIONS A A AEDANS -syn bpy CD cyt C D DA E EPR ET FET GuHCl im LLS ME MP8 NAD• NADH NHE P(r) PD pMDMA Pox Pred RMS ro SDS YNO2 

energy acceptor Alzheimer’s -amyloid peptide 5-((((2-iodoacetyl)-amino)-ethyl)-amino)-naphthalene-1-sulfonic acid -synuclein 2 2 -bipyridine circular dichroism cytochrome compact fluorescent donor donor–acceptor extended electron paramagnetic resonance electron transfer fluorescence energy transfer guanidine hydrochloride imidazole linear least squares maximum entropy microperoxidase-8 nicotinamide adenine dinucleotide, oxidized form nicotinamide adenine dinucleotide, reduced form normal hydrogen electrode distribution of distances Parkinson’s disease p-methoxy-N,N -dimethylaniline oxidized protein reduced protein root mean squared Förster critical length sodium dodecyl sulfate 3-nitrotyrosine

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3 Metal Ion Binding Properties of Proteins Related to Neurodegeneration Henryk Kozlowski,1 Marek Luczkowski,1 Daniela Valensin,2 and Gianni Valensin2 1

Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie 14, 50-383 Wroclaw, Poland <[email protected]> 2

Department of Chemistry, University of Siena, via Aldo Moro, 53100, Siena, Italy

1 INTRODUCTION 2 Cu2+ INTERACTIONS WITH MAMMALIAN PRION PROTEINS AND THEIR FRAGMENTS 2.1 Structures of Mammalian Prion Proteins and the Potential Metal Ion Binding Sites 2.2 Cu2+ Ion Binding to the Mammalian Prion Protein Octapeptide Repeat Fragment 2.3 Binding of Cu2+ to Longer Octarepeat Fragments 2.4 Binding of Cu2+ by Chicken Prion Tandem Repeats 2.5 Cu2+ Binding to the Neurotoxic Prion Protein Fragment 2.6 Interaction of Cu2+ Ions with Globular Domain of the Prion Protein 2.7 The Binding of Cu2+ Ions and Superoxide Dismutase Activity of Prion Protein 2.8 Affinity of the Prion Protein to Bind Cu2+ Ions 3 INTERACTIONS OF METAL IONS WITH THE AMYLOID PRECURSOR PROTEIN AND ITS FRAGMENTS 3.1 Interactions of Metal Ions with the -Amyloid Peptide 3.2 Metal Ion Binding Sites in the Cysteine Region of the Amyloid Precursor Protein

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Edited by Astrid Sigel, Helmut Sigel and Roland K. O. Sigel

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1 INTRODUCTION Many neurodegenerative diseases are characterized by toxic misfolded proteins causing neuronal damage [1]. These noxious proteins display a typical tendency to aggregate and form solid extra- or intracellular deposits as diverse as the plaques of Alzheimer’s (AD) and transmissible spongiform encephalopathies (TSEs), the Lewy bodies of Parkinson’s disease (PD), the nuclear and cytoplasmic inclusions of Huntington disease (HD), the Bunina bodies of familial amyotrophic lateral sclerosis (ALS), and many others [1–4]. Genes implicated in the various disorders are being progressively identified and cellular and animal models are being developed, supporting the generally accepted belief that mutations in the genes yield abnormal processing of the toxic protein with consequent accumulation and deposition of the misfolded protein. However, gene mutations only account for a few percent of the widely occurring neurodegenerative disorders that are mainly determined by impaired homeostatic control of free radicals and metal ions. The human brain is estimated to produce more than 1011 free radicals per day [5]: imbalance in prooxidant vs antioxidant homeostasis results in ‘oxidative stress’ with generation of several potentially toxic reactive oxygen species (ROS). Oxidative stress has been implicated not only in normal brain ageing, but also in many neurodegenerative disorders [5–10]. As a matter of fact, AD, PD, and ALS are all characterized by extensive oxidative damage to cell membranes, proteins, and DNA. ROS are normally implicated in the cell signalling network and are generated by the reaction of oxygen with redox active metal ions through the Fenton reaction. It follows that ROS regulation is tightly linked to the homeostatic control of redox metal ions, namely copper, iron and manganese. The key factors of AD and TSE are neuronal membrane proteins, the amyloid precursor protein (APP) and the prion protein PrPc , respectively. The -amyloid peptide A, the major constituent of the deposit in plaques of AD brain, is in fact a 39–43 residues-long fragment of APP. The biological functions of PrPc and APP are not yet well established, but recent findings indicate they are involved in the homeostatic control of copper (see Chapters 4 and 5 of this volume). PrPc has long been known as a copper-binding protein and recently evidence has been reached about its ability in binding manganese. APP is a transmembrane Met. Ions Life Sci. 1, 61–87 (2006)

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cell surface glycoprotein accommodating binding sites for either copper or zinc ions. Elucidation of the ways copper interacts with both proteins is likely to provide valuable insight into the role that this redox active metal ion may play in the two neurodegenerative disorders. The chemical aspects of such interactions with PrP, APP, and related proteins and peptides are discussed herein.

2 Cu2+ INTERACTIONS WITH MAMMALIAN PRION PROTEINS AND THEIR FRAGMENTS 2.1 Structures of Mammalian Prion Proteins and the Potential Metal Ion Binding Sites Several NMR structures have been obtained for the recombinant mammalian prion proteins in solution [11–15], and a solid state structure has also been reported [16]. The structure is dimeric and results from domain swapping between monomeric structures. Each monomer comprises two distinct domains: (i) a C-terminal globular domain encompassing residues 125–228 in human PrP; and (ii) an N-terminal flexible disordered ‘tail’ (Figure 1) [11–15]. The crystal structure of the globular domain of the sheep prion protein is in very good agreement with the data obtained from NMR studies [17].

230 α3

α1

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Figure 1. Cartoon of the three-dimensional structure of the intact human prion protein, hPrP23-230. (Reproduced by permission from [14]). Met. Ions Life Sci. 1, 61–87 (2006)

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The 3D structures of the C-terminal domain of human (hPrP), murine (mPrP), bovine (bPrP), and Syrian hamster (shPrP) PrP are closely similar to each other, with hPrP(121–230) especially matching bPrP(121–230). As first reported by Hornshaw et al. [18,19], the prion protein tightly binds copper and this ability is physiologically relevant in affecting the brain copper content [20]. Since then, many reports have delineated that PrP accommodates several binding sites for copper and, incidentally, for other metal ions, such as zinc and manganese. As shown in Chapter 4 of this volume, the basic role in copper ion binding, which is biologically relevant, is played by residues 60–91 made of four octapeptide (OP) repeats (PHGGGWGQ) placed within the N-terminal unstructured domain. Residues 51–59 are homologous (PQGGGTWGQ), but lack the His residue. This region, rich in glycines, is the most conserved sequence among mammalian prion proteins [21]. Other metal binding sites are found in the 90–126 region encompassing a neurotoxic fragment and in the C-terminal region (see Sections 2.5 and 2.6).

2.2 Cu2+ Ion Binding to the Mammalian Prion Protein Octapeptide Repeat Fragment The binding of Cu2+ ions to the –PHGGGWGQ– fragment (N-terminus protected) critically depends on the imidazole moiety of the His residue, which acts as an anchoring site. Around physiological pH the octapeptide involves three nitrogen atom donors, an imidazole and two amide donors from two adjacent glycine residues [22–28]. The interesting feature in the Cu2+ interactions with the mammalian octarepeat fragment is the close proximity of the Trp side chain to the metal ion in solution (Figure 2a) [24] as well as in the solid state (Figure 2b) [25]. This interaction is stabilized by a through-water hydrogen-bond network and it may explain how the N-terminal tandem repeat region of human PrP is able to reduce copper(II) and why the Trp residue plays a basic role in this process [29]. The binding mode of the Cu2+ ion with the octapeptide fragment of mammalian PrPs shown in Figure 2 is generally accepted and it also occurs when Gly3 residues are substituted by more bulky amino acids like Ala 3 or Lys3 units [30]. In the latter cases, however, the stability constants of the complex dominating in the neutral pH and the ability to bind Cu2+ ions are lower than in the case of the Gly3 containing peptide. In the case of the simple monomeric octapeptide, the stability constants (binding ability) obtained for the CuH−2 L complex (L = octapeptide) dominating in −  coordination mode (Figure 2), does the pH range 6–8, with the Nimid  2Namide not differ much from those obtained for simple mono-His peptides [31]. This may suggest that the monomeric octapeptide unit may be unable to compete with naturally occurring ligands to bind Cu2+ ions in natural milieu. Met. Ions Life Sci. 1, 61–87 (2006)

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a

b

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G G

G

Figure 2. (a) Molecular structure for the CuH−2 L complex in solution for the Ac-PHGGGWG-NH2 peptide. (Produced with the data presented in [24]). (b) Molecular representation of the crystal structure (07 Å resolution) of the HGGGW segment complexed with Cu2+ . (Reprinted in part by permission from [25]).

Cu2+ binding to the octarepeat is pH-sensitive and a slight decrease in pH, e.g., from 7.4 to 6.5 results in a drastic decrease of CuH−2 L concentration and an increase in free metal concentration (Figure 3). This feature is a critical property for PrP acting as a metal ion transporter from an extra- to an intracellular environment via the endocytosis mechanism suggested by Millhauser et al. (Figure 4) [25,27]. It is noteworthy that substitution of the Gly3 unit by any Met. Ions Life Sci. 1, 61–87 (2006)

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Figure 3. Species distribution profile for the Cu2+ complexes of Ac-PHGGGWGQ-NH2 in the Cu2+ to peptide molar ratio of 1:1; Cu2+ = 0001 M. (Reproduced by permission from [24]).

Figure 4. Working hypotheses for how the prion protein functions to transport Cu2+ through endocytosis. (Reprinted in part by permission from [25]).

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other Xaa3 sequence distinctly decreases the binding ability of the octarepeat peptide towards Cu2+ , suggesting that the glycine-rich fragment may have a critical impact not only on peptide flexibility, but also on its copper binding ability and transport activity of PrP [30].

2.3 Binding of Cu2+ to Longer Octarepeat Fragments There is a basic question concerning binding of Cu2+ ions to PrP within the octarepeat N-terminal region: does metal ion binding to a monomeric octapeptide and a whole N-terminal tandem region have the same thermodynamic and structural features? Many authors have suggested the presence of the cooperative effect [20,27,32–35], which could distinctly increase the ability of the protein to coordinate metal ions in its flexible N-terminus. The nature of the cooperative effect, if it is present, however, is poorly understood. The potentiometric data supported by spectroscopic studies show clear differences between the binding ability of various octapeptide units: monomeric (1-OP), dimeric (2-OP) and full tetrameric 4-OP = PHGGGWGQ4  PrP fragments [36]. Binding of a single Cu2+ ion by 1-OP, 2-OP, and 4-OP differs distinctly from each other. The first major complex in equimolar solutions is the CuL species (L = peptide ligand) in which one, two or four imidazole nitrogen donors are involved in the coordination in a mono-, di- and tetrameric peptide repeat, respectively. The multi-imidazole coordination is clearly seen in the CuL complex structure with a dimeric octapeptide established by NMR (Figure 5) [36]. The metal ion binding is realized by two imidazole moieties with involvement of two different nitrogens, N and N . This binding mode folds the dimeric octapeptide unit in a very specific way, decreasing the flexibility of the peptide chain. The simultaneous binding of four imidazole side chains results in a very stable complex CuL, which dominates over the physiological pH range. The binding of four imidazoles by a Cu2+ ion in CuL species results in folding of at least 32 amino acid residues around the metal ion, thus critically changing peptide conformation and flexibility (Figure 6). The structural studies of mammalian prion proteins, which established that the N-terminal 100 residue domain is unstructured and highly flexible, were performed in the pH range 4.5–5.5 where all His side chains are protonated. At pH values between 6.5 and 7.5, which are more likely to occur at the cell membrane, the octapeptide repeats of human PrP were found to be structured with a backbone conformation similar to that of a Cu2+ -bound peptide unit [37]. Of course, obvious differences occur in the conformation of the aromatic side chains (His and Trp) involved in metal ion binding between the metal-bound and free HGGGW units (Figure 7). Thus, the conformation of the octapeptide loop appears to depend on pH and copper ion binding. In the pH range 4.5–5.8 Met. Ions Life Sci. 1, 61–87 (2006)

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Figure 5. Structural details of the four best structures obtained for the CuL complex of the prion dimeric octapeptide repeat Ac-PHGGGWGQ2 -NH2 . Structure calculation was performed by restrained molecular dynamics with simulated annealing in the torsional angle space. The figure was created with MOLMOL 2K.1.0. (Reproduced by permission from [36]).

Figure 6. Cartoon representing the plausible structure of the CuL complex of the prion tetrameric repeat Ac-PHGGGWGQ4 -NH2 .

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Figure 7. Comparison of the NMR structure of unligated HGGGW and the X-ray structure of HGGGW-Cu2+ . (Reproduced by permission from [37]).

(the expected pH in endosomes) histidine residues are mainly protonated and the octarepeat region is flexible and unstructured, while in the pH range 6.5–7.5 (pH at the cell membrane) histidines are mostly deprotonated stabilizing the HGGGW-loop conformation, which promotes protein aggregation. Binding of copper ions which changes the side chain conformation may modulate the pH dependent aggregation process [37]. The structural features of the metal-free and copper-bound octapeptide repeat (OPR) region should also have a basic impact on a possible cooperative effect. 100

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Figure 8. Distribution profiles of free and complexed fractions of Cu2+ ions in the presence of both Ac-PHGGGWGQ2 -NH2 (solid line) and Ac-PHGGGWGQ4 -NH2 (dashed line) at 25  C and I = 01 M KNO3  Cu2+ = 2 × 10−3 M; ligand:metal:ligand ratio 2:2:1. (Reproduced by permission from [36]). Met. Ions Life Sci. 1, 61–87 (2006)

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The OPR region with four His residues able to coordinate a Cu2+ ion forms very stable CuL species. The stability of the CuL species involving four imidazole donors of an octapeptide tetramer differs by five orders of magnitude from that of monodentate imidazole binding found in a monomeric octapeptide [36]. Binding of four imidazoles by one copper ion brings four potential binding sites close to each other. When the concentration of copper is low (equimolar or lower ratios) the CuL complex dominates within the biologically relevant pH region. High copper concentration (molar excess) leads to multi-copper binding [36]. The binding of the second metal ion by 2-OP and 4-OP is much more effective than by 1-OP and 2-OP, respectively (Figure 8). These results may indicate that longer OP repeats exhibit some type of cooperative effect derived most likely from the fact of multi-histidine binding, which folds the OPR structure allowing easier binding of the successive metal ions [36].

2.4 Binding of Cu2+ by Chicken Prion Tandem Repeats The tandem repeat region of chicken prion consists of hexapeptide units (His-Asn-Pro-Gly-Tyr-Pro, HNPGYP). This region of chicken PrP seems to bind Cu2+ ions as well [18,27,35]. The presence of Pro within the peptide sequence changes the binding ability of chicken hexapeptide repeat when compared to mammalian peptide. The Cu2+ ion binding begins at the His side chain as it was found in mammalian peptides. However, as binding of the Pro amide nitrogen, although likely [38], is less favorable [39], after binding to the His amide nitrogen the third preferential involved donor seems to be the Tyr side chain phenolate oxygen [40]. Comparison of the binding abilities of monomeric (1-HP), dimeric (2-HP) and tetrameric (4-HP) hexa-peptides strongly suggests that the longer fragments are much more effective ligands (Figure 9) than the shorter repeats, as it was observed in the human variant [36,40]. The closer positions of the His residues within the peptide sequence in the chicken PrP repeat region causes an even stronger ‘cooperative effect’ than in the case of human OPR. However, the mammalian octapeptide is a considerably more efficient ligand for Cu2+ ions than the chicken hexapeptide (Figure 10).

2.5 Cu2+ Binding to the Neurotoxic Prion Protein Fragment The fibrillogenic peptide comprising residues 106–126 of the mammalian prion protein is largely used to study the neurotoxic mechanisms related to prion diseases [41–43]. The hydrophobic core sequence (PrP 113–122, AGAAAAGAVV) is basic for the regulation of neurotoxicity although it is not sufficient for a complete neurotoxic effect [44]. There is growing evidence demonstrating that transition metal ions, especially Cu2+ ions, may affect the aggregation process of Met. Ions Life Sci. 1, 61–87 (2006)

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100 Cu2+

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Figure 9. Distribution profiles of competition between 1-Hex = 1-HP (dotted line) and 4-Hex = 4-HP (dashed line) in coordination of four Cu2+  Cu2+ = 5 × 10−4 M; ligand (1-Hex) to metal to ligand (4-Hex) ratio 4:4:1. (Reproduced by permission from [40]).

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Figure 10. Distribution profiles of competition between human octapeptide (AcPHGGGWGQ-NH2 ) 1-Oct = 1-OP and chicken 1-Hex = 1-HP in coordination of one Cu2+ Cu2+ = 1 × 10−3 M; ligand (1-Oct) to metal to ligand (1-Hex) ratio 1:1:1. (Reproduced by permission from [40]). Met. Ions Life Sci. 1, 61–87 (2006)

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PrP 106–126 [45]. His111 was usually assumed to act as the main coordination site for Cu2+ ions. The involvement of methionines (109 and 112) was also suggested [45,46], but all later studies did not reach any evidence of copper binding to the methionine sulfur [27,34,47–49]. His111 acts as the anchoring site for the Cu2+ ion, which may also coordinate to the adjacent amide nitrogen donors [34,47,48]. Neurotoxic properties of the PrP 106–126 fragment are the same for the N-terminal protected and unprotected peptide, but binding abilities towards Cu2+ may be different in these two cases. PrP 106–126 with unprotected N-terminal Lys involves its amino group in metal ion coordination [47,48]. Cu2+ binds the N-terminal and His111 imidazole nitrogen donors and the terminal carboxylate is stabilizing the coordination sphere through ionic interactions (Figure 11) [48]. The involvement of the Lys amino group causes that copper binding to PrP 106–111 is much more effective than to the N-protected version of this peptide. Close to His111 there is a second His residue at position 96. When the PrP 91–105 fragment is added to the neurotoxic PrP 106–126 peptide, the possibility arises of simultaneous binding of His96 and His111 to the same metal ion. However, the fifth Cu2+ ion outside of the octarepeat region was suggested to bind at His96 only [27,34]. The three dimensional likely structure of PrP with five bound metal ions suggested in the latter case is shown in Figure 12. The involvement of both, His96 and His111 in the coordination of one copper ion

Figure 11. NMR structure of the Cu(II) complex of the prion neurotoxic peptide. (Reproduced by permission from [48]). Met. Ions Life Sci. 1, 61–87 (2006)

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Figure 12. Model of the three-dimensional structure of PrP 61–231 with coppers included. (Reproduced by permission from [34]).

(Figure 13) could create potentially the most powerful metal binding site [49], but really convincing thermodynamic and structural data are still missing. The interaction of copper with the PrP 91–115 fragment is suggested to induce the -sheet conformation [49]. It has been shown that the neurotoxic peptide region of PrP is vital for PrP propagation and protein aggregation and it could be linked with copper binding [27,28,34,49,50]. The Mn2+ ions seem to have a critical impact on PrP oxidative chemistry [51]. The neurotoxic peptide seems to interact with Mn2+ ions quite efficiently [48]. However, the binding sites are completely different than those of Cu2+ ions. The Mn2+ ions interact with the neurotoxic peptide via the carbonyl oxygen atoms of Gly124 and Lys125 amide bonds and they approach the carboxylate of Gly126. Mn2+ ions also interact, most likely through hydrogen bonding by a metalcoordinated water molecule, with the imidazole ring of His111 [48]. Thus, even if manganese may not be able to substitute copper ions it could affect oxidative activity of metal-bound PrP using the oxygen donor set of the prion protein.

2.6 Interaction of Cu2+ Ions with Globular Domain of the Prion Protein A globular C-terminal domain of human PrP extending from residues 125 to 228 consists of three helices comprising residues 144–154, 173–194, and 200–228 and the anti-parallel -sheets comprising residues 128–131 and 161–164 [14]. Met. Ions Life Sci. 1, 61–87 (2006)

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Figure 13. Model of PrP 57–231 showing the binding sites of Cu2+ ions. (Reproduced by permission from [49]).

This domain contains several His residues [52], which may act as anchoring sites to bind Cu2+ ions (Fig. 14) [31]. There are some indications that a peptide corresponding to the second helical region of the human PrP shows some ability to form fibrils and it possesses a Cu2+ -mediated neurotoxicity [53]. The abnormal interactions with model membranes may also have some impact on the neurotoxicity of this fragment [54]. His187 acts as an anchoring site for Cu2+ ions and the binding motif seems to be similar to those found in the other fragments of mammalian PrP [55].

2.7 The Binding of Cu2+ Ions and Superoxide Dismutase Activity of Prion Protein Many investigations have indicated that copper interactions with mammalian PrP are closely related to oxidative and antioxidant (superoxide dismutase, SOD) activity [9,56–62]. As a matter of fact, deletion of the octarepeat region impairs the PrP SOD activity [9]. The redox activity of the protein-bound copper ion Met. Ions Life Sci. 1, 61–87 (2006)

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Figure 14. Copper(II) binding sites in the C-terminal domain of the prion protein. (Reproduced by permission from [52]).

suggests the presence of the Cu+ /Cu2+ or Cu2+ /Cu3+ ion pair, which is able to reduce, oxidize or dismutate oxygen species. The SOD activity could rather suggest the possible reduction of Cu2+ to Cu+ during the catalytic cycle. However, in all cases discussed above, in binding PrP fragments, Cu2+ forms stable tetragonal complexes with at least three potent nitrogen donor atoms in the coordination sphere of the metal. This geometry and the donor set involved are hardly accepted by a coordinated Cu+ ion. Thus, the Cu2+ to Cu+ reduction process could need much energy to adopt a new geometry and possibly a new donor arrangement. It follows that the PrP with four Cu2+ ions coordinated within the octarepeat region and one or two more ions within the neurotoxic fragment is not likely to be a very effective SOD-type catalyst. However, speciation studies have shown that for lower copper concentrations (e.g., an equimolar ratio) around physiological pH, the major complex is characterized by Cu2+ coordinated to three or four His side chains, making the metal coordination sphere quite flexible and much easier adjustable for the Cu+ oxidation state [36]. Thus, the catalytic activity could derive from the prion protein not fully loaded with Cu2+ ions.

2.8 Affinity of the Prion Protein to Bind Cu2+ Ions In describing metal ion binding to proteins, biochemists usually use the affinity values. The affinity of metal ion binding to a protein is usually reflected by the dissociation constant Kd . The available data for Cu2+ -PrP systems are in large disagreement, especially those proposed for Cu2+ binding to the octarepeat domain [32,33,35,63,64]. Some studies based on mass spectrometry as well as fluorescence assays demonstrated cooperative binding with Kd in the low Met. Ions Life Sci. 1, 61–87 (2006)

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micromolar to nanomolar range [33,63], while other results obtained with the fluorescence method, in the presence of a competitive glycine buffer, suggest that PrP exhibits a much higher affinity to Cu2+ , comparable to that found in copperdependent superoxide dismutases [64]. Two copper sites exhibit Kd values in the 10−14 − 10−13 range. According to that assumption a biphasic mechanism of Cu2+ binding was proposed with a single copper ion binding with Kd of 8 fM followed by a weaker Cu2+ binding with an affinity of 10 M. There is, however, no direct evidence for two modes of metal ion binding. The CD results obtained by Garnett and Viles are in large disagreement with those suggesting dissociation constants in the fM range; in fact, it was shown that glycine readily removes Cu2+ ions from the octarepeat domain of the protein, thus suggesting an affinity in the micromolar range [35]. The binding affinity of Cu2+ to the other domains of the prion protein has been of minor interest. Again, the data available largely disagree. The affinity suggested for Cu2+ interacting with the fragment 91–120 was reported from micromolar [20,33] through nano- [49] to fentomolar values [64]. Glycine competition assays have suggested that, in contrast with the octapeptide repeat domain, the binding of Cu2+ to extended neurotoxic domain (PrP 91–115) is much stronger [49]. Even histidine was not able to compete effectively with the latter PrP fragment. This finding, if real, could be of crucial importance, indicating that extracellular PrP is able to bind copper under physiological conditions [49]. The high affinity of Cu2+ binding to PrP is strongly suggested by in vivo studies showing that PrP binds indeed extracellular Cu2+ [20].

3 INTERACTIONS OF METAL IONS WITH THE AMYLOID PRECURSOR PROTEIN AND ITS FRAGMENTS The amyloid precursor protein (APP) plays a fundamental role in the development of Alzheimer’s diseases (AD), the most common neurodegenerative disorder. The cause of this disease is closely related to the aggregation of the -amyloid A peptide comprising 39–42 amino acid residues (human variant: 40 or 42 amino acids). There are suggestions that A precipitation and toxicity in AD are caused by abnormal interactions with metal ions like zinc, copper, and iron [65]. APP and its fragments may be also involved in regular metal ion homeostasis (see Chapters 4 and 5 of this volume).

3.1 Interactions of Metal Ions with the -Amyloid Peptide Deposition of amyloid plaques in the parenchyma and vasculature of the brain is characteristic for AD. A peptide is the principal constituent of these plaques and it is believed to be responsible for the neurotoxicity associated with Alzheimer’s Met. Ions Life Sci. 1, 61–87 (2006)

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disease. The formation of peptide aggregates may be mediated by some essential metal ions. Earlier studies have shown that metal ions like Cu2+ or Zn2+ may induce very efficient aggregation of soluble A Zn2+ ions induce aggregation at pH 7.4 in vitro and this reaction is reversible with chelation [66,67], while Cu2+ ions are much more effective in conditions representing physiological acidosis (pH 6.6–6.8) [68]. It was also shown that aggregation is negligible for the rat (mouse) peptide, which differs from the human variant by three substitutions: Arg5 → Gly Tyr10 → Phe, and His13 → Arg. Preliminary data clearly indicated the role of metal interaction with the imidazole donor set of the His side chain in the aggregation process and possible multi-copper binding to A [66–68]. Recent studies based on EPR measurements seem to indicate that hA binds Cu2+ in a mononuclear metal binding site with a metal: peptide ratio of 1:1 both in soluble and fibrillar form [69]. However, a short report has appeared suggesting that Cu2+ ions may inhibit the aggregation process in the wide pH range 5–9 [70]. As none of these studies presented a detailed evaluation of the coordination pattern (speciation versus pH) in the considered systems, it is difficult to compare the relevance of the conclusions reached in these studies [66–70]. The analytical data concerning Alzheimer’s plaque formation are extensive although there was not much analytical information about intact plaques. A recent work using Raman spectroscopy therefore provided a real progress towards the comprehension of the analytical chemistry of Alzheimer plaques [71]. The study has confirmed the composition of isolated amyloid plaque cores, the protein/peptide conformation, and possible metal ion binding sites. The Raman spectra confirmed that also in the intact plaque cores both Zn2+ and Cu2+ ions are coordinated to histidine residues and chelating agents may reverse metal ion binding leading to the loosening of the -structure and possible solubilization of amyloid deposits. The human variant of the A peptide hA contains three histidine residues at positions 6, 13, and 14, which besides the N-terminal amino group can act as the potential anchoring sites for metal ions, especially Cu2+ [31]. The interesting motif typical of hA coordination seems to consist of the His13–His14 pair serving two adjacent imidazoles able to bind the metal ion [72]. The protected hexapeptide fragment hA 11–16 Ac-EVHHQK-NH2  involves both imidazole and amide nitrogen donors to form a very stable complex with the Cu2+ ion [72]. The human variant was found to be much more effective in copper binding than that of the mouse or rat A mA having His13 substituted by Arg. This result indicates that both His residues could be involved in metal ion coordination in vivo affecting the peptide aggregation process. Comparison of the copper binding abilities of hA 11–16 with that of hA 11–28 has shown that in both cases the metal ion coordination donor set is identical but the stability of the complexes formed by the longer peptide are one to two orders of magnitude Met. Ions Life Sci. 1, 61–87 (2006)

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higher compared to those of hA 11–16 [73]. This stabilization may result from some structural organization of a peptide ligand in Cu2+ complexes. The studies with protected amyloid peptide fragments hAc-A-NH2 1–16 and 1–28 have indicated the likely involvement of three His residues in binding one Cu2+ ion in the wide range of pH 5–8 [74]. The mouse variant mAc-A-NH2 may use only two His imidazole side chains to bind a metal ion in the pH range 5–7, forming less stable complexes. The unprotected A 1–16 and 1–28 peptides involve also the N-terminal amino group [74,75]. In hA, the His13–His14 pair seems to be critical for metal ion binding for both protected and unprotected peptides. There is no involvement of Tyr phenolate oxygen in Cu2+ ion binding [74,75] in contrast to what was suggested earlier [76]. The latter work suggested also the formation of the dimeric complex with two metal ions bridged by an imidazole moiety, but these findings were not supported by other studies [69,74,75]. The mechanism of induction of peptide aggregation upon metal ion coordination can be accounted for by different hypotheses. Above pH 9, when the amide nitrogen donors dominate Cu2+ ion coordination [74,75], the metal ion is unable to interact with more than one peptide molecule [77]. However, around pH 6.6–6.8 Cu2+ coordinates to the N-terminal amino and the imidazole nitrogen atoms. Such metal ion binding could allow Cu2+ to form cross-links between different peptide molecules leading to peptide aggregation. In the case of mA Cu2+ binding to His residues is weaker and the amide involvement occurs at lower pH making inter-peptide metal-induced cross-links ineffective. It is also possible that metal ion binding induces changes in peptide conformation and solubility leading to formation of aggregates. Cu2+ coordination will change the overall charge of the peptide molecule. Thus, copper binding will have a profound impact on the electrostatic behavior of A having a strongly charged N-terminal and the hydrophobic C-terminal, which may result in the aggregation process [75]. CD, EPR, and preliminary NMR studies on hA and its variants in which His residues were substituted by Ala have strongly supported the critical involvement of imidazole nitrogens in metal ion coordination [75]. Cu2+ binds to the N-terminus of A and three His (Figure 15). His13 seems to be the critical residue for metal ion coordination. Due to the lack of this residue in mA, copper does not induce peptide aggregation [75]. CD spectra suggest that binding of Cu2+ does not induce a typical -sheet conformation found in A fibrils. Although the stability [74] and affinity constants for the Cu2+ -hA system are known, it is still difficult to evaluate the physiological significance of metal ion binding to hA. The reported affinities differ very much from each other [75] and the stability constants are difficult to be compared with the ‘physiological complexes’. There is, however, general agreement that hA has sufficient affinity to bind copper at physiological levels of Cu2+ [75]. The details about the binding ability of Zn2+ ions are largely understood. The major binding sites in hA for Zn2+ seem to be three His residue [76]. Met. Ions Life Sci. 1, 61–87 (2006)

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Figure 15. Model of A coordinating to Cu2+ . (Reproduced by permission from [75]).

The N-terminal region of metal-free hA 1–28 lacks a stable structure, but its C-terminal is better defined including the helical region starting from Ala21 [78]. The rat variant, rA 1–28, exhibits a more extended helical structure and it is better defined (Figure 16) [79]. Since the helical to -strand transition of the human peptide results in amyloid formation, the more ordered conformation of rA may protect the amyloid formation in aged rats [79]. In the case of rA the binding sites for Zn2+ have been suggested to be His6, His14 and Arg13 [79].

Figure 16. The solution structure of rat A 1–28. (Reproduced by permission from [79]). Met. Ions Life Sci. 1, 61–87 (2006)

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Figure 17. (left) The solution structure of rat A 1–28 and (right) its zinc(II) complex. (Reproduced by permission from [79]).

A good fit of the NMR titration curves was obtained for 1:1 stoichiometry which was suggested earlier for rA 1–40 using Scatchard analysis [80]. The affinity of Zn2+ ion binding to rA is much weaker 4 × 102 M−1  [79] than that found for hA 9 × 106 M−1 . The binding of Zn2+ to rA 1–28 reduces the flexibility of the N-terminal peptide region leaving the C-terminal well folded (Figure 17) [79]. In the brain, A can fold into a well soluble random coil and helical structures or aggregating -sheet structures [82,83]. Thus, the rA 1–28 peptide with its longer helical segment than it is found in hA 1–28 is protecting aggregation in the aged rat brain [79]. Aggregation of hA may be facilitated in aged brain when zinc concentration in the brain is increasing, while in rats the cerebral zinc concentration may not be high enough to induce peptide aggregation [79].

3.2 Metal Ion Binding Sites in the Cysteine Region of the Amyloid Precursor Protein Although the normal biological function of the amyloid precursor protein is badly understood, the discovery of metal Zn2+ and Cu2+  binding sites allows to suggest a role of APP in metal ion homeostasis [84–87]. A copper Met. Ions Life Sci. 1, 61–87 (2006)

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binding site was located within residues 135–155 of the cysteine-rich region of APP [88]. Evidence has been reached for a copper-binding APP superfamily with and without conserved His at positions 147, 149, and 151 [89]. The copper binding domain in the proteins of the higher species indicates higher reducing activities and lower affinity to Cu2+ . The unique redox properties of the APP family could have critical impact on still unknown biological functions [89]. The amino acid sequence between Cys144 and Cys158 of human APP comprises three His residues: –CETHLHWHTVAKETC–. The potentiometric and spectroscopic studies with the short fragment Ac-His-Leu-His-Trp-His-NH2 have shown that all three His residues can be involved in Cu2+ binding via their imidazole side-chains [90]. This coordination mode is characteristic for the CuL complex that is a major species in the pH range 5–6. Around the physiological pH the dominant complex is CuH−2 L in which the three imidazoles and two amide nitrogens are involved (Figure 18) [90]. A coordination mode with five nitrogen donors is very effective in binding of copper ions. Very similar results were obtained for the longer (145–155) Ac-Glu-Thr-His-Leu-His-Trp-His-ThrVal-Ala-Lys-Glu-Thr-NH2 peptide [91]. The whole copper-binding domain (CuBD) which comprises residues 124–189, however, has a well ordered structure with only three N-terminal residues unstructured (Figure 19) [92]. Preliminary studies on copper ion interactions with the whole CuBD indicated that only two His residues (147 and 151) are involved in metal ion binding (Figure 19) [92]. The additional coordination of Tyr168 via phenolate oxygen and Met170 via thioether sulfur is also assumed. This tentative structure could be an effective site for both Cu2+ and Cu+ ions creating an efficient redox center [92].

Figure 18. Structural model of the Cu2+ complex of the pentapeptide fragment of the APP copper binding domain (Ac-HLHWH-NH2 ). (Reproduced by permission from [91]). Met. Ions Life Sci. 1, 61–87 (2006)

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Figure 19. A model of Cu(I) coordinated in a tetrahedral configuration to His147, His151, Tyr168, and Met170. (Reproduced by permission from [92]).

4 CONCLUDING REMARKS The coordination pattern in the Cu2+ -PrP fragments are relatively well understood, although several questions still remain including the basic problem of a cooperative effect in metal ion binding. It is also not clear whether PrP really needs to be completely loaded with copper ions to be biologically relevant. In all cases the His residue is an anchor and a basic binding site for the Cu2+ ion and several His can be involved in the coordination of one copper ion but the relations between the specific binding site and its biological implications are still not understood. The other basic chemical question, still open, concerns the SOD-like activity of the Cu2+ -PrP system and a clear answer is needed to the question: Which binding site and species are relevant for the redox activity of prion bound copper? The role of His residues is also critical for the binding ability of Cu2+ to other proteins like APP fragments, including the A peptide. The formed complexes are thermodynamically not very potent and there is the very biological question to be answered. How do Cu2+ ions and particular proteins meet in the natural milieu? The binding of metal ions has a critical impact on protein conformation and it is likely that metal ions induce in the protein structure (e.g., -structure) the ability to aggregate. This problem still needs more data to make clear relations between metal ion binding and neurological disorders. Met. Ions Life Sci. 1, 61–87 (2006)

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ACKNOWLEDGMENTS Financial support of the Polish State Committee for Scientific Research (KBN 4 T09A 054 23) and MURST COFIN 2001 is gratefully acknowledged. M. L. would like to thank the Foundation for Polish Science (FNP) for a Domestic Grant for Young Scientists.

ABBREVIATIONS aa AD ALS APP A CD CuBD hA HD HP hPrp mA mPrP OP OPR PrPc rA ROS shPrP SOD TSE

amino acid Alzheimer’s disease amyotrophic lateral sclerosis amyloid precursor protein -amyloid peptide circular dichroism copper-binding domain human -amyloid peptide Huntingdon’s disease hexapeptide (= Hex) human prion protein murine -amyloid peptide murine prion protein octapeptide (= Oct) octapeptide repeat prion protein rat -amyloid peptide reactive oxygen species Syrian hamster prion protein superoxide dismutase transmissible spongiform encephalopathy

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4 Metallic Prions: Mining the Core of Transmissible Spongiform Encephalopathies David R. Brown Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK

1 INTRODUCTION 2 HISTORICAL CONNECTIONS BETWEEN COPPER AND TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES 3 COPPER BINDING TO PRION PROTEIN 4 COPPER COORDINATION BY PRION PROTEIN 5 COPPER UPTAKE AND PRION PROTEIN INTERNALIZATION 6 PRION PROTEIN AS AN ANTIOXIDANT 7 MANGANESE BINDING 8 TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES AND METALS 9 CONCLUSIONS ABBREVIATIONS REFERENCES

89 92 93 95 97 101 104 107 109 109 110

1 INTRODUCTION Transmissible spongiform encephalopathies (TSEs) have cast a shadow of fear over the world especially for those who like a good piece of beef steak [1]. However, the reality of the variant Creutzfeldt–Jakob disease (CJD) epidemic is

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that it has not happened [2]. Nevertheless, the possible transmission of a disease by a protein has sparked considerable interest in these diseases despite their very low occurrence. In addition the availability of animal models to study prion diseases has meant that they have become an exemplar for the study of neurodegenerative diseases in general [3]. The family of TSEs or prion diseases include both animal and human forms. The first studied human disease became known as Creutzfeldt–Jakob disease after the German neuropathologists who first described the changes in human patients [4]. Kuru, a disease of natives of New Guinea was linked to the consumption of human brain that carried CJD [5]. Eventually, inherited human prion diseases such as Gerstmann–Sträussler–Scheinker syndrome [6,7] and fatal familiar insomnia were also described [8]. The inherited forms of disease are linked to point mutations or insertions in the gene that encodes the prion protein (prnp). The three most common animal forms of TSE are bovine spongiform encephalopthay (BSE) of cattle [9], scrapie of sheep [10] and chronic wasting disease (CWD) of deer and elk [11]. BSE is still widespread in Europe despite attempts to eradicate it by preventing cattle from eating possibly infectious food sources. The origin of BSE remains unknown and attempts to link it to scrapie have proven to be an inadequate explanation [12]. Similary, scrapie and CWD have no clear cause and appear to develop sporadically, often associated with particular environmental locations [13]. Experimental models of TSEs depend upon the inoculation of mice or hamsters with extracts from the brains of animals with one of the TSEs. This is therefore a form of experimental infection. Although such experimental transmission can occur in the laboratory, TSEs are not contagious diseases. Transmission between individuals largely does not occur, although it is possible that a small number of vCJD cases have been passed to other patients through blood transfusions [14]. CJD is the most common form of human prion disease. It is different to variant CJD in that this latter form affects mostly young people with a specific genotype. To date, all patients with vCJD have two copies of the 129Met (coding methionine at codon129) allele of prnp. All prion diseases are linked to the deposition of an abnormal isoform of the prion protein in the central nervous system of the affected individual [3]. This abnormal isofrom PrPSc  is accepted as the infectious agent of the diseases and is the probable cause of the pathology associated with the disease [15]. PrPSc is highly rich in -sheet structure, is highly resistant to protease digestion and aggregates to form fibrils in vitro. Although central to the disease process, it is still not completely clear that this form of the protein is the one that can cause transmission of disease between individuals or that it is all that is essential for the disease. Without expression of the protein prion disease cannot develop because mice that have been transgenically altered to lack expression of PrP are resistant to TSEs [16]. The ‘protein-only’ hypothesis remains largely accepted and has been supported by recent studies that show that recombinant PrP can Met. Ions Life Sci. 1, 89–114 (2006)

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be used to initiate a prion-like disease in mice and that this new disease can be transmitted to other mice. Yet, other researchers still argue that the protein itself is insufficient for a true TSE. Although it is unclear what exactly causes prion disease and the mechanism of conversion of the normal cellular isoform PrPc  to PrPSc remains undescribed [17], recent research has made strong inroads into understanding the nature of the substrate for prion diseases, the normal isoform of the prion protein. PrPc is a glycoprotein [18] expressed by many cell types, but especially by neurons [19]. The protein is concentrated at synapses [20] and is therefore thought to have some special role in neuronal function. It is produced in cells as a single monomeric polypeptide of around 250 amino acids in length. The regulation of its expression is still poorly characterized, but it is highly regulated with not only a promoter, but also by regulator regions in exons 1 and 2 encoded by the messenger RNA that are not transcribed into protein [21]. The third exon of PrP encodes the whole open reading frame and contains highly conserved regions. Prion protein knockout mice develop normally and show little in the way of behavioral difference to wild-type [22]. Nevertheless, there have been a large number of reported differences in the mice and especially there are distinct difference in the response of neurons cultured from the brains of the mice. These differences suggest that PrPc aids to protect neurons from cellular responses to stress [23]. The essential requirement of host prion protein expression for both, the transmission of the disease and the triggering of neuronal death, implies that understanding the nature of the normal isoform of the prion protein is inseparable from discovering the mechanism of disease transmission and progression. The mechanism of conversion of the protein remains unknown, but is thought to involve the formation of seeds or small aggregates of PrP which can then catalyze rapid conversion to further host protein to the abnormal isoform. This issue is further complicated by the existence of strains [24]. Strains of prion diseases (especially scrapie) are characterized by different characteristics of the resulting disease when applied to the same species of host. Thus, the characteristics will remain the same when transmitted to different individuals of the same species of animal or even the same breed of mouse. These characteristics include location and severity of neuronal loss and gliosis, extent and location of PrPSc deposition, incubation time for the disease and duration of symptoms. These characteristics for BSE in cattle may change when the disease is transmitted to mice, but within mice or cattle the characteristics are the same in different individuals after subsequent passages. This secondary information suggests that if the protein is the sole cause of the disease, then somehow this is encoded by the way the conformation is altered during conversion of PrPc to PrPSc . There is currently no explanation for how this information could be encoded by the conformational change. Prion diseases have only been identified in mammals. However, other vertebrates express homologues of PrP. These include birds, reptiles, amphibians Met. Ions Life Sci. 1, 89–114 (2006)

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and fish [25–29]. The sequences identified in reptiles and birds show very high homology to mammals, except that in the N-terminus mammals have four or more repeats of an octamer while birds and reptiles have five or more repeats of a hexameric region. Amphibians and fish express one or more homologues of PrP, but essential domains are quite different and it is unclear if these homologues would serve the same function in these lower vertebrates. The hexameric or octameric repeat regions are the suggested binding place for copper to PrPc . Investigation of the possible function of these regions leads to the suggestion that these sites could be copper bindings sites [30,31]. Early studies of peptides related to these regions were the first to demonstrate interaction of copper occurs at these sites. These findings triggered a wave of interest in the potential of PrPc to be a metalloprotein. This review will deal with current understanding of metal interactions with PrPc and their possible consequence for the function of the PrPc and disease progression in TSEs.

2 HISTORICAL CONNECTIONS BETWEEN COPPER AND TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES The prion protein was first isolated by Prusiner and colleagues in 1982 [15]. The gene for PrP was also first identified in 1985 [32]. The relationship between this protein and prion disease dominated the field for many years. Scientists continue to try to unravel the mechanism behind the conversion of PrP from the normal to the abnormal isoform. The relation between copper and TSEs dates back to before the discovery of PrP. The first patients with the human disease CJD were studied by the German neuropathologist Creutzfeldt. Creutzfeldt’s original reports of patients included cases which later turned out to be Wilson’s disease. Wilson’s disease is a neurological condition linked to mutations in a copper-transporting ATPase. The pathology in the brain caused by the disease often results in spongiform changes similar to prion disease. CWD, the TSE of deer and elk was first described in the 1960s. It was originally believed to be caused by copper deficiency. Deficiencies in trace elements such as copper and selenium have long been known to cause neurological or other diseases in animals. In particular, regions of the world low in copper have been known for some time to be associated with scrapie. The regions of Colorado where CWD first existed were known to be regions where copper supplementation was necessary. Many similarities were noted by researchers looking at sheep with scrapie or copper deficiencies in sheep such as sway-back. Researchers looking at both scrapie and the toxicity of the copper chelator cuprizone [33–37] noticed a similarity between the two diseases (1960–1970s). Biochemists including Kimberlin identified a number of similarities and the level of metabolites that were similar between cuprizone toxicity and scrapie [36,37]. Unfortunately at that time Met. Ions Life Sci. 1, 89–114 (2006)

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experiments combining cuprizone and scrapie failed to identify any significant effect or relationship [35]. At this point there was little further research on this topic until after purification of the prion protein. In 1992 Pan and colleagues [38] reported a method of purification of prion protein from hamster brain. The technique was based on immobilized metal affinity chromatography. In this technique copper was immobilized on a column and the copper binding proteins were trapped on the copper. Selective elution with salt led to the isolation of the prion protein. It was also found that metabolically cleaved prion protein lacking the N-terminus did not bind to this column. At about the same time it was suggested that copper interaction with the N-terminal repeats might catalyse the conversion of PrPc to PrPSc [39].

3 COPPER BINDING TO PRION PROTEIN The sites at which copper binds to PrPc are still a matter of debate, but there is no doubt that the octameric repeat region of the protein is the main site of copper binding. The number of copper binding sites in this domain appears to equal the number of histidines available to bind the metal [40,41]. However, some studies have suggested that there are more than four sites for copper binding [42]. A putative fifth copper binding site is, however, a matter for discussion. There are now a number of views on this issue. Some authors have presented evidence that copper is bound in the copper(II) form within the C-terminal domain of the protein. This proposed type-2 site is disrupted by a mutation at amino residue 198 suggesting that the histidine involved in binding might be that at amino residue 187 [42–44]. In contrast, it is suggested that the site of copper binding is within the region of the toxic peptide PrP 106–126 [45]. The histidine within the peptide would then be involved in the binding site with additional coordination from other nitrogen atoms in the vicinity and also requiring two peptides to create the site. The initial suggested fifth site was based on studies of a fragment from amino residues 23–98 [40]. Therefore, possibly a histidine at amino residue 95 or 96 (depending on animal species of the protein’s origin) might be involved in the binding of copper at the fifth site. Caution should be adopted when considering these sites and suggesting that this site might be involved in the conversion of the protein. These regions of the protein are not as highly conserved as the sequence within the octameric repeat region and coordination of copper at these sites would be different from species to species. Most of these studies have also used fragments of the protein rather than the whole protein as synthesized in cells. Another such study based on X-ray solution scattering has proposed yet another fifth site. This site is not within the globular domain of the protein and was detected in the region spanning residues 91–120. This was determined in a mutant form of the protein lacking amino Met. Ions Life Sci. 1, 89–114 (2006)

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residues up to 90 in the human sequence [46]. Recent progress in X-ray scattering methods have allowed low-resolution structures of a number of proteins and their complexes to be established from scattering data alone. The technique (XAFS) was applied to obtain such a structure for PrP 90–231. The interaction was studied at low concentrations of copper so that any interaction was specific for the highest affinity site. However, it should be noted that it is quite possible that deletion of the N-terminus fragment from such a protein might alter the affinity that copper would have for this site. For example, the site might be out-competed by the N-terminal octameric repeats. Alternatively, this fifth site might be a point of transfer for copper from this site to the N-terminal repeats. Analysis of this protein was carried out at both high and low temperatures and both studies indicated that two histidines were involved. These were the histidines at amino residue 96 and 111 of the human sequence. Other studies with the mouse sequence support the idea that these two histidines form the fifth site [47]. Although the involvement of two histidines is unusual, there is evidence for this from other proteins such as azurin and superoxide dismutase. Based on X-ray data the authors propose that the two histidines come within 4 Å of each other. This model suggests the possible interaction of the copper with oxygen molecules in glutamine-98 and water surrounding the sulfur of methionine-109. NMR studies of this protein suggested that this site has an affinity of 4 fM [48]. It was proposed [46] that a drop in pH from neutral on the outside of the cell to the acidic environment inside endosomes would result in a drop of 8 orders of magnitude in the affinity. This would imply that copper bound to this site could be released inside the cell. Thus, the site would comply with the proposal that PrPc transports copper into the cell [49]. However, such high affinity would also be associated with this site with the golgi, implying that the site would be filled before the protein reaches the cell surface. Therefore, although this is an interesting idea, the model clearly needs further thought. These analyses all assume that fragments lacking the octameric repeat region are valid models of copper binding. This can only be true if these sites have higher affinity in the full-length molecule than the sites in the octameric repeat region. This has not been shown to be true. Thus, at present the relevance of such studies is not entirely clear. Unfortunately, although there have been quite a number of studies of the copper affinity for the octameric repeat region, most of these studies have also been based on fragments. The highest affinity observed was that reported by Jackson et al. [48]. Again, they reported a femtomolar affinity for copper binding to a peptide based on the octameric repeat region. This study had the advantage of using copper in a physiologically relevant form, chelated to glycine. Other studies carried out with larger fragments did not report such high affinities [40]. Other studies using free copper found slightly higher affinities [50] but, as these experiments were carried out under distinctly nonphysiological conditions, their relevance is not clear. Our recent work has been the first to Met. Ions Life Sci. 1, 89–114 (2006)

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use both full length recombinant PrP and physiological conditions. Two separate techniques confirmed that the affinity of copper for PrP is in the femtomolar range for the first site, picomolar for the second and third site and nanomolar for the fourth site. In contrast, the supposed fifth site showed only low micromolar affinity [51]. Interestingly, we also showed that truncation of the molecule resulted in a significant increase in the copper affinity for this site to nanomolar affinity [47,51], but not to the same level as observed by Jackson et al. [48].

4 COPPER COORDINATION BY PRION PROTEIN The first study attempting to determine the coordination of copper to the octameric repeats was that of Viles et al. [52]. This work showed the pH dependence of copper binding in that region. Although a peptide based on the octameric repeat region showed reduced copper binding at acidic conditions below pH 6, studies with full-length protein [53] indicated that copper was retained at pH 5.5. In the absence of copper octarepeat peptides showed no structure in solution as determined by circular dichroism (CD). However, on binding copper the peptides showed CD changes characteristic of turns and structured loops. Another study using Raman spectroscopy suggested a helical structure was formed [54]. However, the NMR studies of Viles et al. [52] were probably more accurate. Additionally, as suggested by Brown et al. [40] binding of copper to the repeats showed cooperativity. This implies a higher affinity for an individual atom of copper when more than one bind. Cu(II) can adopt a range of coordination geometries in peptides and will coordinate to nitrogen, oxygen and sulfur ligands. The electron spin resonance (ESR) spectra of PrP peptides are typical for the type-2 class of Cu(II) sites in proteins or complexes. These have tetragonal coordination geometry, either square-planar or square-planar with weak axial ligands. The g and a values obtained from ESR give insights into the type of ligands involved. These factors were all identical for each atom of Cu(II) added to the octarepeat peptide which can bind four atoms of Cu. This implies the sites have identical coordination. There was great similarity between ESR and CD spectra for either two or four repeats. The model proposed by Viles et al. [52] suggested that each copper atom was coordinated by the nitrogens of two imidazole rings from two histidines and a nitrogen in a proline residue as well as an interaction with the oxygen of a water molecule. Further analysis has suggested that this model is not quite correct. AronoffSpencer et al. [55] used similar techniques to study other peptides based on the octarepeats of the prion protein. Their electron paramagnetic resonance (EPR) spectra were composed of two components, the first of these arising from three nitrogen atoms and one oxygen while the second component consisted of two nitrogens and two oxygens. The first component comes from studies of peptides Met. Ions Life Sci. 1, 89–114 (2006)

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that were equal to or longer than a single octarepeat. The sequence HGGGW gave a pure spectrum of this kind, suggesting that this fragment binds Cu(II) in a fashion that is nearly equivalent to that of the multi-repeat peptides and therefore comprises the fundamental binding unit. Thus, each metal ion of the four bound by the octameric repeat region may bind as a single atom to a single HGGGW region. In this three nitrogen coordination model Aronoff-Spencer et al. [55] suggest that the first nitrogen is contributed by the histidine imidazole ring and the other two come from deprotonated backbone amide groups. That each copper ion is bound mainly by residues within an octarepeat is further supported by titration experiments on 1, 2 and 4 octarepeat containing peptides and has shown that the ratio of copper binding is 1:1. These findings are different to those of Viles et al. [52] who proposed that Cu interacted with two imidazole rings. An imidazolate bridge between a single pair of Cu(II) ions leads to an exchange interaction and the resulting EPR transitions take place solely among the twospin triplet levels. If, in addition the exchange interaction is large compared to the kT, the Boltzmann factor will favor the singlet ground state, thereby decreasing the integrated EPR absorption signal relative to that expected from uncoupled spins. The work of Aronoff-Spencer et al. [55] found no evidence for this, suggesting that exchange coupling does not occur in binding of copper to multirepeat peptides. The finding that one repeat binds one copper ion agrees with the findings of Miura et al. [54] who also suggested from Raman studies that HGGG is the fundamental binding unit. For a fully Cu(II) loaded protein the model of Aronoff-Spencer et al. [55] suggests that the metal ion binding sites in the N-terminal domain are like beads on a string where each bead is a Cu-HGGGW segment separated by intervening Gly-Gln-Pro links. Interestingly Gly and Pro often participate in -turns and thus the intervening links may provide a mechanism for allowing the Cu binding segments to fold and perhaps collapse together. Coordination dominated by a single histidine would make copper binding to PrPc pH sensitive, supporting the idea that it may act as a copper transporter. Following on from the work described above the group of Millhauser continued studies on the copper binding ability of the HGGGW sequence. Burns et al. [56] have produced the first crystal structure of copper binding to this region of the protein. The binding of copper to this segment as determined by crystal structure analysis are consistent with EPR and other spectroscopic studies of copper binding to the complete single octameric repeat peptide. In the crystal structure the HGGGW-Cu complex includes six ordered water molecules. Thus, the Cu(II) is configured in a pentacoordinate environment with equatorial ligation from the 1 nitrogen of the imidazole ring and deprotonated amide nitrogens of the next two Gly residues. The second Gly also contributes its amide carbonyl oxygen. Except for the His backbone nitrogen and  carbon, all atoms from the His through to the nitrogen of the third Gly lie approximately in the equatorial plane and the copper lies just above this plane as consistent with a pentacoordinate Met. Ions Life Sci. 1, 89–114 (2006)

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complex. The Trp indole also participates in the coordination environment, but in a rather unusual fashion. The indole nitrogen from the Trp side chain is 30 Å from the oxygen of water bound axially to the Cu(II) suggesting the presence of a hydrogen bond. This arrangement places the plane of the indole ring above the copper such that it is nearly parallel with the equatorial plane. Two additional water molecules hydrogen bond to the axial water, forming a network extending from the backbone carbonyl proceeding the His to the carbonyl of the third Gly. There is no equatorial water involved in this model. Examination of intermolecular contacts found in the crystal structure reveal a potentially important docking interaction between HGGGW-Cu units that may explain previously observed cooperative binding of copper to PrPc . However, there is still some controversy about this cooperativity as this was not evident from the work of Jackson et al. [48]. Similarly, further studies by Garnett and Viles [57] suggest that the coordination of copper does not involve the backbone of the peptide, that it requires proline and that binding can only occur with cooperation between two of the octameric repeats. Once again it should be noted that these studies are all based on peptides and perhaps these results are subject to artefacts that would be eliminated by the use of large fragments or full-length proteins. Therefore, the true nature of the coordination remains unresolved.

5 COPPER UPTAKE AND PRION PROTEIN INTERNALIZATION Analysis of the levels of copper bound to PrPc purified from the brains of mice suggests that on average there are three copper atoms bound per molecule [58]. This would suggest that one site, possibly with nanomolar affinity for copper, is available for copper binding at the cell surface. Further studies with cultured cerebellar granule cells from mouse brain suggested that under copperdepleted conditions PrPc reaches the cell surface with as little as one copper binding site filled per molecule [58]. The uptake of copper into neuronal cells is regulated by both a high-affinity, low-capacity and low-affinity, high-capacity mechanism [59]. This uptake is largely believed to be mediated by the copper transporting receptor (CTR1) but, as PrPc is expressed at high levels in neurons it is also possible that internalization could result in uptake of copper into these cells. The potential for PrPc to mediate the uptake of radioactive copper Cu67  was investigated. It was first shown that it aids uptake of copper in 1997 [60]. Further investigation indicated that copper uptake is reduced in neurons lacking expression of PrPc [49]. It has been postulated that no free copper exists in biological systems [61]. Mediated internalization of copper was only observed when copper was provided in a chelated form [49]. As well as neurons, astrocytes also have shown evidence of PrPc mediated Cu uptake [62]. Astrocytes have a greater Met. Ions Life Sci. 1, 89–114 (2006)

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capacity to store copper and therefore uptake of Cu by astrocytes is far greater than by neurons despite the lower level of PrPc expression by these cells. The half-life of PrPc at the cell surface has been shown to be in the order of an hour or less. One consequence of copper binding at the cell surface is therefore internalization of the PrP-Cu complex. However, it has also been suggested that binding of copper initiates a higher rate of internalization [63]. This response was initially only observed with high 500 M nonphysiological concentrations of copper [63] and therefore dismissed as an artefact. Similar experiments carried out over a longer time period did indicate that copper bound at the cell surface, but did not result in increased internalization of the PrP-Cu complex [64]. Both sets of experiments were carried out with nonchelated copper. As indicated above, the use of nonchelated copper does not reflect the form copper is available in vivo and is therefore not appropriate for this kind of study. As already mentioned, it has been shown that PrPc -mediated internalization of Cu requires copper to be provided in a chelated form [49]. Two factors have been suggested that are important for the internalization of PrPc . The first of these is an N-terminal sequence of the protein not including the octameric repeat region [65–67]. Internalization driven by the N-terminus causes PrPc to move out of lipid raft domains to membrane regions that will form coated pits [68]. This response seems to be critically dependent on the basic amino acids KKRPKP at the N-terminus [68]. It is possible that this domain interacts with other proteins [69], but there is also evidence that this domain interacts with another internal domain within the hydrophobic region [70]. As indicated above, the second factor suggested to regulate internalization is binding of metal ions [63,65]. However, internalization of xenopus PrPc (a PrPc that has been suggested not to bind Cu) occurs in a similar way to mammalian PrPc [67], which would imply that internalization may not be copper dependent, though this remains unproven. Copper binding to PrPc at the cell surface causes the protein to enter endosomes [71]. This would clearly deliver copper into the cell. A further suggestion is that internalization may be simply a propensity of possessing a GPI anchor [72], though given the evidence already considered this would seem unlikely. As well as being internalized there is some evidence that PrPc is also secreted from the cell [73]. Possibly, PrPc can be transferred between cells by this mechanism [62]. There is evidence that metal ions such as copper can induce this release [62] and that such shedding is a result of the action of metalloproteases [74]. However, there is also a metal-independent shedding process [74]. Inherited mutations in the prnp gene protein coding region can also modulate the membrane association of the protein [75–77], and can prevent the protein from reaching the plasma membrane at all [78]. A definitive answer to the question of what regulates internalization has come from a recent paper [79]. In this work copper was applied in a chelated form with glycine. Under these conditions as little as 100 nM copper was able to stimulate Met. Ions Life Sci. 1, 89–114 (2006)

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significant internalization of PrPc . This concentration of copper is equivalent to that found in cultured medium [58]. The implication is that under normal culture conditions internalization is driven by copper binding at the cell surface. This is clearly in contrast to previous work suggesting that binding of another protein to the very N-terminal of the protein is necessary for internalization. In this study, a mutant protein, lacking amino acid residues 23–38 of the mouse sequence could still be internalized by addition of copper (Figure 1). In contrast, mutant proteins lacking either the octameric repeat region (amino acid residues 51–89) or the hydophobic palendrome with the sequence AGAAAAGA (amino acid residues 112–119) showed no internalization of PrPc and no response to copper. However, the expression of a single octameric repeat was sufficient to restore internalization in response to copper. The implication of these findings is that copper binding to the octameric repeat region regulates internalization of PrPc [79]. As conversion of PrPc to PrPSc requires internalization of the

Figure 1. Copper-mediated internalization of PrP. Cells were transfected with DNA constructs that allowed expression of PrP and mutants fused to GFP. GFP has no effect on the localization of the protein. The cells expressing wild-type (WT) GFP-PrP were then treated with 100 M Cu complexed with glycine. The same cells were visualized at time of addition (0) or at 10 min or 2 h later. Decreased GFP signal indicated internalization of the protein and its rapid degradation. Cells expressing mutant PrPs were similarly treated; 23–38, lacking the very N-terminus of the protein still responded to glutamate while the mutants 51–89 (lacking the copper binding site) or 112–119 (lacking the hydrophobic palendrome) showed no response to copper, indicating that these domains were essential for internalization of the protein in response to copper. Met. Ions Life Sci. 1, 89–114 (2006)

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PrPc –PrPSc complex, then this suggests that availability of copper to bind to PrP might limit protein conversion during disease. Neurons showing PrPc -mediated Cu67 uptake have been shown to re-release Cu gradually or following depolarization [49]. As suggested above, PrPc can also be shed by cells. There is also evidence that PrPc produced by neurons is taken up by astrocytes [62]. The implication of this is that neurons release PrPc into the extracellular environment. Secreted PrPc has been considered to be a small percentage of the total protein expressed by cells [73,80]. In addition to full-length PrPc , astrocytes appear to take up a fragment of PrPc . This fragment is equivalent in size to the N-terminus of PrPc , but as the N-terminus of mouse PrP lacks methionine this is unlikely unless it is longer than that caused by cleavage at amino acid residue 112. Analysis of brain samples from human patients has indicated a fragment this size exists in the brain [81] and furthermore, the N-terminus can be isolated by metal affinity chromatography from hamster brain [39]. It is unknown whether metabolic breakdown of PrPc to C-terminal and N-terminal fragment occurs entirely within the cell or whether it can occur in the extracellular milieu. It is possible that saturation of Cu binding sites might cause the release of its N-terminus or a larger fragment. Alternatively this could be a metabolic breakdown product generated in astrocytes from full length PrP. This would result in release of Cu bound to PrPc into the extracellular space. Neurons loaded with Cu67 have been shown to transfer a percentage of this copper to astrocytes when co-cultured [62]. The transfer of copper between neurons and astrocytes could be inhibited by an anti-PrP antibody. The implication of this is that transfer of PrPc results in the transfer of Cu to the astrocytes. Transfer of PrPc between cells did not occur when the astrocytes did not express PrPc . It has been suggested that PrP can interact with itself [82]. Therefore, PrPc might act as its own receptor. Alternatively, lack of PrPc expression might result in down regulation of another protein that acts as the receptor for PrPc in astrocytes. Transfer of PrPc between neurons and astrocytes has a further implication. It suggests that astrocytes can take up extracellular PrP, whatever its origin. Mice have been genetically engineered to express PrPc driven by the GFAP promoter [83]. This results in PrPc being expressed only in astrocytes. Mice, expressing PrPc in astrocytes only, can be infected with mouse scrapie. This implies that PrPSc is able to interact with PrPc expressed by astrocytes. Other studies have suggested that astrocytes are the first cells in the brain in which PrPSc can be detected [84]. Potentially, the ability of astrocytes to take up PrP and the possibility that any form of PrP can interact with PrPc at the surface of astrocytes implies that astrocytes might be the point at which PrPSc and PrPc first interact. Prion disease progression is dependent on formation of PrPSc from host PrPc . The most recognized hypothesis for how this occurs is the seeding hypothesis [17]. Once seeds, or small aggregates of PrPSc form, then further Met. Ions Life Sci. 1, 89–114 (2006)

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generation of PrPSc occurs rapidly. Therefore, it is possible that the formation of these seeds, being the slow step in the reaction, occurs in astrocytes. However, confirmation of this would require further investigation.

6 PRION PROTEIN AS AN ANTIOXIDANT It has been demonstrated that both recombinant and brain-derived PrPc have superoxide dismutase (SOD)-like activity when bound to copper [53,58]. There are currently three known superoxide dismutases in mammals [85]: the cytosolic Cu/Zn SOD-1, mitochondrial MnSOD or SOD-2 and extracellular EC-SOD or SOD-3. The former two are found in all cells at varying concentrations and often show increased expression under conditions of oxidative stress. EC-SOD exists in three different isoforms, binds one atom of copper per molecule and is either released into the extracellular matrix or remains bound at the cell surface. In brain tissue, the expression of SOD-3 is very low [86], although it is elevated in PrP knockout mice [58]. It is interesting to correlate these observations with the in vivo expression of PrPc , considering its SOD activity. The expression of PrPc is highest in the brain and particularly abundant at synapses. It is also present at neuromuscular junctions [87]. Thus it has been proposed that PrPc may serve as a synaptic SOD. Superoxide is known to inhibit synaptic transmission and the presence of SOD activity in these regions of the nervous system may have a protective role. The possible protective role of PrPc against oxidative stress was confirmed in cell culture using PC12 rat tumor cells, which can be differentiated into neurons using nerve growth factor (NGF). It was also observed that PrPc expression increased in PC12 cell cultures on exposure to oxidative stress [88]. The depletion of PrPc from cell extracts results in a lower SOD activity in the extract [89] and also when PrPc converts to PrPSc , its SOD function is abolished [90]. That PrPc adds to the total SOD activity of the cell was recently confirmed [91]. Another publication studying transgenic mice modified to lack expression of SOD-1 or SOD-3 examined whether there was any increased contribution of another SOD (i.e., PrPc ) to the total SOD activity measured in the brain [92]. This study reported no contribution of PrPc to brain SOD activity. However, careful analyses of the methods used by the authors indicated that the high-affinity copper chelator, EDTA, was used in the SOD assays at millimolar concentrations. It has been shown before [93] that similar concentrations can completely remove bound copper in brain extracts. As PrPc requires Cu for SOD activity, then these authors’ experiments were carried out under condition where PrPc could not act as an SOD. These findings should therefore be discounted when considering the function of PrPc . Recombinant PrPc rPrPc  purified from E. coli expression systems and refolded with copper, can be used in quantitative assays designed to measure superoxide dismutase activity (Figure 2). Experiments showed that both chicken and mouse rPrPc could catalyze the dismutation of the superoxide radical at a Met. Ions Life Sci. 1, 89–114 (2006)

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Figure 2. In-gel assay of PrP’s SOD-like activity. An in-gel assay was used to provide a visual demonstration of the SOD-like activity of wild-type PrP and some of the active mutants. 5 g of protein was electrophoresed on a native polyacrylamide gel and stained for SOD-like activity. Shown are 1, wild type PrP protein; 2, PrP 35-45; 3, PrP 112-136; 4, PrP23-171; 5, PrP 112-119, and 6, PrP45-231. This experiment shows that deleting the hydrophobic domain of PrP inactivates this activity. This domain is important to the active site of the protein.

rate equivalent to one tenth of SOD-1. SOD-1 is a very potent enzyme which catalyzes the reaction at around 100 000 times the spontaneous rate of superoxide degradation [94]. It was therefore concluded that PrPc had significant superoxide dismutase activity and this was also confirmed for native protein purified from mouse brain [58]. Strict controls were used to ensure that the prion protein SOD activity was a real enzymatic activity and not simply due to Fenton chemistry arising from the copper ligation within the protein. The deletion of the specific octameric repeats of the N-terminal region abolished the SOD activity, despite there still being copper bound at the C-terminal domain [53]. In addition, a peptide based on the octarepeat region with copper bound to it had no SOD activity. When rPrPc was refolded without copper and the copper subsequently added to the refolded protein, the mixture did not demonstrate SOD activity to the level of the coordinated copper in the protein [53]. It is therefore clear that regions outside the N-terminal octarepeat domain are required for the SOD activity of the PrPc . Further evidence for this comes from amino acid analysis of rPrPc after copper binding. It was found that methionine residues were oxidized in rPrPc with copper incorporated during refolding [95] and this is characteristic of certain antioxidant copper binding enzymes such as SOD-1 [96]. Thus it could be concluded that the observed catalytic SOD activity was not due to the presence of copper alone, but was more likely due to a true enzymatic activity and it Met. Ions Life Sci. 1, 89–114 (2006)

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would appear that the dismutation of superoxide could indeed be one of the normal functions of the prion protein. At least two copper atoms per molecule of PrPc were necessary to endow the protein with SOD activity. Binding of the copper also induced a more ordered structure of the molecule. On binding of two more copper atoms, a further increase in SOD activity was observed and an additional molecular ordering [54,97]. Mutational analyses have been carried out to assess what components of the protein are necessary for the SOD-like activity [98]. In particular the hydrophobic domain central in the protein was shown to be necessary for this activity. The implication is that two domains of the protein must interact for the activity. Finally the globular domain of the protein appears to play a role in regeneration of the protein to an active form. It is of particular interest to note that the two domains identified in this study as being important to antioxidant activity are identical to those found to be important to internalization. Therefore, from two different perspectives, the AGAAAAG palindrome and the octameric repeat regions are emerging as the regions with the highest functional significance (Figure 3). PrP Features Octameric Repeats (5) N-terminal Cleavage Signal Peptide

GPI Anchor Signal Peptide

Sugar chain attachment

Hydrophobic core

N 1

23

51

90

110

135

S

S

231

252

Disulfide bridge

Functional Domains

Figure 3. Schematic representation of PrP. Numbers are based on the mouse sequence. This protein is anchored to the cell membrane by a GPI anchor. The signal peptide for entry into the endoplasmic reticulum and the GPI signal peptide are cleaved off before the protein reaches the cell surface. Glycosylation can occur at one, two or none of the asparagine residues indicated. A hydrophobic region envelops a cleavage point where the protein is cleaved during normal metabolic breakdown. A disulfide bond links two regions of the protein which form separate -helices in the three dimensional structure of the protein. The complete octarepeats can bind up to four Cu atoms. Most mammals also have an incomplete repeat prior to this. The diagram shows the two most important functional domains. These two regions are essential for copper uptake, internalization of PrP and the SOD-like activity of the protein. Met. Ions Life Sci. 1, 89–114 (2006)

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Studies of the structure have suggested that the C-terminal domain with its globular domain of three -helices is structurally conserved between different species [99]. Therefore, it seems likely that this domain of the protein would have a clear and definable function. However, there is currently no clear evidence for a function for this domain.

7 MANGANESE BINDING Interaction between PrP and other metals has also been studied but to a lesser degree (Figure 4). In addition to metal binding it has also been suggested that interactions between metals can also increase the aggregation and conformational change of PrP. In particular, it has been suggested that copper and manganese can accelerate the formation of aggregated, protease resistant protein [41,100–102].

Figure 4. PrPSc binds manganese. Metal affinity chromatography using a manganesecharged column was used to isolate manganese binding proteins from cultured cells. The cells used were either cells without scrapie infection or scrapie-infected cells (SMB). The bound proteins were eluted and then analyzed by western blot. In both cases PrP from the cells bound to the column. In the case of the infected cells protease resistant PrP also bound to the column. PrPSc is resistant to protease K (PK) treatment 200 g/mL 3 h. This result shows that PrPSc can be isolated by its affinity for manganese. Met. Ions Life Sci. 1, 89–114 (2006)

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Metals have a high potential to interact with -sheet structural elements and therefore have the potential to exacerbate aggregation. It was found that copper has the potential to enhance the infective potency of scrapie infection [103]. In contrast, treatment with penicillamine, a copper chelator, lengthens the incubation period of scrapie, indicating a protective effect [104]. It has been found that hydrogen peroxide causes oxidation of methionine residues in PrP [105]. In addition, hydrogen peroxide can cause the cleavage of PrP at an alternative site in the presence of copper [106], but hydrogen peroxide can also be generated when PrP or fragments of PrP interact with metal such as copper and iron [107]. These data indicate that interactions between PrP and metals need not be beneficial (Figure 5).

Figure 5. Two different protein fates from metal binding to PrP. Alternative interactions of Cu with prion protein that cause either protective activity or conversion to a toxic species. Met. Ions Life Sci. 1, 89–114 (2006)

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Although most studies have examined the binding of copper to PrP, a small number of studies have also examined the potential of the protein to bind other metals. Studies with peptides have shown very little binding of any other metal besides copper [30,31,57]. Studies with full-length recombinant protein have suggested that three other metals could bind to the protein, manganese, nickel and zinc [40]. Of these nickel has the greatest similarity to copper, but measurements of its affinity in comparison to copper have suggested that this affinity is very poor. There has been more interest in zinc and PrP but again, there is no strong evidence for metal substitution, only some suggestions that the expression of PrPc at the cell surface might alter zinc entry into the cell [108]. However, initial studies indicated that manganese could compete equally with copper for binding to PrP [41]. Since then, further studies have verified that manganese can bind to PrP [47,109], but it appears that the exact binding site of Mn to PrP is different to that of copper, with a preference to the proposed fifth metal binding site in the region of the two histidines at amino acid residues 95 and 110. Manganese binding has one fundamental difference to that of copper. The binding of manganese to the protein leads to conversion of the protein to a protease resistant isoform that is a different conformation [41]. Acquisition of this -sheet-rich conformation requires time, but could occur within minutes with exposure to infrared radiation [109]. We observed that PrP with manganese bound to it could form extensive fibrils when exposed to a broad range of the near-infrared spectrum. Additionally, PrP expressing astrocytes were also found to express a protease resistant form of PrP when grown in a high concentration of manganese. PrP with manganese bound was also shown to be capable of catalyzing superoxide dismutation, indicating that the metal bound could undergo redox cycling [41]. However, this activity was lost following the conformational change from -helix to -sheet. Further analysis indicated that when copper is bound to the protein, the copper is largely protected from interaction with water molecules, indicating that the redox cycling of copper is tightly regulated [109]. The structure of copper-bound PrP was found to be very stable, more stable than the protein without metal bound. However, PrP with Mn bound was very unstable and the Mn could easily interact with water, increasing the probability that the Mn would heavily oxidize the protein. This oxidation is likely to be responsible for the conformational change and aggregation of the protein. Manganese-bound PrP converted to certain conformations is able to catalyze conversion of further PrP molecules to polymerize [110]. This polymerization was not autoaggregation, as the substrate protein did not form aggregates, even after several weeks of incubation. This indicates that Mn-PrP was truly able to act as a seed for PrP polymerization. Therefore, the investigation of manganese mediated PrP polymerization is a potentially important model for understanding the conversion process that generates PrPSc . Met. Ions Life Sci. 1, 89–114 (2006)

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8 TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES AND METALS An immunoaffinity technique has been developed to isolate PrPc from brain [58]. This same technique can be applied to isolated PrP from brains of patients with CJD. This isolated protein contains both PrPc and PrPSc but the majority of the protein is in the form of PrPSc . Metal analysis of PrPSc isolated from the brain of CJD patients showed that this protein lacked significant copper binding and that substitution with manganese and zinc had occurred [111]. Other researchers have also suggested that PrP in CJD patients’ brains might bind metals other than copper [93]. The SOD-like antioxidant activity associated with PrPc was lost completely from the purified PrPSc . This finding confirms the notion that prion disease causes a loss of PrPc function, which is directly related to the ability of the protein to bind copper. Brain tissue from CJD patients has a disadvantage in that it is the end stage of the disease. It is thus difficult to determine from studying such tissue what changes lead up to the final state described above. As tissue cannot be biopsied from the brains of living patients, other models must be found. Experimental mouse scrapie is an effective model to study prion disease during its time course. With such a model it is possible to study changes during the asymptomatic incubation period of the disease before the accumulation of PrPSc in the brain. There are many strains of scrapie as defined by the sheep-derived innoculum used to infect the mice originally. These strains have names such as RML, ME7 and 79A. The RML-induced prion disease has been studied by many groups and the time course of the disease is well characterized. In RML infected mice PrP was immunoaffinity purified from the brains and the metal associated with the protein [90]. When the levels were compared to that bound to PrPc from age match control mice it was found that copper binding was reduced from 60 days into the disease and onward (long before the onset of symptoms at around 127 days). In parallel, there was an increase in the level of manganese bound, implying that substitution of copper with manganese occurs during the disease process. Manganese was the predominant metal bound by the onset of symptoms. This implies that prion disease is associated with a major alteration in the metal associated with PrP. It remains to be determined whether this change is a consequence of prion ‘infection’ or is causative to the development of the disease. The SOD-like activity of PrP purified from the brains of the infected mice was also examined and compared to that prepared from control mice [90]. There was a considerable loss of SOD activity from the protein after 60 days post infection. These results show that changes in antioxidant defence may occur during prion disease. However, in particular changes in the metabolism of the metals copper and manganese may lie at the heart of these diseases. Met. Ions Life Sci. 1, 89–114 (2006)

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If loss of prion protein function has consequences for disease progression in TSEs then one would expect that the earliest changes in prion disease would be seen at the synapse. Recent studies of changes in neurons in experimental prion disease have identified loss of dendritic spines occurring before any other change in prion disease [112]. However, such changes, although fitting with the hypothesis that loss of prion protein function contributes to neurodegeneration in prion disease, do not prove the connection. As already mentioned PrPc expression is necessary if not sufficient for prion disease [16]. Animals lacking PrPc expression do not develop a spontaneous form of prion disease. Nevertheless, such animals do have a phenotype indicative of a disturbance and neurons lacking PrPc expression in particular are more sensitive to oxidative stress [23]. PrPc -deficient cells have diminished cellular activity of SOD and diminished copper content. Recently studies of transition metals in prion diseases have begun to emerge. Studies of the brain of CJD patients have shown that the levels of copper in their brains are decreased when compared to controls which do not have CJD [111]. In addition, there was a striking elevation in manganese. The severity of these changes appeared to change with the prnp genotype of the patients. Those patients homozygous for methionine at codon 129 showed the largest changes. Accompanying the metal perturbations in CJD were changes to the levels of activity of antioxidant proteins. Although Cu/Zn SOD was only mildly reduced in activity, MnSOD showed a three fold increase in activity in CJD patients. Also the brains of CJD parients showed large increases in the level of lipid peroxidation and carbonylation of protein that could be detected. These changes indicate that the brains of CJD patients show signs of ongoing oxidative stress and oxidative damage. Changes in the levels of metals in the brain and other tissues of scrapie-infected mice have also been studied [90]. There was a decrease in copper in the brains of RML-infected mice that reached a maximum at the onset of clinical signs. At the same time there was an increase in the level of manganese, but other metals did not change. In the liver there was only an increase in copper, but no change in any other metal examined. In blood there was a small increase in blood copper after onset of clinical signs but there was an elevation of manganese that occurred within the first 30 and 60 days after the intracerebrallar injection with the RML inoculum. Muscle showed some elevation in manganese but no other changes. These variations in metal ion content were accompanied by a decrease in the activity of Cu/Zn SOD in the brains. However, this change only occurred after the onset of clinical signs, suggesting it was secondary to the changes in the metal ions. These changes in metal ions for scrapie-infected mice are similar to the findings in CJD brains, but suggest that changes in metal ion content occur in parallel with changes in the levels of PrPSc . Maintaining functional PrPc is clearly advantageous and there is evidence to suggest that it can protect against prion disease in PrP-knockout mice that have Met. Ions Life Sci. 1, 89–114 (2006)

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been modified to express hamster PrP, via a GFAP promoter, only in astrocytes. These mice are susceptible to infection with hamster scrapie and develop prion disease [83]. Wild-type mice are highly resistant to hamster scrapie because of specific differences between the protein sequence of hamster and mouse PrPc . However, if wild-type mice are made transgenic to express hamster PrPc in astrocytes they cannot be infected with hamster scrapie. The implication of this is that mouse PrPc , which cannot be converted to mouse PrPSc by hamster PrPSc , protects against prion disease. This suggests that where there is sufficient functional PrPc then neurons may be protected from neuronal death caused by prion disease. In years to come strategies that protect or restore the normal copper-dependent functions of PrPc might be useful therapeutics to treat or prevent prion disease.

9 CONCLUSIONS Although there has been a historic link between copper and TSEs, it has only been in recent years that interest on this aspect has gained popularity. Now that it is accepted that PrPc is a copper binding protein, there has been a great expansion on research carried out on this subject. There is now a clear definite link between prion diseases and metal metabolism. Manganese is emerging as potential player in the conversion process or at least a contributor and potential surrogate marker for disease state itself. The exact nature of the disturbance that causes prion disease remains elusive, but exposing this alluvial seam of possibilities has potentially opened a shaft direct to the core of solving these enigmatic diseases.

ABBREVIATIONS BSE CD CJD CTRI CWD EC-SOD EDTA EPR ESR GFAP GFP GPI NGF

bovine spongiform encephalopathy circular dichroism Creutzfeldt–Jakob disease copper transporting receptor chronic wasting disease extracellular SOD ethylenediamine-N,N,N ,N  -tetraacetate electron paramagnetic resonance electron spin resonance glial fibrillary acid protein green fluorescent protein glycosylphosphatidylinositol nerve growth factor Met. Ions Life Sci. 1, 89–114 (2006)

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NMR PrP PrPc PrPSc rPrPc SOD TSE vCJD WT XAFS

nuclear magnetic resonance prion protein cellular PrP scrapie isoform of PrP recombinant prion protein superoxide dismutase transmissible spongiform encephalopathy variant CJD wild-type X-ray absorption fine-structure spectroscopy

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5 The Role of Metal Ions in the Amyloid Precursor Protein and in Alzheimer’s Disease Thomas A. Bayer1 and Gerd Multhaup2 1

2

Universität des Saarlandes, Klinik für Psychiatrie, Abteilung für Neurobiologie, D-66421 Homburg, Germany

Institut für Chemie/Biochemie, Freie Universität Berlin, Thielallee 63, D-14195 Berlin, Germany <[email protected]>

1 INTRODUCTION 2 AMYLOID PRECURSOR PROTEIN AND BRAIN COPPER HOMEOSTASIS 3 AMYLOID PRECURSOR PROTEIN AND Cu,Zn-SUPEROXIDE DISMUTASE-1 4 GENERAL CONCLUSIONS ABBREVIATIONS REFERENCES

115 117 119 120 121 122

1 INTRODUCTION Alzheimer’s disease (AD) is characterized pathologically by synaptic and neuronal degeneration, deposition of amyloid plaques and development of neurofibrillary tangles in the brains of AD patients. Amyloid deposits are primarily composed of the A amyloid peptide of which A1–40 is the predominant

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soluble species in biological fluids and A1–42 is a major species found in developing plaque deposits. The amyloid deposits in AD brains originate from the larger amyloid precursor protein (APP). APP and the secreted -cleaved ectodomain of APP are involved in the viability, growth and morphological, functional plasticity of nerve cells and in learning or memory processes. APP belongs to a multigene family including amyloid-like precursor proteins (APLP1 and APLP2) [1], which share a number of motifs and functions such as heparin and metal binding domains and neurotrophic activity. For an overview see Figure 1. One such a domain is the N-terminal Cu binding domain (CuBD-I) of APP which binds Cu with nanomolar affinity [2] and a secondary CuBD-II, which appears in A after proteolytic processing of APP [3]. N-terminal CuBDs of APP family paralogs and orthologs were found to have antioxidant activities whereas the recently evolved human APP exerts unique redox properties suggesting an overall conservation in its function or activity [4]. The underlying reaction, a reduction of Cu(II) to Cu(I), and the structural homology of the CuBD-I of

A CHO CHO

TM

Signal peptide

P

P

CS-GAG

Aβ Cu(II) Zn(II) Heparin

KPI exon 7

exon 15 Heparin

Cu (II) Zn(II)

B Amyloidogenic pathway

sAPPβ

Aβ β-

Non-amyloidogenic pathway

sAPPα

C-99 γ-secretase C-83

α-secretase

Figure 1. APP domains and processing. (A) Schematic representation of domain structure and important binding sites of APP. (B) Amyloidogenic and nonamyloidogenic processing of APP. - and -secretase activity generates secreted sAPP and A (A40 and 42 in length) and the membrane bound C-99 fragment. Nonamyloidogenic processing generates secreted sAPP and the membrane bound C-83 fragment. Abbreviations: P: phosphorylation; CHO: glycosylation; KPI: Kunitz-type protease inhibitor consensus sequence; CS-GAG: chondroitin sulfate glucosaminoglycan attachment site; TM: transmembrane region. Met. Ions Life Sci. 1, 115–123 (2006)

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APP to Cu chaperones suggests APP to function as a Cu(I) binding neuronal metallochaperone. Potentially, APP-Cu(I) complexes may reduce hydrogen peroxide by forming an APP-Cu(II)-hydroxyl radical intermediate or may modulate neurotoxicity and APP fragmentation when Cu is allowed to accumulate beyond cellular needs. In addition, Cu has been reported to bind to A (CuBD-II) and increase its aggregation in vitro, potentiating its neurotoxic effects (reviewed in [5]). These seemingly contradictory observations are being discussed in the light of the enigmatic role and function of the binding of Cu to APP and A. The possible mechanism of APP-mediated oxidative stress in AD has been discussed elsewhere [6].

2 AMYLOID PRECURSOR PROTEIN AND BRAIN COPPER HOMEOSTASIS Recent research has revealed that APP is actively involved in balancing Cu concentrations in cells. In APP and APLP2 knockout mice, increased Cu levels were found in cerebral cortex and liver [7], whereas overexpression of APP was reported to result in significantly reduced Cu levels in the Tg2576 line [8]. Depending on the conservation of the CuBD, Cu was also found to influence APP processing in cells when Cu greatly reduced the levels of amyloid A and caused an increase in the secretion of the APP ectodomain [9]. Increased brain Cu levels were achieved by crossing APP transgenic mice with TxJ ‘toxic milk’ mice with a mutant CuATPase7b transporter [10]. Aged TxJ/APP double transgenic mice exhibited a 45% reduction in A plaque load and Cu levels of the central nervous system (CNS) in aged APP transgenic mice and in postmortem AD brain tissue were significantly reduced compared to controls. These observations support the notion of an inverse relationship between elevated Cu levels and A accumulation. Remarkably, the TxJ mutation also increased survival of APP transgenic mice and lowered murine A levels prior to detectable A plaques. The CuATPase7b transporter is a P-type ATPase associated with the trans Golgi network. While the TxJ mutation contains a Cu-selective domain, Cu is elevated with 100%, Zn levels also rise about 20%. Phinney et al. [10] discuss the possibility whether the beneficial effect caused by the TxJ mutation is due to elevated Cu or Zn. The smaller percentage increase in Zn and the pro-amyloidogenic effect in synaptic Zn levels argue against a contribution of Zn. In good agreement with Phinney et al. [10], we have shown that dietary Cu elicits the same effects as genetic modulated Cu levels [11]. To investigate the influence of bioavailable environmental Cu, transgenic APP mice received an oral treatment with Cu-supplemented sucrose-sweetened drinking water. Chronic overexpression per se reduced superoxide dismutase-1 (SOD-1) activity in transgenic mouse brain, which could be restored to normal levels after Cu treatment. A significant increase of brain Cu indicated its bioavailability on Cu treatment Met. Ions Life Sci. 1, 115–123 (2006)

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in APP transgenic mice, whereas Cu levels remained unaffected in littermate controls. Cu treatment lowered A before a detectable reduction of amyloid plaques. This beneficial influence of Cu on APP metabolism is best explained by a role for APP in Cu efflux. Two reports have been published that do not agree with the latter observations. One study has shown that treatment of 21-months-old Tg2576 mice with the antibiotic clioquinol inhibited plaque formation and concomitantly increased soluble brain Cu and Zn levels [12]. This increase of metal ions in brain observed upon clioquinol treatment might either be attributed to an inefficiency of the chelator with weak affinities for Zn log K1 = 70 and for Cu log K1 = 89 or even more likely, due to a facilitated uptake in brain of clioquinol-Cu complexes, similarly to Cu-nitrilotriacetate (Cu-NTA)-treated mice. Moreover, lowered insoluble A levels (by ∼49%) and increased soluble A levels (by ∼50%) were accompanied with elevated Cu levels (and Zn). Interestingly, the prion protein like APP is also a Cu-binding protein and a well characterized cause of an experimental rodent model for spongiform encephalophathy is the application of the Cu chelator cuprizone [13]. Novel observations indicate that clioquinol mediates Cu uptake and counteracts Cu efflux activities of APP [14]. These data uncover a novel biological role of APP and APLP2 in Cu efflux and provide a new conceptual framework of formerly diverging theories of Cu supplementation and chelation in the treatment of AD. In the second study Sparks and Schreurs [15] challenged rabbits with a highcholesterol diet and reported that the intake of minor concentrations of Cu (0.12 mg/L) in the drinking water impaired the behavioral phenotype. Both intraneuronal and extracellular (neuropil deposits) A levels were elevated. Unfortunately, no brain Cu levels were shown. This provocative finding implies that tap water has sufficient Cu contamination to influence the pathology of AD. However, the following caveats have to be considered: Cu intake from drinking water by the rabbits was (given a body weight of 2.2 kg) 18–36 g Cu/kg body weight from water. The estimated Cu ingestion from rabbit food is about 1.1–3.5 mg/day, exceeding the amount from Cu-spiked drinking water used in this experiment. Laboratory rabbit food normally contains only trace amounts of cholesterol. A high-cholesterol diet is used in studies of cardiovascular disease in rabbits, as well as in the rabbit model for AD, which in the context of rabbits as vegetarian animals is questionable. The rabbits’ metabolism may not be able to cope with excessive amounts of dietary cholesterol. Moreover, the plaques both lack surrounding gliosis (an important marker for degeneration) and neuritic involvement. It could very well be that the cholesterol/Cu challenge endangers an acute stress (comparable to delirium), in contrast to igniting a chronic degenerative AD-like condition comparable to dementia [16]. While serum Cu levels were reported to be elevated in AD patients, which correlated with poorer neuropsychological performance [17,18], postmortem Cu levels in CNS of AD patients is decreased [19]. This reverse relationship may Met. Ions Life Sci. 1, 115–123 (2006)

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Table 1. Correlation between A phenotype and copper levels Source

APP/A Phenotype

In vitro Cu treatment of APP transfected CHO cells

↑ of intracellular APP ↑ of sAPP ↑ Cu induced ↓ of A

[9]

Dietary Cu treatment of APP transgenic mice

↓ of PBS soluble A

↑ Cu induced

[11]

Genetic elevation of brain Cu levels in APP transgenic mice

↓ of soluble A ↓ of plaque A

↑ Cu induced

[10]

APP transgenic mouse brain (APP overexpression)

↑ of A

↓ Cu detected

[8,11]

APP knockout mouse brain (no APP expression)

No APP/A

↑ Cu detected

[7]

C-100 transgenic mouse brain (C-terminal fragment overexpressed)

No plaques No enhanced soluble A

↓ Cu detected

[8]

Postmortem brain tissue of patients with Alzheimer’s disease

↑ of plaque A

↓ Cu detected

[19]

Serum Cu levels in patients with AD

↓ of soluble Aa

No change in Cu ↑ Cu detected

[18] [17]

Oral treatment with Cu chelator clioquinol in APP transgenic mice

↑ of soluble A ↓ of plaque A

↑ Cu induced

[12]

Rabbits treated with trace amounts of Cu in water

↑ of intracellular A ↑ of plaque A

No Cu levels measured

[15]

a

Copper level

Reference

Lower serum A levels correlate with AD

be attributed to elevated Cu efflux through the blood–brain barrier in AD brain and correlates with APP overexpressing transgenic mice that also have lower Cu levels in brain. Table 1 gives an overview of the above discussed results.

3 AMYLOID PRECURSOR PROTEIN AND Cu,Zn-SUPEROXIDE DISMUTASE-1 Early studies have shown that APP can bind and reduce Cu(II) to Cu(I), leading to oxidative modification of APP [4]. Subsequently, we have demonstrated that adding Cu to Chinese hamster ovary (CHO) cells greatly reduced the level of A and increased the levels of sAPP and intracellular APP [9]. These observations Met. Ions Life Sci. 1, 115–123 (2006)

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suggested the existence of two distinct regulating mechanisms, one acting on A production and the other on APP synthesis. However, the steady-state levels of sAPP remained unchanged in mouse brains expressing APP and a mutant Cu-transporter (leading to elevated Cu levels). As discussed above, dietary Cu treatment had a modulating effect on brain Cu levels and a normalizing effect on SOD-1 activity in these animals compared to nontransgenic littermate control mice [11]. Several studies link APP and SOD-1 function in vivo. Overexpression of APP in brain is associated with postnatal lethality. Co-expression of high levels of human cytosolic superoxidescavanging activity protected against the lethal effects of APP and rescued the reduced life duration [20]. Moreover, SOD-1 rescues cerebral endothelial vascular dysfunction in mice overexpressing APP [21]. The drinking water therapy with Cu supplementation seems to provide sufficient bioavailable Cu to APP transgenic mice. The recent observation that overexpression of APP in Tg2576 transgenic mice also resulted in significantly reduced brain Cu levels prior to the appearance of amyloid pathology suggests that the APP ectodomain is involved in reducing brain Cu levels. This is further supported by the observation that APLP2 knockout mice, like APP knockout mice, have increased brain copper levels. APLP2 has a Cu binding site that shares structural and functional homology with the APP Cu binding ectodomain. Thus, deleterious effects of APP or APLP2 overexpression are likely due to an interference with Cu homeostasis and intracellular Cu trafficking. More evidence that APP is directly or indirectly involved in intracellular Cu homeostasis is provided by the recently revealed structural homology of its CuBD to intracellular Cu chaperones and the link to the Cu chaperone for SOD-1 (CCS). The neuronal adaptor protein X11 interacts with the cytoplasmic domain of APP and CCS. Overexpression of X11 inhibits SOD-1 activity through binding to CCS, which delivers and inserts Cu into SOD-1 [22]. SOD-1 activity is a marker for intracellular Cu levels [23]. Therefore, it is interesting to note that SOD-1 and ceruloplasmin activity are found to be reduced in plasma of AD patients compared to age- and sex-matched controls with no change in Cu and ceruloplasmin protein levels [24]. The Cu status (SOD-1 and ceruloplasmin activity) was seen to be influenced by long-term Cu intake in young men [25], which indicates that environmental Cu modulates body Cu status of humans.

4 GENERAL CONCLUSIONS APP is involved in Cu homeostasis in mouse and man. In vitro observations and in vivo data obtained from APP mouse models at least provide strong evidence that APP overexpression enables intracellular Cu to be transported out of the cell. The increased Cu efflux seems to lead to a Cu deficiency and a subsequently Met. Ions Life Sci. 1, 115–123 (2006)

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A Cu

Cu

sAPPα

physiological role: • cell adhesion • synaptic transmission • export of Cu

B

sAPPβ Aβ

+

Aβ

pathological role: • neurodegeneration • plaques • dementia

Figure 2. Model for the hypothesized mechanism of the physiological and pathological relation between binding of Cu and APP. (A) In healthy brain most of the APP molecules are Cu loaded within the cell. APP exports Cu via secretion of sAPP. Cu may be transferred to other Cu proteins and/or excreted via bile and liver. (B) In Alzheimer’s disease brain Cu deficiency leads to abundant Cu-free APP molecules, which may experience another three-dimensional structure. Subsequently, APP is cleaved predominantly by the amyloidogenic pathway, secreting increased amounts of sAPP and A, the latter being deposited in the neuropil forming senile plaques.

reduced SOD-1 activity. Studies have shown that a disturbed metal-ion homeostasis with elevated serum Cu levels occurs in Alzheimer and Down’s patients and lowered levels in post-mortem AD brain. We conclude that bioavailable Cu has beneficial and specific effects in an Alzheimer’s disease mouse model, and suggest that our observation should be regarded as a proof-of-concept for a prophylactic approach to overcome the observed CNS Cu deficiency in the brain of AD patients (Figure 2).

ABBREVIATIONS A AD APLP2

-amyloid peptide Alzheimer’s disease amyloid precursor-like protein-2 Met. Ions Life Sci. 1, 115–123 (2006)

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APP CCS CHO cells CNS CuBD Cu-NTA PBS sAPP SOD-1

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amyloid precursor protein Cu chaperone for SOD-1 Chinese hamster ovary cells central nervous system copper binding domain Cu-nitrilotriacetate phosphate-buffered saline secreted APP Cu,Zn-superoxide dismutase-1

REFERENCES 1. T. A. Bayer, R. Cappai, C. L. Masters, K. Beyreuther, and G. Multhaup, Mol. Psychiatry, 4, 524–528 (1999). 2. L. Hesse, D. Beher, C. L. Masters, and G. Multhaup, FEBS Lett., 349, 109–116 (1994). 3. C. S. Atwood, R. C. Scarpa, X. Huang, R. D. Moir, W. D. Jones, D. P. Fairlie, R. E. Tanzi, and A. I. Bush, J. Neurochem., 75, 1219–1233 (2000). 4. G. Multhaup, A. Schlicksupp, L. Hesse, D. Beher, T. Ruppert, C. L. Masters, and K. Beyreuther, Science, 271, 1406–1409 (1996). 5. A. I. Bush, Trends Neurosci., 26, 207–214 (2003). 6. G. Multhaup, S. Scheuermann, A. Schlicksupp, A. Simons, M. Strauss, A. Kemmling, C. Oehler, R. Cappai, R. Pipkorn, and T. A. Bayer, Free Radic. Biol. Med., 33, 45–51 (2002). 7. A. R. White, R. Reyes, J. F. Mercer, J. Camakaris, H. Zheng, A. I. Bush, G. Multhaup, K. Beyreuther, C. L. Masters, and R. Cappai, Brain Res., 842, 439–444 (1999). 8. C. J. Maynard, R. Cappai, I. Volitakis, R. A. Cherny, A. R. White, K. Beyreuther, C. L. Masters, A. I. Bush, and Q. X. Li, J. Biol. Chem., 277, 44670–11676 (2002). 9. T. Borchardt, J. Camakaris, R. Cappai, C. L. Masters, K. Beyreuther, and G. Multhaup, Biochem. J., 344, 461–467 (1999). 10. A. L. Phinney, B. Drisaldi, S. Lugowski, S. Schmidt, H. Bonek, Y. Liang, P. Home, L. Yang, J. Sekoulidis, J. Coomaraswarmy, D. Cox, P. M. Mathews, R. A. Nixon, G. A. Carlson, P. St George-Hyslop, and D. Westaway, Proc. Natl. Acad. Sci. USA, 100, 14193–14198 (2003). 11. T. A. Bayer, S. Schäfer, A. Simons, A. Kemmling, T. Kamer, R. Tepest, A. Eckert, K. Schüssel, O. Eikenberg, C. Sturchler-Pierrat, D. Abramowski, M. Staufenbiel, and G. Multhaup, Proc. Natl. Acad. Sci. USA, 100, 14187–14192 (2003). 12. R. A. Cherny, C. S. Atwood, M. E. Xilinas, D. N. Gray, W. D. Jones, C. A. McLean, K. J. Barnham, I. Volitakis, F. W. Fraser, Y. Kim, X. Huang, L. E. Goldstein, R. D. Moir, J. T. Lim, K. Beyreuther, H. Zheng, R. E. Tanzi, C. L. Masters, and A. I. Bush, Neuron, 30, 665–676 (2001). 13. W. W. Carlton, Exp. Mol. Pathol., 10, 274–287 (1969). 14. C. Treiber, A. Simons, M. Strauss, M. Hafner, R. Cappai, T. A. Bayer, and G. Multhaup, J. Biol. Chem., 279, 51958–51964 (2004). Met. Ions Life Sci. 1, 115–123 (2006)

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15. D. L. Sparks and B. G. Schreurs, Proc. Natl. Acad. Sci. USA, 100, 11065–11069 (2003). 16. A. I. Bush, C. L. Masters, and R. Tanzi, Proc. Natl. Acad. Sci. USA, 100, 11193–11194 (2003). 17. R. Squitti, D. Lupoi, P. Pasqualetti, G. Dal Forno, F. Vernieri, P. Chiovenda, L. Rossi, M. Cortesi, E. Cassetta, and P. M. Rossini, Neurology, 59, 1153–1161 (2002). 18. C. Gonzalez, T. Martin, J. Cacho, M. T. Brenas, T. Arroyo, B. Garcia-Berrocal, J. A. Navajo, and J. M. Gonzalez-Buitrago, Eur. J. Clin. Invest., 29, 637–42 (1999). 19. M. A. Deibel, W. D. Ehmann, and W. R. Markesbery, J. Neurol. Sci., 143, 137–142 (1996). 20. G. A. Carlson, D. R. Borchelt, A. Dake, S. Turner, V. Danielson, J. D. Coffin, C. Eckman, J. Meiners, S. P. Nilsen, S. G. Younkin and K. K. Hsiao, Hum. Mol. Genet., 6, 1951–1959 (1997). 21. C. Iadecola, F. Zhang, K. Niwa, C. Eckman, S. K. Turner, E. Fischer, S. Younkin, D. R. Borchelt, K. K. Hsiao, and G. A. Carlson, Nat. Neurosci., 2, 157–161 (1999). 22. D. M. McLoughlin, C. L. Standen, K. F. Lau, S. Ackerley, T. P. Bartnikas, J. D. Gitlin, and C. C. Miller, J. Biol. Chem., 276, 9303–9307 (2001). 23. T. B. Bartnikas and J. D. Gitlin, J. Biol. Chem., 278, 33602–33608 (2003). 24. J. Snaedal, J. Kristinsson, S. Gunnarsdottir, M. Olafsdottir, M. Baldvinsson, and T. Johannesson, Dement. Geriatr. Cogn. Disord., 9, 239–242 (1998). 25. J. R. Turnlund, R. A. Jacob, C. L. Keen, J. J. Strain, D. S. Kelley, J. M. Domek, W. R. Keyes, J. L. Ensunsa, J. Lykkesfeldt, and J. Coulter, Am. J. Clin. Nutr., 79, 1037–1044 (2004).

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6 The Role of Iron in the Pathogenesis of Parkinson’s Disease Manfred Gerlach,1 Kay L. Double,2 Mario E. Götz,3 Moussa B. H. Youdim,4 and Peter Riederer5 1

2

Department of Child and Adolescence Psychiatry and Psychotherapy, Clinical Neurochemistry, University of Würzburg, Füchsleinstrasse 15, D-97080 Würzburg, Germany <[email protected]>

Prince of Wales Medical Research Institute, Barker St., Randwick, Sydney, NSW, 2031, Australia 3

Department of Pharmacology, Universitätsklinikum Kiel, Hospitalstrasse 4, D-24105 Kiel, Germany <[email protected]>

4

Eve Topf and National Parkinson Foundation Centers of Excellence for Neurodegenerative Diseases Research, and Department of Pharmacology, Technion-Faculty of Medicine, Efron St., PO Box 969-7, Haifa 31096, Israel

5

Department of Psychiatry and Psychotherapy, Clinical Neurochemistry, National Parkinson Foundation Centers of Excellence for Neurodegenerative Diseases Research, University of Würzburg, Füchsleinstrasse 15, D-97080 Würzburg, Germany

1 INTRODUCTION 2 IRON IN THE ETIOLOGY OF PARKINSON’S DISEASE 2.1 Epidemiological Findings 2.2 Genetic Disorders Implicated with Misregulation of Iron Metabolism and Parkinsonism 2.3 Brain Iron in Parkinson’s Disease 3 SOURCES OF INCREASED IRON IN PARKINSON’S DISEASE 3.1 Ferritin 3.2 Neuromelanin

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4 CONSEQUENCES OF IRON OVERLOAD IN PARKINSON’S DISEASE 4.1 Oxidative Stress-induced Neuronal Damage 4.2 Protein Aggregation 5 GENERAL CONCLUSIONS ACKNOWLEDGMENTS ABBREVIATIONS AND DEFINITIONS REFERENCES

139 139 140 141 142 142 143

1 INTRODUCTION Iron is one of the most abundant metals in the human body and is an essential nutrient in virtually all cells. It is essential for the normal healthy function of the brain and is involved in mitochondrial electron transport, DNA and protein synthesis and degradation, myelination of axons and enzyme function, including those involved in neurotransmitter production and metabolism [1,2]. The local concentration of iron in the brain varies according to the region examined and can be surprisingly high; in some brain regions of basal ganglia, a part of the motor system related to the neuronal operation of movement, iron content is greater (for example, globus pallidus 21.30 and substantia nigra, SN, 18.46 mg iron/100 g fresh weight) than that found in the liver (13.44 mg iron/100 g fresh weight), the primary peripheral iron storage depot [3]. Iron deposition in the brain is mainly in organic storage forms such as ferritin, but not hemosiderin [4] with relatively little in a free and reactive form. In the mature brain, the blood– brain barrier (BBB) is considered to be closed to iron: that is, the movement of iron into the brain is tightly regulated by a variety of iron mobilization proteins (ferritin, transferrin). Despite these physiological systems designed to achieve iron homeostasis, increased concentrations of brain iron have been demonstrated in a range of neurodegenerative diseases [5–7]. Parkinson’s disease (PD, synonyms: idiopathic Parkinson syndrome, paralysis agitans), first described as a clinical entity by James Parkinson, is the second most common neurodegenerative disease after Alzheimer’s disease (AD), affecting approximately 2% of the human population aged 65 and above [8]. On the basis of etiological factors the frequently encountered idiopathic form is distinguished from the various less frequently occurring symptomatic forms, and from those disease presentations that are accompanied by multi-system degeneration (Table 1). The cardinal clinical features of PD are resting tremor, rigidity, and bradykinesia or motor slowing. However, signs of postural instability, autonomic dysfunctions, psychiatric symptoms such as depression and dementia are also present in a large percentage of patients. Met. Ions Life Sci. 1, 125–149 (2006)

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Table 1. Etiological classification of different types of parkinsonism [9]. Parkinson’s disease (synonyms paralysis agitans, shaking palsy, idiopathic parkinsonism) Symptomatic parkinsonism Drug-induced (neuroleptics, reserpine, calcium channel blockers such as cinnarizine and flunarizine) Toxin-induced (MPTP, manganese, carbon monoxide, cyanide, methanol, lead- and sulfur-compounds, pesticides, TaClo) Infectious (postencephalitic, luetic) Vascular lesions Brain tumors Posttraumatic (for example, following head injury or boxing) Heterogeneous system degenerations Progressive supranuclear palsy (Steele–Richardson–Olszewski syndrome) Multisystem atrophy (Striatonigral degeneration) Olivopontocerebellar degeneration Dementia (amyotrophic lateral sclerosis/parkinsonism/dementia of Guam, Creutzfeld–Jakob disease, Alzheimer’s and Pick’s disease) Hereditary disorders (for example, Wilson’s disease and movement disorders depicted in Table 2) MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; TaClo, 1-trichloromethyl-1,2,3,4-tetrahydrobeta-carbolin.

Pathologically, PD is characterized by a preferential loss of neuromelanin(NM)containing dopamine neurons in the pars compacta of the SN, with intracellular proteinaceous inclusions named Lewy bodies and a reduction in striatal dopamine [10,11]. This ongoing loss of nigral dopaminergic neurons mainly leads to clinical diagnosis due to occurrence of motor symptoms such as rigidity, tremor and bradykinesia, which results from a reduction of about 70% of striatal dopamine [12,13]. This is the rationale for the dopamine-substitution therapies, including treatment with l-DOPA (l-3,4-dihydroxyphenylalanine, levodopa) and peripheral aromatic amino acid decarboxylase- and catecholamineO-methyltransferase (COMT) inhibitors, dopaminergic agonists, selective monoamine oxidase (MAO) type B inhibitors and drugs which indirectly improve dopaminergic functions (for example, glutamate antagonists). Despite numerous attempts at elucidation, the cause of PD remains unclear. It is hypothesized that the cause of neurodegeneration in PD is multi-factorial in terms of both etiology and pathogenesis. Genetic factors are known to cause PD in a small number of patients with a familial form of the disorder. Mutations in different genes (for example, -synuclein and parkin) have been identified, and PD subtypes have been linked in addition to different chromosomal loci [14]. In contrast, twin studies suggest that while genetic factors may be important in young-onset patients, they do not play a primary role in the majority of Met. Ions Life Sci. 1, 125–149 (2006)

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individuals who experience a sporadic form of the disorder, most likely related to environmental factors [15]. Indeed, current thinking favors the hypothesis that most sporadic cases are caused by a complex interplay between different genetic and environmental factors. Several biochemical factors appear to be involved in the pathogenetic cascade of events leading to cell dysfunction and death in PD, including a mitochondrial complex I deficiency, a disturbed iron metabolism, free radicals, excitotoxicity, a disturbed calcium homeostasis, microglia activation and protein aggregation [5,16–18]. This chapter will review the current evidence indicating a role of iron as a cause of neuronal cell death in PD. In particular, neurochemical and histochemical findings that were obtained at autopsy from analyses of the brain from patients with PD will be discussed. Special attention will be paid to clarifying the possible implication of the observed changes in the etiology of PD. For a detailed treatment of iron metabolism and its importance in brain, the interested reader is referred to the corresponding Chapters 1, 10 and 11 in this volume.

2 IRON IN THE ETIOLOGY OF PARKINSON’S DISEASE 2.1 Epidemiological Findings Several epidemiological studies suggest that long-term occupational and dietary metal exposure is associated with the occurrence of PD [19–22]. In particular, significant associations of PD with manganese, copper as well as combinations of lead with copper and iron, and iron with copper for workers with more than 20 years of occupational contact were reported [21,22]. In addition, it appears that an increased risk for patients with family history of PD exists [23]. A combination of manganese, iron, and aluminum might favor the development of PD after 30 years of exposure [24]. The only positive dose response relationship was, however, found between mercury exposure and PD, but not for other metals [25,26]. In a recent study, a moderate association between iron intake from foods and PD and an apparent joint effect of iron and manganese was reported [20]. These epidemiological findings suggest transition metals are a risk factor for the development of PD, but cannot explain the occurrence of most cases of sporadic PD.

2.2 Genetic Disorders Implicated with Misregulation of Iron Metabolism and Parkinsonism There are several movement disorders that result from mutations in the ironregulatory pathways (Table 2). Patients with these mutations showed marked clinical heterogeneity and the symptoms of neurological complications include Met. Ions Life Sci. 1, 125–149 (2006)

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cognitive decline and extrapyramidal dysfunctions including dystonia and parkinsonism [27]. The disrupted expression or function of proteins involved in iron metabolism increases the concentration of iron in the brain (Table 2). How these mutated genes operate and interact to induce abnormal brain iron metabolism, transport and accumulation in the central nervous system (CNS) and how iron in turn interacts with proteins involved in parkinsonism (for example, -synuclein), causing them to aggregate into toxic inclusions is the focus of much current Table 2. Movement disorders associated with neurodegeneration, and genetic abnormalities in iron regulation resulting in iron deposition in the brain (according to [27]). Disorder

Gene mutation or locus

Clinical symptoms

Resulting pathology

Pantothenate kinase-associated neurodegeneration (PKAN)

PANK2 Gly-411 → Arg Thr-418 → Met

Dystonia, parkinsonism, chorea, and other movement disorders with retinal pigmentary changes

Depletion of coenzyme A, accumulation of cysteine (cofactor of iron), abnormal iron deposition in the basal ganglia

Hypoprebetalipoproteinemia, acanthocytosis, and retinitis pigmentosa with pallidal degeneration (HARP)

Parkinsonism, PANK2 dystonia, and Arg-411 → stop mutation Met- choreoathetosis 327 → Thr

Acanthocytosis and low levels of pre-lipoprotein, abnormal iron deposition in the basal ganglia

Hallervorden–Spatz syndrome

Parkinsonism NBIA-1 locus on chromosome 20p13, and dystonia no mutation in the PANK2 gene

Abnormal iron deposition in the basal ganglia

Neuroferritinopathy

Mutation in FTL (ferritin light chain) gene on chromosome 19q13.3 (single adenine insertion between nucleotides 460 and 461 in exon 4)

Choreoathetosis, dystonia, spasticity, parkinsonism, and rigidity

Low serum ferritin levels, reddish-brown discoloration of basal ganglia and ferritin-positive inclusions, abnormal iron deposition in the basal ganglia

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Table 2. (Continued) Disorder

Gene mutation or locus

Clinical symptoms

Resulting pathology

Aceruloplasminemia

Mutations in the exons 3,7,13,15, and 18 of the ceruloplasmin gene on chromosome 3q23-24

Retinal degeneration, diabetus mellitus, and neurological symptoms in the form of ataxia, blepharospasm, dystonia, tremor, parkinsonism, and chorea

Paradoxal decrease in the serum iron, iron overload in the basal ganglia, retina, liver, and pancreas, neurodegeneration in the striatum and substantia nigra

Hemochromatosis (rare cases)

Mutations in the HFE gene on chromosome 6p21.3 Cys-282 → Tyr

Central nervous system superficial siderosis and vasculitis, systemic hemosiderosis, parkinsonism (rare cases)

Iron overload in the visceral organs

Friedreich’s ataxia

Triplet repeat expansion GAA on the first intron of the frataxin gene on chromosome 9q13-q21

Ataxia and sensory neuropathy in the lower limbs, tremor, dystonia, myoclonus and chorea

Increased brain iron as a result of defective iron transport from mitochondria

research. Increased brain iron triggers a cascade of deleterious events that lead to neurodegeneration (see the following section).

2.3 Brain Iron in Parkinson’s Disease Increased regional total brain iron has been identified in a variety of neurodegenerative disorders such as PD, movement disorders associated with parkinsonism, Huntington’s disease and the dementia syndromes, including AD [5,7,28]. The most striking feature of these changes is that increased brain iron is confined Met. Ions Life Sci. 1, 125–149 (2006)

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to those brain regions most affected by the degeneration characteristic of the particular disorder; for example, in the movement disorders total iron levels are increased in the basal ganglia, while in AD the increased iron is associated with the pathological hallmarks of the disease in the vulnerable cortical regions [29,30]. Further, the number of regions affected also parallels the pattern of degeneration seen in each disease, so that in PD significantly increased iron levels are found only in the SN in the midbrain [5,7], while multi-system atrophy and progressive supranuclear palsy are characterized by increased iron not only in the SN, but also in the degenerating caudate nucleus and the putamen [31,32]. The topographical distribution of the increased iron thus suggests a direct relationship with the disease state. The presence of increased total iron levels in SN in PD was first demonstrated 1924 by Lehermitte et al. [33]. It has been confirmed by others using different histochemical, biochemical and physicochemical methods to measure iron concentrations ([5,7] and references cited therein). The increase in nonheme chelatable ferric iron Fe3+  was not found in patients with mild neuropathological changes in the SN [34,35]. However, total iron content – determined following reduction of Fe3+ to the ferrous form Fe2+  by using the Fe2+ chelator ferrozine and granulated ascorbic acid as a reductant for Fe3+ – increased only in the SN in more severe cases of PD, but not in incidental Lewy body disease, a neuropathologically classified disorder which is assumed to reflect presymptomatic cases of PD being devoid of clinical signs of parkinsonism but exhibiting cell loss in the SN and diffuse Lewy bodies postmortem [36,37]. In a further study using iron chelators and spectrophotometry increased total iron and Fe3+ contents in the SN pars compacta from parkinsonian, but not from AD subjects were reported [38]. Histochemical iron staining of paraffin sections using Perl’s stain of Fe3+ after pretreatment with ferrocyanide demonstrated Fe3+ in astrocytes, microglia, the walls of arterioles and veins in putamen and pallidum, but only rarely in neurons [39]. In the SN pars compacta Fe3+ was localized in microglia, in astrocytes often localized next to neurons, and in single nonpigmented neurons. A selective increase in total iron in the parkinsonian SN pars compacta has also been reported in studies using inductively coupled plasma spectroscopy and energy dispersive X-ray microanalysis ([5,7] and references cited therein). Other research groups however, using inductively coupled plasma spectroscopy, atomic absorption and emission, or Mössbauer spectroscopy, could not find significant changes in total iron content in the SN of parkinsonian patients ([5,7] and references cited therein). Explanations for the contradictory findings of different groups may be based on the usage of brain tissue of different stage of neurodegeneration at the time of death as well as different dissection and tissue handling protocols. Moreover the sensitivity of these various methods vary considerably. For example, Mössbauer spectroscopy only measures 57 Fe, a low-abundance isotope in brain tissue [40,41]. Other more sensitive methods, Met. Ions Life Sci. 1, 125–149 (2006)

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such as energy dispersive X-ray microanalysis and laser microprobe mass analysis (LAMMA), provide detailed microstructural information but cannot measure total iron levels. Using energy dispersive X-ray microanalysis on an electron microscope working in the scanning transmission mode, NM-bound iron was detected only in pigmented neurons of the SN pars compacta of parkinsonian patients [42]. Similarly, using LAMMA, prominent iron and aluminum signals were found associated with NM granules [43]. Probe sites directed to nonmelanized portions of cytoplasm of these cells or to the adjacent neuropil revealed lower concentrations of both metals with energy dispersive X-ray microanalysis and LAMMA. Further, the use of X-ray absorption fine structure and cryoelectron transmission microscopy confirmed the increased iron in parkinsonian SN and the lateral globus pallidus and revealed that ferritin was more heavily loaded with iron in PD when compared with age-matched controls [44]. When iron levels are correlated with dopamine concentrations the most significant negative association was found between dopamine and Fe3+ in the putamen and not in the SN in severe PD, although in putamen total iron levels are not significantly different from controls [5]. In the globus pallidus there is a subregional alteration of iron levels in the lateral versus the medial part [31,33,44]. These findings may be indicative of retrograde degeneration of catecholaminergic neurons in PD. However, unlike the presence of oxidative stress and neurochemical changes reported for SN pars compacta, where iron is increased, the striatum is relatively unaltered biochemically. The increase in iron levels in SN identified biochemically in the postmortem brain from parkinsonian patients appears to be confirmed and associated to disease severity in the living patient as assessed by magnetic resonance imaging (MRI) [45,46]. Changes in the magnetic field induced by tissue attributes have an important effect on image contrast of water proton resonance in MRI. The prominent contrast changes seen on T2 -weighted images correlate with tissue Fe3+ content, resulting in decreased T2 relaxation times in MRI experiments. T2 -weighted images demonstrate prominent low signal areas in the red nucleus and the SN pars reticulata which may be regarded as indicative of a high Fe3+ content, since iron concentrations in the adult are much higher than those of paramagnetic manganese or copper [47]. Further, several studies have suggested that transcranial ultrasound may reflect alterations in brain metal constituents since PD patients exhibit a substantially increased signal called hyperechogenicity in the SN [48]. In more than 90% of PD patients, the SN is superimposed by extended white signals, reflecting increased echogenicity, which predominate contralateral to the clinically more affected body side [6,49,50]. A recent postmortem study using brains from normal subjects at different ages suggests an association of SN echogenicity with higher levels of iron, L-ferritin and H-ferritin and reduced NM concentration [51]. This molecular constellation of different iron species may describe a noxious cellular milieu promoting the generation of reactive oxygen species (ROS) and cell Met. Ions Life Sci. 1, 125–149 (2006)

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damage. It may explain the increased susceptibility of subjects with SN hyperechogenicity for nigral injury as demonstrated by positron emission tomography (PET) studies. In summary, the reported data on iron deposits suggest brain iron in PD to be associated with neurite degeneration proceeding in a retrograde manner to neuronal cell death as disease is progressing. A causative role of iron in neurodegeneration, however, cannot be assumed as results of the analytical approaches used to date are not sufficient for testing causal relationships.

3 SOURCES OF INCREASED IRON IN PARKINSON’S DISEASE The question then arises as to the source of the increased iron. There are several possibilities and iron misregulation in the brain may have genetic (see Section 2.2) and nongenetic causes ([52] and references cited therein). Firstly, local alterations in the BBB might result in an increased entry of peripheral iron; for example, a change in the normal iron regulatory systems, such as a local increase in transferrin receptor number could result in an increase in SN iron. Results from studies investigating the density and distribution of the transferrin binding site in the midbrain in post-mortem PD suggested that transferrin receptor number, while increased in the caudate and putamen, are actually decreased on the perikarya of melanised neurons in the SN [53–55]. This agrees with results reported in the periphery where transferrin (and, in agreement with the ferritin results of Dexter et al. [56]) levels were decreased in serum in PD [57]. Further, serum iron is reported to be either unchanged [58] or even decreased in the parkinsonian patients compared with controls, even when iron intake is equivalent [57]. Such results point to a general change in iron regulation in PD, which is not restricted to the brain. In contrast, another iron-binding glycoprotein, lactotransferrin, is reported to be increased in surviving neurons in the SN and ventral tegmental area in the PD brain [59]. The increase in lactotransferrin is associated with increased numbers of lactotransferrin receptors on neurons and microvessels in the parkinsonian SN [60]. The observed increases in this iron mobilization system could represent one mechanism by which iron might concentrate within the PD SN. Another potential source of increased iron is from peripheral influx through a disturbed or open BBB in the SN. This has been suggested from studies in rats, in which the dopaminergic neurons were destroyed by infusion of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle and higher iron concentrations were measured in SN using both histochemical and neurochemical methods [61]. Indeed, recently BBB dysfunction in the midbrain of PD patients was demonstrated by using radiolabelled verapamil hydrochloride and PET [62]. Verapamil is a specific substrate for the P-glycoprotein multidrug Met. Ions Life Sci. 1, 125–149 (2006)

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resistance system in the cell membrane. P-glycoprotein functions as an efflux pump, and verapamil does not cross the BBB. Kortekaass et al. [62] found a high level of uptake of verapamil in the midbrain of patients with PD, but no uptake in age-matched healthy controls. These data suggest a dysfunction of the P-glycoprotein system in vulnerable brain regions in PD, suggesting the BBB might render the midbrain accessible to serum iron. Another possibility for increased iron in PD SN is that iron might be transported intraneuronally from iron-rich areas into the SN. Many areas of the basal ganglia normally contain high concentrations of iron, as described above; the globus pallidus, in particular contains the highest concentration of iron in the brain and is directly connected to the SN pars compacta via afferent -aminobutyric acid (GABA) neurons. To date, however, there is no known mechanism which would explain the translocation of iron from one area of the brain to another, although such a phenomenon has been demonstrated in the immature rat where the BBB, however, is not fully developed [63]. In addition, there is no evidence for decreased iron concentrations in iron-rich areas in PD. A third possibility is that the increased iron levels might result from the redistribution of intracellular iron. Early work attributed the increased iron primarily to nigral glial cells [64]; glial cells are known to store iron and the gliosis occurring in the parkinsonian SN is associated with the degenerating dopaminergic neurons [42,64]. The migration of iron-containing activated microglia and macrophages into the degenerating SN represents a normal immune response to the degenerative process, but could also pose another source of increased ROS production in the SN. Significantly, the glial cells contain ferritin, the major iron binding protein within the brain.

3.1 Ferritin Ferritin is a 450 kDa protein with 24 subunits forming a cavity which can store up to 4500 atoms of Fe3+ . Ferritin-bound iron is compartmentized within the brain so that it cannot participate in redox reactions and acts as a protective mechanism against iron-induced oxidative damage [65]. Ferritin is highly expressed within the glial compartment, predominantly in oligodendrocytes, but also in microglia and astrocytes [29], while ferritin staining of neurons is absent in both the young and aged brain [66]. Jellinger et al. reported an increase in the number of ferritinimmunoreactive microglia in the PD SN [64], the presence of an abundance of these scavenger cells in the degenerating brain region might be expected. The logical consequence of the migration of ferritin-expressing glial cells into the degenerating SN would be an increase in total ferritin in this region. The concentration of ferritin in the parkinsonian basal ganglia has been reported to be slightly increased [34] or reduced (by 25–53% [56]) or unchanged when compared with controls [67] depending on the method of measurement and quantification of L- and H-ferritin, respectively, used. These latter findings are, Met. Ions Life Sci. 1, 125–149 (2006)

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however, surprising, given that intracellular iron levels regulate ferritin levels; an increase in intracellular iron would normally result in an up-regulation of ferritin expression, rather than a decrease, suggesting that normal iron regulatory systems are dysfunctional in PD. The work of Connor et al. [29,68] supports this hypothesis; this research group has studied changes in iron regulatory systems in both normal aging and in disease in detail and have reported changes in both PD and AD which are at variance with the changes occurring in normal aging. This suggests that iron homeostasis may be disrupted in both the AD and PD brain, but the focus of these changes appears to differ between the two diseases. AD is characterized by a decrease in the iron mobilization protein transferrin [29], in contrast, PD is characterized by a decrease in iron storage capacity. Normal aging is reported to be accompanied by an up-regulation of ferritin expression for reasons that are unknown; in PD this normal up-regulation response appears to fail [68]. At the post-transcriptional level cellular iron uptake and storage are regulated by cytoplasmic factors, iron-regulatory protein 1 and 2 (IRP-1 and IRP-2). When intracellular iron levels fall, IRPs bind to iron-responsive elements (IREs) in the 5 -untranslated region of ferritin mRNA and the 3 -untranslated region of the transferrin receptor mRNA, inhibiting the translation of ferritin RNA to decrease iron storage capacity and stimulates the translation of the transferrin receptor mRNA by stabilization of the mRNA to up-regulate iron uptake. When sufficient intracellular iron is present the opposite situation develops to down-regulate intracellular iron levels (reviewed in [69]). These proteins have received recent attention because of the observation that IRPs can be regulated by ROS (reviewed in [70]), suggesting that these proteins may represent an oxidant-mediated mechanism by which iron regulation can be altered. Of particular interest is the fact that the activation of IRP increases the cell’s potential to take up iron [71]. IRPs, predominantly IRP-1, have been described in the human brain [72], and changes in IRP-2 are reported to be associated with the pathological hallmarks of AD, suggesting that changes in this iron regulatory system might be linked to the disease process [73]. Possible changes in this system in PD are yet to be investigated. Iron can be released from ferritin by various exogenous and endogenous substances via reductive mechanisms [74–76]. Of particular interest are mechanisms that might be physiologically relevant. Glial cells produce significant amounts • of superoxide • O− 2  and also nitric oxide NO  from L-arginine, and both of these free radicals are reported to release iron from ferritin stores [77–80]. Fur• − • ther, as depicted in Figure 1, • O− 2 and NO also interact to produce ONOO , • another free radical species which can result in the production of NO2 and the hydroxyl radical • OH [81]. This may contribute to oxidative damage and is of particular interest because of the accumulation of activated microglia associated with pigmented dopaminergic neurons in the parkinsonian SN [82]. Met. Ions Life Sci. 1, 125–149 (2006)

Protein aggregation

α-synuclein/ iron interaction

?

Disease triggers

Parkinson’s disease

Neuronal death in substantia nigra

?

Lewy body formation

Source? Timepoint?

Increased nigral iron

+ e– · –

O2

H2O2 + Fe

2+

H2O2

+ e– · 2H2O Mitochondrial respiration OH + –OH + 2H+

Fenton reaction

DOPAC + NH3 + H2O2 Dopamine metabolism

+ e–

MAO-B, SOD activity glutathione peroxidase activity catalase activity glutathione

Changes in antioxidant systems

· 3+ OH + –OH + Fe

MAO-B

+ 2H+

+ e–

Oxidative stress

Mitochondrial dysfunction

Dopamine + O2

O2

Neuromelanin/ iron interaction

i.e. transferrin, ferritin, ceruloplasmin, neuromelanin-iron-binding

Dysregulation of iron homeostasis

Unknown but probably includes both genetic and environmental factors

Figure 1. The role of iron in the pathogenesis of Parkinson’s disease (PD). This scheme summarizes pathochemical findings in PD and attempts to explain possible synergistic molecular mechanisms that finally lead to dopaminergic neuronal cell death. DOPAC, 3,4-dihydroxyphenylacetic acid; MAO-B, monoamine oxidase type B; SOD, superoxide dismutase.

Toxic metabolic dopamine products

Increased dopamine autoxidation

Changes to blood-brain barrier

Age-related changes

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We have further demonstrated that a variety of catechol-based substances, including the dopaminergic neurotoxin 6-OHDA, can release iron from ferritin in vitro [83]. The release of ferritin-bound iron by 6-OHDA is associated with lipid peroxidation, a response abolished by the addition of an iron chelator; thus we have suggested that this release is important for 6-OHDA toxicity. Interestingly, we showed that the native neurotransmitter dopamine is also capable of releasing iron from ferritin, although whether this release is elicited by dopamine itself or after its oxidation to 6-OHDA is unclear. Comparative studies demonstrated that the release of ferritin-bound iron is dependent upon the substance containing an ortho-dihydroxyphenyl structure and upon the redox potential of the substance [83]. Such in vitro work is of interest as significant amounts of 6-OHDA can be formed in vitro from the oxidation of dopamine by H2 O2 [84], and it has been suggested that 6-OHDA can also be formed in vivo under conditions of oxidative stress and may contribute to degeneration in PD [85,86]. Certainly ‘6-OHDA-like substances’ have been identified in the urine of PD patients [87]. However, as discussed above, ferritin is located in the glial compartment in the brain; neurons do not stain positive for ferritin [66]. As free radicals are highly reactive, it is unlikely that glial-derived free radicals diffuse across the intracellular space in sufficient quantities to damage neuronal constituents. If intracellular iron release contributes to neuronal damage it seems more probable that an intraneuronal iron source is responsible for oxidant-mediated damage. Such an iron source is the intraneuronal pigment NM.

3.2 Neuromelanin Neuromelanin is a dark pigment produced in dopaminergic and noradrenergic neurons of the human SN and locus coeruleus, respectively. Traditionally, NM is thought to be an inert cellular by-product, produced via a simple autoxidation pathway, a hypothesis supported by the failure to link tyrosinase, the rate-limiting enzyme of peripheral melanin synthesis, to NM. Recent evidence (reviewed in [88]) however, suggests a regulatory pathway for NM production and a possible physiological role in the cell. The loss of NM-containing dopaminergic neurons and the resulting pallor of the SN is one of the most striking features of PD [9–11]. A relationship between the loss of the dopaminergic SN cells and their NM content has been reported [89], suggesting a role of NM for neurodegeneration in PD. The function of NM has yet to be established, but it is considered as an endogenous iron-binding molecule in pigmented neurons (reviewed in [52,88]). It may therefore play a physiological role in intraneuronal iron homeostasis. Support for this theory comes from changes in NM in the PD brain where Met. Ions Life Sci. 1, 125–149 (2006)

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significantly less iron is bound to NM than that seen in the normal brain [90]. This suggests that changes in iron-binding to NM result in increased levels of intraneuronal free iron and the subsequent cell damage observed in PD. 7% of isolated NM is reported to consist of inorganic components, including iron, copper, zinc, and chromium [91,92]. Isolated human NM consists of 2.8% iron as estimated by Mössbauer spectroscopy [41], while the concentration of Fe3+ in the SN has been estimated using electron paramagnetic resonance at 6780 ng iron/mg intact SN tissue or 11300 ng iron/mg isolated NM [93]. This measurement is in agreement with the estimate of 9700 ng iron/mg isolated NM using total reflection X-ray fluorescence [92]. Iron binding studies using NM isolated from the human SN demonstrated that NM contains high Kd = 718 ± 108 nM and low-affinity binding sites Kd = 9431 ± 655 nM for Fe3+ [94]. Our recent data demonstrates that a purely Fe3+ signal can be measured from intact frozen SN tissue using Mössbauer spectroscopy [94]. These data support reports that iron is directly bound to NM granules in the SN [42,43,92,95] and that this signal is increased in PD [96]. The interaction of iron with NM is of interest because the behavior of NM changes in the presence of iron; instead of inactivating free radicals, it begins to act as an effective pro-oxidant. A NM–iron interaction-mediated increase in free radical production has been demonstrated in vitro [97,98]. It is unclear whether iron bound to NM can contribute to free radical-producing mechanisms or whether the presence of NM within the dopaminergic cells represents a pool of iron which, under certain circumstances, can be released to interact in free radical-producing pathways. Nevertheless such mechanisms are of interest as they represent an intraneuronal source of free radicals that could increase the oxidative load within the vulnerable dopaminergic neurons. While the physiological relevance of these proposed mechanisms is unclear, we have demonstrated the functional consequences of the influence of NMs on the cell and its modulation by iron in vitro [99]. In the absence of iron, isolated human NM significantly decreased membranal damage in rat cortical homogenates in vitro as measured by lipid peroxidation. Further when NM was added together with iron, the amount of lipid peroxidation measured was significantly less than that induced by iron alone. These results support the hypothesis that NM has antioxidant properties and can protect the cell from radical-induced damage. It is possible that NM may serve a similar function in binding iron in neurons, as does ferritin in the glia, thus representing an as yet unrecognized mechanism by which the cell can protect itself against oxidative damage. In contrast, when iron-saturated NM was added to the membrane homogenate, cell damage was significantly increased to 264% of that induced by NM alone; this damage was significantly attenuated by the addition of the iron chelator desferoxamine [99]. These results support the hypothesis that NM can have a protective influence on the cell, but can be detrimental when iron levels rise above a certain level. Met. Ions Life Sci. 1, 125–149 (2006)

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4 CONSEQUENCES OF IRON OVERLOAD IN PARKINSON’S DISEASE 4.1 Oxidative Stress-induced Neuronal Damage ‘Oxidative stress’ is an expression used for a process that implicates reactions of O2 or derived substances with biomolecules [100]. Among all oxygen species • − • • the free radicals • O− 2  OH NO , and ONOO as well as H2 O2 are supposed to be the most abundant ROS in biological systems. Depending on the redox state of iron, iron stimulates the production of free radicals via several pathways (Figure 1). Due to the high reactivity of free radicals, covalent modifications of lipids, proteins, and DNA are likely to occur if radicals are not trapped by scavengers such as tocopherol, ascorbate or glutathione, which can lead to neuronal damage. Lipid peroxidation gives rise to cytotoxic aldehydes that have to be detoxified by glutathionylation. Oxidized proteins are metabolized by proteases and radical-mediated DNA-base modifications may be repaired by specific glycosylases. Incomplete repair of DNA and proteins may, however, result in altered transcriptional response and protein aggregation. Over the last decade, many theories for the etiology of PD have been proposed, but oxidative stress has remained a constant theme, and unifies the two most discussed mechanisms, increased ROS production and mitochondrial dysfunction (which may also result in increased free radical formation). The dopaminergic SN is particularly vulnerable to the production of ROS because of its neurochemical environment, including a normally high level of tissue iron. Both the enzymatic metabolism of dopamine by MAO-B and autoxidation of dopamine results in the formation of H2 O2 , and H2 O2 stimulates MAO-B via positive feedback to further increase H2 O2 production. H2 O2 is capable of reacting with free Fe2+ via the Fenton reaction described in Figure 1 to produce • OH, a particularly active radical species that can react with and damage cellular constituents. In addition, a disturbed NM–iron interaction has been discussed as contributing to the nigral oxidative load in vivo [101–104] as shown diagrammatically in Figure 1. Indeed, we have demonstrated in a recent study that discrete asymptomatic lesions were produced by the injection of iron-laden NM (2.5 nmol iron) into the rat SN and that lisuride attenuated the iron-induced dopaminergic cell death by a decrease in oxidative load within the iron-treated tissue [105]. By binding metals, NM may potentiate free radical formation [102] or assist with protective scavenging of • OH [104] via metal sequestration [103,106,107]. Reductions in NM in PD are likely to render the cell more susceptible to oxidative damage. Indeed, the level of redox activity detected in NM aggregates was significantly increased +69% in parkinsonian patients and was highest in patients with the most severe neuronal loss [108]. This change was not observed in tissue in the immediate vicinity of melanized neurons. Met. Ions Life Sci. 1, 125–149 (2006)

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Indirect evidence supports the hypothesis that the SN experiences oxidative stress in PD. Two of the enzymatic antioxidant systems which act to protect tissue against oxidative damage are unaltered in PD (catalase and glutathione peroxidase) [109,110], but superoxide dismutase activity is increased in the parkinsonian SN [109,111], and both total and reduced glutathione levels are decreased [112–114], changes consistent with an increased production of ROS. Further heme oxygenase-1, a cellular stress protein, is upregulated [115] also suggesting that the tissue is undergoing chronic oxidative stress. More direct evidence can be gained from the measurement of increased oxidative damage to membrane lipids (lipid peroxidation) in the SN in PD [116,117], as well as oxidative damage to proteins [118,119] and DNA [120], and oxidative changes in the mitochondrial respiratory chain that can also result in increased production of free radical species (reviewed in [121]).

4.2 Protein Aggregation One of the primary neuropathological criteria for a confirmed postmortem diagnosis of PD is the presence of cytosolic filamentous inclusions known as Lewy bodies and Lewy neurites in some surviving dopaminergic nigral neurons. The major fibrillar material of these inclusion bodies is an ubiquitous presynaptic protein of unknown function, -synuclein [122,123]. Mutations in -synuclein cause a form of familial PD in the human [124,125] and a parkinsonian-like syndrome in experimental animals [126,127]. Abnormal filamentous aggregates of misfolded wild-type -synuclein protein are the main components of Lewy bodies [122,123] with iron deposits in the rim [128], perhaps indicating a pathogenic role for -synuclein in PD. However, the mechanism of formation of mutations or transformation of unfolded normal -synuclein protein to aggregated fibrillar -synuclein is not completely understood. One of the prevailing hypotheses is that mutated or oxidatively damaged -synuclein first forms soluble oligomers, followed by insoluble fibrils such as those found in Lewy bodies. These protein aggregates are refractory to ubiquitin–proteasome system-mediated proteolysis, thus ultimately leading to neuronal cell death via a disturbance of the cytosolic environment [129]. Mutated -synuclein naturally aggregates [130], and in PD, and other disorders such as dementia with Lewy bodies, the protein aggregates into the filamentous structures present in Lewy bodies. The reason for this aggregation is unknown, but it has been shown that even small amounts of di- and trivalent metals, including iron, increase the rate of -synuclein fibrillation [131]. An analysis of Lewy bodies in the parkinsonian SN demonstrated that these pathological inclusions contain redox active iron [128], suggesting that this mechanism may also occur in vivo. Recently it was shown that in the presence of iron, Met. Ions Life Sci. 1, 125–149 (2006)

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-synuclein stimulated the production of H2 O2 in vitro via the Fenton reaction [132], possibly via the binding of Fe2+ to the protein [133]. This effect was not seen for - and -synuclein which are not associated with neurodegenerative disease [132]. While the relationship between the aggregation state of the protein and free radical production is unclear, this may represent an iron-mediated mechanism by which • OH may be produced inside the SN neurons. Oxidative stress is reported to induce the aggregation of -synuclein [134], leading to fibril formation [135]. In addition, aggregation of -synuclein may be promoted by advanced glycation end products (AGEs), major structural crosslinkers which cause the transformation of soluble cytoplasmic proteins to insoluble deposits, including neurofibrillary tangles and Hirano bodies in AD as well a Lewy bodies in PD [136–138]. Interestingly, formation of AGEs is accelerated by iron since the reaction products formed (Amadori products) from reducing sugars with primary or secondary amino groups are further oxidized. Aggregated -synuclein in diseased brains displays evidence of oxidative damage [139], suggesting a mechanistic link between iron, oxidative stress, protein aggregation and cell death in PD and other synucleinopathies (Figure 1). More recently, it has been demonstrated that soluble -synuclein oligomers, protofibrils, are more toxic than insoluble fibrils [140,141]. Further, catecholamines related to dopamine stabilize oligomers of -synuclein by covalent oxidative ligation in test tubes [142]. Thus, iron-catalyzed oxidation of catechols may lead to -synuclein protofibrils. These protofibrils bind to and permeate lipid vesicles or mitochondrial membranes and then form insoluble fibrils [140,141], and these disturbances might result in activation of neuronal cell-death cascades. However, the question of whether -synuclein is neurotoxic or neuroprotective is still open, as the formation of Lewy bodies and accumulation of -synuclein could also be a compensatory process to enable the neuron to protect itself from toxic species.

5 GENERAL CONCLUSIONS Histopathological, biochemical, and in vivo brain imaging techniques, such as magnetic resonance imaging and transcranial sonography, revealed a consistent increase of brain iron in PD. Increased iron deposits in the SN in PD may have several reasons that may reside in disturbances of iron uptake, storage, and transport as neurodegeneration progresses. Major iron stores are ferritin and hemosiderin in glial cells as well as NM in neurons. Age- and disease-dependent overload of iron storage proteins may result in iron release upon reduction. Consequently, the low molecular weight chelatable iron complexes may trigger redox reactions leading to damage of biomolecules. Met. Ions Life Sci. 1, 125–149 (2006)

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These processes may promote disease progresssion by stimulating aggregation of susceptible proteins such as -synuclein. Additionally, upon neurodegeneration there is strong microglial activation which can be another source of high iron concentrations in the brain. Although the current evidence suggests that increased brain iron may be a secondary result of neuronal degeneration and reactive gliosis in several neurological disorders affecting the basal ganglia including PD, progressive supranuclear palsy and multi-system atrophy, the question of whether iron-associated degenerative pathways are a significant factor driving progressive neuronal death in these disorders is yet to be definitively answered.

ACKNOWLEDGMENTS K.L.D. is the recipient of a R. D. Wright Research Fellowship from the National Health and Medical Research Council of Australia. The work was funded by the National Health and Medical Research Council of Australia and the BrainNet Europe. The support of the Deutsche Parkinson-Gesellschaft e.V. is acknowledged.

ABBREVIATIONS AND DEFINITIONS AD AGEs basal ganglia

BBB COMT

CNS l-DOPA GABA 6-OHDA IRE

Alzheimer’s disease advanced glycation endproducts a part of the motor system related to the neuronal operation of movement that consist of several large, anatomically distinct masses of gray matter situated in the core of the cerebral hemispheres. These constitute the striatum – the caudate and the putamen – and the pallidum – the internal and external portions of the globus pallidus blood–brain barrier catechol-O-methyltransferase, an extraneuronal enzyme that methylates catecholamines such as dopamine on the 3-hydroxy group in the catecholamine ring central nervous system synonym levodopa, l-3,4-dihydroxyphenylalanine; the blood– brain barrier permeable precursor of dopamine -aminobutyric acid, an inhibitory neurotransmitter in the central nervous system 6-hydroxydopamine, a dopaminergic neurotoxin iron-responsive elements

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IRP Kd

LAMMA MAO

microglia MRI NM

PD PET ROS

SN

143

iron-regulatory protein dissociation constant – Kd values were obtained from Scatchard analysis of saturation binding experiments. This value indicates the capacity of a radioactive ligand to displace a selective ligand for a receptor subtype from the receptor; if a receptor has a high affinity for the ligand, the Kd value, will be low laser microprobe mass analysis monoamine oxidase, an enzyme that is located on the outer membrane of the mitochondria and oxidatively deaminate catecholamines such as dopamine to their corresponding aldehydes immune cells of the brain magnetic resonance imaging neuromelanin, melanin in the brain share some similarities in appearance and structure to cutaneous melanins, and has thus been named neuromelanin Parkinson’s disease positron emission tomography reactive oxygen species, are produced by the mitochondrial respiratory chain and in the cytoplasma of dopaminergic nerve cells. In excess, they can cause intraneuronal and mitochondrial damage, which promotes neuronal cell death substantia nigra (from the Latin meaning ‘black body’), a small area of the human midbrain that contains neuromelanin and dopamine-producing neurons, the axons of which innervate the striatum and thereby control body movements

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7 In Vivo Assessment of Iron in Huntington’s Disease and Other Age-Related Neurodegenerative Brain Diseases George Bartzokis,1234 Po H. Lu,13 Todd A. Tishler,135 and Susan Perlman1 1

2

Department of Neurology, Alzheimer’s Disease Center, The David Geffen School of Medicine at UCLA, 710 Westwood Plaza, Los Angeles, CA 90095-1769, USA

Laboratory of Neuroimaging, Department of Neurology, Division of Brain Mapping, UCLA, Los Angeles, CA 90095, USA 3

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Greater Los Angeles VA Healthcare System, Department of Psychiatry, West Los Angeles, CA 90073, USA

Department of Psychiatry, Charles R. Drew University of Medicine and Science, Los Angeles, CA 90043, USA 5

Neuroscience Interdepartmental Graduate Program, The David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA

1 INTRODUCTION. PUZZLING CHANGES IN CELL NUMBERS IN HUNTINGTON’S DISEASE BRAIN 2 HUMAN BRAIN DEVELOPMENT AND DISEASE PHENOTYPES 2.1 Developmental Underpinnings of Degenerative Brain Diseases 2.2 Parallels Between the Spread of Myelination and Huntington’s Disease Neurotoxicity 2.3 Specific Toxicity of Myelinated Projection Neurons

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Edited by Astrid Sigel, Helmut Sigel and Roland K. O. Sigel

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3 OLIGODENDROCYTES AND IRON IN BRAIN DEVELOPMENT AND DEGENERATION 3.1 Oligodendrocytes as the Iron Storage Cell in Development 3.1.1 Gray and White Matter Oligodendrocyte Density and Iron Levels in Degenerative Brain Diseases 3.1.2 Iron and Oligodendrocytes in Aging and Huntington’s Disease 3.2 Oligodendrocyte Vulnerability: The ‘Achilles’ Heel’ of Our Brain 3.3 Huntingtin and Myelin Integrity 3.4 Huntingtin and Brain Iron Homeostasis 3.5 Triplet Length and Age at Onset 3.6 Elevated Iron and Exacerbation of Excitotoxicity 4 TRANSITION METAL METABOLISM AND PROTEINOPATHIES 5 IN VIVO MEASUREMENT OF BRAIN IRON 5.1 Measuring Ferritin Iron in Vivo 5.2 Measuring Ferritin Iron in Neurodegenerative Diseases 5.3 Ferritin Iron as a Biomarker 6 NOVEL TREATMENT CONSIDERATIONS 7 CONCLUSIONS ACKNOWLEDGMENTS ABBREVIATIONS REFERENCES

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1 INTRODUCTION. PUZZLING CHANGES IN CELL NUMBERS IN HUNTINGTON’S DISEASE BRAIN Huntington’s disease (HD) is a genetic disease caused by the repetition of a CAG trinucleotide sequence encoding for a polyglutamine tract at the N terminal of the gene coding for a protein named huntingtin (for review see [1,2]). The number of polyglutamine repeats in healthy unaffected individuals varies between 6 and 35. Repeats of 36 or above define the HD allele and repeat expansions above 60 characterize juvenile onset cases (for review see [2]). Despite our understanding of the genetics of HD, the physiologic role of huntingtin and the ‘normal’ variation of 6 to 35 CAG repeats remains unclear as do the pathologic processes triggered by 36 or more repeats that results in the observed HD phenotypes. Met. Ions Life Sci. 1, 151–177 (2006)

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The inadequate understanding of the pathogenesis has encumbered the search for therapeutic interventions and the disease continues to lack effective treatment. The search for viable therapies is also hampered by the apparently unique manifestation of this disease in humans. The uniquely human aspect of this disease is demonstrated by the difficulties encountered in creating a mouse model of HD that mimics the human disease with fidelity. Despite expressing mutant huntingtin protein and developing significant neurologic symptoms the mouse models have failed to develop the striking and precise neuronal degeneration observed in humans (for review see [1]). This situation is reminiscent of similar difficulties in developing accurate animal models of other uniquely human agerelated degenerative diseases such as Alzheimer’s disease (AD) (reviewed in [3]). Several other aspects of HD that have received relatively little attention are also worthy of mention in regard to the human pathogenesis of this disease. First is the observation that early-stage and presymptomatic HD brains show a puzzling increase (doubling) in the density of oligodendrocytes in the striatum years before atrophy or loss of neurons occurs [4,5]. Second, postmortem and in vivo imaging studies confirm that in HD, brain degeneration may begin with and clearly involves white matter (see Figure 1 top in Section 2.3 [6]) [7–17]. Third, HD is associated with increased brain iron levels [8,13,18,19] which also occur early in the disease process and are more pronounced than those seen in AD and Parkinson’s disease (PD) ([20,21], reviewed in [22]). Finally, HD is a neuropsychiatric disorder that in humans manifests with cognitive decline and behavioral symptoms such as depression decades before the better-known motor and dementia symptoms [23,24]. Brain disorders with uniquely human manifestations such as HD may benefit from being examined within a conceptual framework that is based on the unique biology of the human brain (reviewed in [3]). We believe that the unique pattern and timing of human brain myelination can help explain many phenotypic aspects of neuropsychiatric disease as well as the striking patterns of spread of their pathognomonic lesions across the brain in predictable symmetric bilateral patterns (reviewed in [25]) (see Section 2.2). We will therefore examine HD as well as other age-related neuropsychiatric diseases in the context of a myelin-centered model of the human brain. The model’s central premise is that the trajectory of myelin development and breakdown is essential to our very uniqueness as a species (reviewed in [3,25,26]). In its widest perspective, the model primarily delineates a myelin hypothesis of human brain evolution and normal development and is ‘secondarily’ useful in conceptualizing a wide range of age-related neuropsychiatric diseases such as schizophrenia, AD, and PD. The unique vulnerabilities of oligodendrocytes and the highly protracted and extensive developmental process of human brain myelination delineated in the model are directly pertinent to many uniquely human brain functions and neuropsychiatric diseases that we believe include HD and its puzzling and striking increases in oligodendrocytes and brain iron. Met. Ions Life Sci. 1, 151–177 (2006)

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2 HUMAN BRAIN DEVELOPMENT AND DISEASE PHENOTYPES 2.1 Developmental Underpinnings of Degenerative Brain Diseases The human brain is unique in its extensive and pervasive myelination process that supports its high information processing capacity ([3], reviewed in [26–28]). Myelination increases axonal transmission speed, reduces action potential refractory time, and improves synchrony of high-frequency bursts of signals across the distributed neural networks of the brain. Thus, if the brain were to be compared to the Internet [29], myelination not only increases its speed (transforming it from a telephone-line based system to a T1-line based system) but also its ‘bandwidth’ increasing the actual amount of information that can be transmitted per unit time. Human behavior and cognitive function is dependent on high-throughput information processing, which in turn depends on the uniquely extensive myelination of our brain and the maintenance of its functional integrity over our lifespan (reviewed in [3,26,28,30]). Neither brain size nor a disproportionate over-development of specific brain regions such as the frontal lobes is uniquely human (reviewed in [26]). Recent data indicate that, compared to other primates, the human frontal lobes are not disproportionately larger than other brain regions when controlling for body size and do not contain more gray matter [27,31]. The human frontal lobe does however have disproportionately greater percentage of white matter volume (approximately 20–30%) compared to the other higher primates [27,31] and the percentage of brain dry weight accounted for by myelin in our brain (35%) is substantially (30%) higher when compared to rodents [32]. In this context, the difficulty in creating transgenic rodent models that reproduce human disease with fidelity supports the suggestion that like AD [3], the pathogenesis of HD may involve the unique human myelination trajectory and its associated physiologic changes such as iron accumulation ([3], reviewed in [26,33]) (see Section 3.1). This myelin-centered model of the human brain is not intended to diminish the importance of other brain developmental processes such as neurogenesis that are primarily intrauterine, but whose disruption can cause catastrophic abnormalities that are usually evident at birth or in early infancy [34]. The abnormal HD gene as well as genes involved in other neuropsychiatric disorders such as AD and PD do not cause developmental abnormalities in utero despite their presence and expression from inception. The focus on myelin is also not intended to dismiss the drastic processes of neuronal, synaptic, axonal, and dendritic pruning and elimination that appears to reduce the ‘connectivity’ of the infant and young child’s brain by as much as two thirds [34,35]. These processes, which have been hypothesized to impact several childhood disorders, are generally completed Met. Ions Life Sci. 1, 151–177 (2006)

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by puberty or mid-adolescence [34,35] and likely serve to create the necessary intracranial space needed in order for myelination to continue into adulthood [28]. With the exception of a small subset of juvenile cases (see Section 3.5), HD manifests primarily in early adulthood and midlife, during which time the principal developmental process in the human brain is myelination [reviewed in [28,29]). In this context HD is primarily a developmental disorder with an abnormal myelination trajectory [4,5] that ultimately leads to neurodegeneration and thus distinctly differs from AD where degeneration begins primarily in old age after a normal developmental trajectory [25].

2.2 Parallels Between the Spread of Myelination and Huntington’s Disease Neurotoxicity The normal lifelong trajectory of human brain myelination has a quadratic-like (inverted U) shape with increasing myelin content that peaks in middle-age and subsequently breaks down and declines in older age (reviewed in [25,28,36]). The myelin-centered view of the human brain defines our entire lifespan and especially the first six decades as a period of development as myelinated white matter volume increases [29,37]. At birth oligodendrocytes each myelinate single axon segments in the motor and sensory circuits and then continually increase the number of segments each cell myelinates throughout development such that by middle-age, each cell myelinates up to 50 axon segments in certain cortical regions. The early-myelinating oligodendrocytes myelinate fewer axon segments with thicker myelin sheaths containing many wraps per segment in the motor (including basal ganglia) and sensory circuits in order to enable primary motor and sensory functions. Myelination then proceeds all the way to the cortex where, in humans, it has a very predictable heterochronous pattern (i.e., different regions myelinate at different times) [35,38]. Cortical myelination begins with the motor and sensory regions and then continues through middle-age to involve association regions (frontal, temporal, parietal lobes), with the medial temporal lobe region being myelinated last (reviewed in [26,28,36,39,40]). Concurrently with ‘connecting’ and bringing the entire cortex ‘on line’ myelination proceeds into the cortex with the lower cortical layers becoming most heavily myelinated [28,29,36]. The earlier and more heavily myelinated circuits seem to be preferentially impacted in HD, which is characterized by the loss of neuronal cells that send out myelinated projection axons while almost totally sparing local interneurons (for review see [1,2]). This neuronal degeneration pattern is a pathognomonic feature of HD, is strikingly predictable [41], and parallels the spreading pattern of brain myelination described in the previous paragraph. It begins with striatal (caudate and putamen) GABAergic medium spiny neurons that constitute 95% of all striatal Met. Ions Life Sci. 1, 151–177 (2006)

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neurons. These neurons project to the substantia nigra and globus pallidus that in turn send reciprocal glutaminergic excitatory inputs back to these striatal neurons (for review see [1,2,41]). In HD the loss of striatal cells and volume is followed by substantial neuron loss in the lower cortical layers (especially layer VI) that project to the thalamus [41,42]. The thalamus in turn sends reciprocal glutaminergic inputs back to the cortex (reviewed in [42]). Even within cortical regions, the spread of the myelination process and neuronal destruction seem to follow similar patterns. Area 9, which is adjacent to but myelinates before area 46 [43], sustains more and earlier neuronal degeneration than area 46 [42]. Cortical neuronal loss and thinning progresses from the earlier-myelinated sensorimotor areas to frontal and temporal association areas [44], consistent with early deficits in perceiving sensory stimuli [23]. Furthermore, the medial temporal lobe region that is the last to myelinate (reviewed in [26,36,39,40] is relatively spared even in the later stages of HD [45,46]. With respect to this progression of its pathognomonic neurodegenerative lesions, HD has the opposite pattern of spread to AD. The degenerative lesions in AD (neuritic plaques and neurofibrillary tangles) progress in the opposite pattern of myelination, beginning in the medial temporal lobe regions, progressing to the association areas, and not involving motor regions until very late in the disease (reviewed in [26,47]). Together with the much younger age at onset, the pattern of progression of HD paralleling myelination strongly suggests that the pathogenesis of HD may involve a defect in myelin itself.

2.3 Specific Toxicity of Myelinated Projection Neurons At very early and even presymptomatic stages of HD there is evidence for myelin breakdown and loss (see Figure 1 top [6] and [7,9,10,13–17]) (see also Figure 3 in Section 3.1.1). The spreading pattern of myelin breakdown seems to follow the spreading pattern of neurotoxic damage described above. Earliermyelinating axons such as those in the occipital system show more evidence of myelin breakdown than later-myelinating axons in frontal regions [17] and this same spreading pattern is also noted in the progression of cortical atrophy [44] which may be a reflection of intracortical myelin loss [48]. A primary dysfunction of oligodendrocyte development seems an unlikely cause for the pathogenesis of HD as initial brain development and oligodendrocyte proliferation is intact [4,5]. Recent data suggest that the abnormal huntingtin protein of HD may reduce trophic factors necessary for myelin stability (see Section 3.3 below). Thus, an indirect effect of dysfunction on the risk of neurotoxicity is possible and supported by the evidence of myelin breakdown [6,7,9,10,13–17]. A myelin defect resulting in its dysfunction could also activate normal compensatory mechanisms that promote continued Met. Ions Life Sci. 1, 151–177 (2006)

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myelination [37], resulting in the abnormally high numbers of oligodendrocytes observed in HD [4,5]. Although mechanisms for how abnormal huntingtin may cause myelin breakdown/dysfunction are not fully worked out (see Section 3.3 below), myelin dysfunction can contribute to the pathogenesis of the disease and help explain the striking specificity of HD neurotoxicity for certain groups of projection neurons (for review see [1,2,41]). The affected neurons all extend myelinated projection axons, are part of reciprocal circuits, and depend on the adequate function of their myelin sheaths. Myelin dysfunction (failure of myelin sheaths to support saltatory conduction) can result in excitotoxic neuronal death (for review see [26,49]). We propose that after a reciprocal projection circuit is established, myelin dysfunction of vulnerable projection neurons reduces their efferent output to their target neurons. As signals fail to stimulate their target neuron with the appropriate intensity and/or timing, the target neuron in the circuit would attempt to reestablish homeostasis and up-regulate its transmission back to the projection neuron whose myelin is dysfunctional (reviewed in [3,26]). Such upregulated feedback to the projection neuron would result in toxic levels of activation and death of the projection neuron whose dysfunctional myelin prevents it from appropriately responding to its efferent input (see Figure 1 bottom). Much data support the idea of an excitotoxic mechanism for projection neuron degeneration in HD and excitotoxic damage has been an HD model of striatum cell death for over 30 years (for review see [49]). Animal as well as human postmortem studies support the supposition of metabolic dysfunction with concomitant oxidative damage [50–54] and/or oxidative damage secondary to excitotoxic neurotransmission [55,56]. The striatum as well as the lower cortical layers receive dense excitatory glutaminergic input. Glutamate, glutamate receptor agonists, and especially N -methyl-D-aspartate (NMDA) receptor agonists produce toxic effects in the striatum very similar to the lesions seen in HD. We suggest that the underlying cause of the excitotoxicity in HD is the initial disruption of such circuits due to myelin breakdown resulting in failure of saltatory conduction. The subsequent compensatory excitatory feedback back to the projection neurons results in excitotoxicity and their eventual degeneration. This process would impact primarily projection neurons that are dependent on adequate myelination to maintain proper circuit function (Figure 1 bottom). We further propose that the same underlying cause (myelin breakdown) [6,7,9,10,13–17] also results in compensatory attempts to remyelinate, causing the overproduction of oligodendrocytes and the doubling of their numbers [4,5]. By itself, the excitotoxic mechanism does not fully explain the observed pattern of neuronal loss however. NMDA receptors are equally dense in the neocortex superficial layers, striatum, hippocampus, and other regions, while HD neurotoxicity is localized primarily in the striatum and lower cortical layers and Met. Ions Life Sci. 1, 151–177 (2006)

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Figure 1. Top: myelin breakdown in Huntington’s disease brain. Postmortem myelin stain (myelin is black). On the right side a coronal plane section through the frontal and temporal lobes of a normal brain. On the left side a section at the same level taken from a patient with Huntington’s chorea that is magnified approximately ×2. Note striking myelin breakdown and white matter atrophy in the HD brain (left) [167]. Bottom: reciprocal circuits of neurons. This oblique coronal MRI image from a healthy individual captures in one image the striatum (C and P), G, T, and SN (structures outlined in black). The circuits drawn in white between striatum and SN (left side of image) and between cortex and thalamus (right side of image) depict reciprocal circuits. Dotted lines represent axons whose myelin fails to support saltatory conduction and solid white lines represent glutaminergic feedback to striatal and cortical neurons that undergo excitotoxicity (see Section 2.3). C = caudate; G = globus pallidus; P = putamen; SN = substantia nigra; T = thalamus. (Top: Reproduced by permission from [167]). Met. Ions Life Sci. 1, 151–177 (2006)

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leaves areas such as the hippocampus relatively unaffected [45,46]. Therefore, in addition to NMDA-related excitotoxicity another synergistic factor must also be involved in the toxic effects to reproduce the pattern of neurotoxicity observed in HD. A huntingtin-related defect in myelination can explain the myelin dysfunction and the compensatory overproduction of oligodendrocytes described above, as well as this second synergistic risk factor that we proposed to be iron accumulation [13]. As will be detailed below, heavily myelinated regions such as the basal ganglia and lower cortical layers also contain high iron levels [8,57– 59] and iron promotes free radical damage as well as proteinopathies (abnormal deposits of proteins) ([60,61], for review see [62]).

3 OLIGODENDROCYTES AND IRON IN BRAIN DEVELOPMENT AND DEGENERATION 3.1 Oligodendrocytes as the Iron Storage Cell in Development Oligodendrocytes have the highest iron content of all brain cell types [63–66]. These cells are unique in their capacity to obtain their iron directly through binding the iron storage protein ferritin [67], and may be directly involved in brain iron regulation [68]. In HD, oligodendrocyte production is upregulated and results in increased density of these cells before any clinical symptoms appear [5]. During development, brain ferritin binding coincides with the onset and progression of myelination [33,57,67,69–71] and increasing brain iron levels are a necessary [64,72,73] and integral part of myelin development and differentiation in childhood and early adulthood [8,21,57,74,75]. With age, myelin breakdown becomes an increasing problem, even in healthy individuals [25,29,36,76,77] and continued repairs and remyelination result in increased numbers of oligodendrocytes in old age [37]. This ‘normal’ aging process is essentially what we postulate for the process observed in HD (see Section 2.2) except that abnormal huntingtin causes the process to occur prematurely and thus preferentially impacts early-myelinating regions. Iron can be translocated between brain regions [78,79]. It is therefore possible that iron released by myelin breakdown [80,81] ultimately contributes to the iron increases observed in gray matter regions and the age-related increased risk of toxicity in these regions (for review see [3,26]). In healthy individuals certain brain regions such as the striatum continue accumulating ferritin iron throughout the lifespan [8,57,59,61] and these increases occur as ferritin iron decreases in white matter regions [61]. In disease states such as HD and AD where myelin breakdown is exacerbated, evidence of lower white matter iron levels are observed in conjunction with abnormally increased iron levels in the striatum (see Figures 2 and 3 in the next section). Met. Ions Life Sci. 1, 151–177 (2006)

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3.1.1 Gray and White Matter Oligodendrocyte Density and Iron Levels in Degenerative Brain Diseases Quantitative studies have shown that human brain gray matter contains substantial numbers of oligodendrocytes. These cells represent approximately 15% (cortex) to 30% (striatum) of total cell bodies in gray matter [4,82,83]. In adults, oligodendrocyte precursors represent an additional 3–5% of gray matter cells [84], but the precursor percentage is likely much higher at younger ages since in humans, childhood and adulthood represent a continuum of oligodendrocyte development and differentiation [29,37]. In white matter regions oligodendrocytes make up the vast majority (approximately 90%) of cell bodies [85] and the age-related changes as well as the absolute levels of ferritin iron in these regions probably closely reflect changes in the density of oligodendrocytes and their associated myelin as the remaining tissue is composed primarily of axonal cytoplasm [86]. When compared to age-matched normal controls the basal ganglia show increased iron deposits in AD and this increase is even more striking in HD (Figure 2). A large portion of brain iron is associated with myelin [87,88]. It is therefore remarkable that in both HD and AD the iron levels in frontal white matter are lower than in their matched control groups; the opposite pattern from the one seen in the basal ganglia (Figure 2). The reduction in white matter iron is more striking in HD, reaches statistical significance, and supports published data that suggests a process of myelin breakdown [6–17]. When relaxation rates (a measure of white matter integrity) are examined in the same subject samples that had brain iron levels measured, it is clear that both AD and HD are characterized by loss of myelin integrity (Figure 3). In HD, this observation of myelin breakdown early in the disease process has recently been confirmed using diffusion tensor imaging measures [16].

3.1.2 Iron and Oligodendrocytes in Aging and Huntington’s Disease The striatum is a site of high iron concentrations that increase with age from very low levels at birth ([8,57,59,61], for review see [62]). Several postmortem studies have found markedly increased iron levels in the striatum of patients with HD, suggesting a role for iron in the HD process [8,18,19]. This increase has been confirmed by an in vivo study that also showed the levels to be increased early in the disease process [13]. In early-stage (presymptomatic) HD an increase in the density of oligodendrocytes is seen in the striatum before atrophy or loss of neurons [4,5]. Thus the high-iron striatal environment is likely also created early in the disease process [13] since adequate iron levels are essential for oligodendrocyte Met. Ions Life Sci. 1, 151–177 (2006)

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a) 8 7

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Figure 2. Abnormal ferritin iron levels in (a) Huntington’s and (b) Alzheimer’s disease versus matched healthy control subjects. FDRI = field dependent relaxation rate increase (an in vivo MRI measure of ferritin iron – see Section 5.1); N = healthy normal control (white); HD = Huntington’s disease (light grey); AD = Alzheimer’s disease (dark grey). Fwm = frontal lobe white matter; C = caudate; P = putamen; G = globus pallidus. ∗ p < 005∗∗ p < 001∗∗∗ p < 0001 (from [13,21]).

differentiation and oligodendrocytes and their myelin contain large amounts of iron (reviewed in [26,33,57,67,69–71,87,88]). Myelin contains ferritin mRNA [89] that is expressed at myelination onset [90] and a large portion of brain iron is associated with myelin [87,88]. As myelin undergoes a process of age-related breakdown [25,29,36,76,77] the released iron may increase risk of toxicity as iron promotes free radical damage ([60,61], for review see [62]) (see Sections 3.5, 3.6, and 4 below). Met. Ions Life Sci. 1, 151–177 (2006)

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Figure 3. Myelin integrity in Alzheimer’s and Huntington’s disease versus matched healthy control subjects. Fwm = Frontal lobe white matter; R2 = transverse relaxation rate (an in vivo MRI measure of myelin breakdown – see Section 5.1); N = healthy normal control (white); HD = Huntington’s disease (light grey); AD = Alzheimer’s disease (dark grey). ∗∗∗ p < 0001 (from [13,21]).

3.2 Oligodendrocyte Vulnerability: The ‘Achilles’ Heel’ of Our Brain The accumulation of high iron levels, along with the metabolic demands of producing and maintaining the myelin sheaths, contribute to the exceptional susceptibility of oligodendrocytes and their myelin to a variety of insults. Many normal as well as pathological processes (anoxia, oxidative stress, etc.) that have been shown to damage oligodendrocytes (for review see [3,26]) can release iron from ferritin [60,91–94]. Oligodendrocytes may be more vulnerable than other cells to such iron releases since in addition to containing the highest iron stores of any brain cell, their particular ferritin subunit composition makes iron available with greater ease than in other cells [64,65,95]. In addition, oligodendrocyte precursors are especially vulnerable to oxidative damage, making actively myelinating intracortical and subcortical regions especially vulnerable ([3], for review see [26,96–98]). Both post mortem and in vivo studies have demonstrated that substantial myelin breakdown occurs during normal aging [25,29,36,76,77,99–102]. This breakdown could release some of the considerable iron stores in myelin and oligodendrocytes [63–66,87,88]. The released iron can be translocated and contribute to iron increases in gray matter regions (see Section 3.1.1) and promote toxicity in these regions (for review see [3,26,61]). In addition, synergism between iron-mediated toxicity and excitotoxicity may also contribute to the observed neuronal loss in HD (see Section 2.3). Met. Ions Life Sci. 1, 151–177 (2006)

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3.3 Huntingtin and Myelin Integrity Huntingtin appears to be essential for embryogenesis, since deletion of the huntingtin gene in mice results in early embryonic lethality (for review see [1,2]). This early lethality is due to a critical role huntingtin plays in extraembryonic membrane function, presumably in vesicular transport of nutrients including transport of neurotrophins and especially brain-derived neurotrophic factor (BDNF) [103,104]. Wild-type huntingtin increases vesicular transport of BDNF and mutant polyglutamine expansion huntingtin interferes with this transport [103,104]. BDNF increases peripheral [105] as well as central nervous system (CNS) expression of myelin basic protein [106,107]. The reduction of BDNF could thus contribute to myelin breakdown as this essential myelin component (myelin basic protein) is decreased due to reduced BDNF. Such a scenario is likely in light of both post mortem and in vivo imaging studies assessing individuals in earlier-stages of the disease and confirming that in HD brain degeneration may begin with and clearly involves white matter [6,7,9–17]. Given the unique vulnerability of oligodendrocytes and their myelin described in Section 3.2, other effects of huntingtin in addition to its effects on BDNF may contribute to the premature myelin breakdown observed in this disease.

3.4 Huntingtin and Brain Iron Homeostasis In addition to the possibility of the abnormal huntingtin compromising myelin integrity described above, huntingtin may also impact oligodendrocyte function and development through its impact on iron metabolism. Huntingtin is an ironresponsive protein that is involved in brain development and regulates receptors for transferrin, a key iron metabolism protein [108,109]. It could therefore have an indirect role in the striatal iron accumulation observed in this disease (Figure 2) and the neurotoxicity associated with HD as the destructiveness of free radical damage is greatly enhanced by the catalytic effects of iron [60]. A more severe dysregulation of the iron regulatory function of huntingtin [108,109] may be expected when the CAG repeat number is larger, as occurs in juvenile-onset HD. In these young individuals the toxicity may be more heavily dependent on the risk associated with iron [13] (see following section).

3.5 Triplet Length and Age at Onset In AD and PD age-related brain iron increases act as a risk factor that impacts age at onset of the disease with earlier age at onset being associated with increased iron levels (reviewed in [110]). A similar interaction between age-related Met. Ions Life Sci. 1, 151–177 (2006)

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increases in striatal iron levels and age at onset may occur in HD with huntingtin polyglutamine length acting to influence this interaction. In adult-onset HD it is well-established that in general less severely affected individuals have a lower polyglutamine repeat length and often have a later age at onset. Observations in the other end of the spectrum (juvenile-onset HD) suggest that an iron–polyglutamine length interaction impacts the age at onset. Iron accumulation is deposited earlier and reaches higher levels in the globus pallidus and substantia nigra than in the rest of the extrapyramidal system [8,57,59,61,75]. Dysregulation of iron metabolism in these structures has been implicated in the pathophysiology of Parkinson’s disease that often manifests with rigidity. Rigidity is preponderant in patients with juvenile-onset HD as opposed to the choreoathetoid movements observed most often in those with adult-onset HD. This observation may be explained by a role for enhanced catalytic effects of iron on free radical and excitotoxic processes described in previous sections. The more severe abnormalities in huntingtin (e.g., higher repeats in juvenile-onset cases) may manifest with damage in these earlier-myelinating, higher-iron regions [7,13]. Thus, in these young individuals the location of the toxicity and thus the clinical manifestations may be dependent primarily on the presence of a higher iron level and its earlier accumulation in these regions.

3.6 Elevated Iron and Exacerbation of Excitotoxicity The striatum has relatively high iron levels, while the hippocampus does not [61], supporting the possibility that both NMDA (see Section 2.3) and iron need to be elevated for the full neurotoxic effect of HD to manifest. The globus pallidus, on the other hand, is an example of a structure that is less severely affected than the striatum, despite having higher iron concentrations [57] (Figure 2). However, this structure could be less susceptible in adult HD because it has minimal NMDA enervation [46]. In addition, cerebral cortex iron levels are highest in the deeper layers [58], which are most heavily myelinated [28,36] and where neuron loss is most noticeable in HD [41,42]. Thus, once again iron levels and myelination are implicated since, by itself, NMDA enervation which is more abundant in superficial cortical layers [111] cannot be the sole explanation for the observed differential pattern of toxicity in the various cortical levels (reviewed in [13]). The involvement of iron deposition in HD neurotoxicity is also supported by the fact that the manifestation of HD is age-dependent and, with advancing age, brain iron levels increase [57,112] in conjunction with myelin breakdown [25,29,76,77]. In the striatum, this age-related iron deposition progresses in a dorsoventral, mediolateral, and posteroanterior direction [113] which is similar to the progression of HD neurotoxicity [114]. Met. Ions Life Sci. 1, 151–177 (2006)

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4 TRANSITION METAL METABOLISM AND PROTEINOPATHIES Although essential for cell function, increased tissue iron can promote tissue oxidative damage, to which the brain is especially vulnerable (for review see [26,60]). In addition to HD, iron (as well as other transition metals such as zinc and copper) contributes to the development of proteinopathies (abnormal deposits of proteins) associated with several prevalent neurodegenerative diseases such as AD, PD, and dementia with Lewy bodies (DLB) (for review see [3,26,115–117]). Iron levels are abnormally elevated in these diseases suggesting, that as is the case in HD, increased iron levels may contribute to the age risk-factor of degenerative brain diseases in general ([3], for review see [26,118]). The importance of transition metal metabolism in the pathophysiology of degenerative brain diseases has been highlighted by recent studies suggesting central roles for such metals in several steps leading to amyloid  A-, tau-, -synuclein Syn-, and prion-associated brain degeneration (for review see [3,26,115,116,119–121]). The promotion of degeneration by transition metals may occur through multiple mechanisms as will be briefly delineated using A-induced toxicity as an example. Increased iron is associated with increased production of amyloid precursor protein (APP) through an untranslated region that contains an iron responsive element [122] and soluble A (the initial A form produced from APP cleavage) can act as an iron chelator and oppose the age-related elevation of brain iron levels [123,124]. Thus a normal function of APP and A increases may be to protect against iron-mediated toxicity [26,115]. The age-related increases in iron could contribute to AD risk by promoting A production, resulting in increased A levels with age (for review see [26,125,126]). The subsequent oligomerization of A into fibrils and their eventual precipitation into neuritic plaques results in the decreased levels of A that occurs as individuals develop AD and DLB and as the diseases progress [125–127]. The process of A oligomerization is promoted by transition metals such as zinc (and to a lesser extent iron and copper) [115,116,123,128–131], and oligomerized A is the toxic entity associated with free radical damage and AD [119,129,132,133]. Iron as well as copper (which in brain has 3–10-fold lower concentration than iron [115,134,135]), mediate the oxidative toxicity of soluble A oligomers (for review see [26,115,116]). In AD the oxidative toxicity of soluble A oligomers predates A deposition in the form of plaques [136]. Abnormal aggregation of other proteins such as -synuclein (a cytoskeletal protein whose aggregation results in Lewy bodies) and tau (a cytoskeletal protein whose aggregation results in neurofibrillary tangles) are also promoted by iron [119–121], and are often observed to coexist in multiple degenerative brain disorders [137–140], including the prototypical disorder defined by brain Met. Ions Life Sci. 1, 151–177 (2006)

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iron accumulation: neurodegeneration with brain iron accumulation type 1 (Hallervorden–Spatz syndrome) [141,142]. Like huntingtin’s impact on transferrin function [108,109] and A’s impact on free iron levels [123,124], other neuropathogenic proteins such as prion protein are involved in metabolism of transition metals such as copper and zinc [143,144], and iron-mediated toxicity is again involved in the age-related manifestations of prion-associated diseases [116,145]. Several additional rare genetic degenerative diseases of the central nervous system also involve abnormalities in proteins involved in transition metal metabolism either in cell cytoplasm or mitochondria [108,146]. We therefore suggest that the process of abnormal protein aggregation in general may be dependent on the promotion of protein oligomerization and cytotoxic effects of transition metals such as iron (for review see [26,147]). This possibility is supported by evidence from animal models with genetic iron regulatory protein deficiencies that result in adult-onset neurodegenerative diseases [148]. Whether iron is involved in aggregation of huntingtin and the effects of this aggregation on neurons and glia remains to be elucidated.

5

IN VIVO MEASUREMENT OF BRAIN IRON

5.1 Measuring Ferritin Iron in Vivo The bulk of brain iron is stored in ferritin molecules [66,149,150] and an in vivo MRI method called field-dependent relaxation rate R2  increase (FDRI) can obtain specific measures of the iron content of ferritin molecules (ferritin iron) [59,74,118]. Ferritin, a spherical protein in which upwards of 90% of tissue nonheme iron is stored [149–151], can sequester iron (as well as other transition metals such as zinc) and may function as a general metal detoxicant [152,153]. Although not a direct measure of other transition metals, FDRI measures of ferritin iron may be pertinent to the overall transition metal storage capacity of different brain tissues. In brain, iron is by far the most abundant transition metal [115,134,135,154] and in this context, the age-related increases of brain ferritin iron levels in normal individuals likely has implications for the homeostatic mechanisms of other transition metals [116,117,152,153,155]. The FDRI method takes advantage of the fact that ferritin changes the magnetic properties of tissues in a way that is dependent on the magnet (field) strength of the MRI instrument [156] to produce highly specific and reproducible measures of this tissue iron store [59,74,118]. Briefly, FDRI is the difference in measures of brain tissues relaxation rates R2  obtained with two different field-strength MRI instruments. In the presence of ferritin, R2 increases with increasing magnetic field-strength [59,74,75,118,157–160]. This field-dependent R2 increase is specifically associated with the total iron contained in ferritin Met. Ions Life Sci. 1, 151–177 (2006)

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molecules [118,158] and has been shown to be independent of the amount of iron loading (number of iron atoms per molecule of ferritin) [159] and to increase linearly with field-strength [156,158–160]. Thus, FDRI is a specific measure of the total iron contained in ferric oxyhydroxide particles that form the mineral core of ferritin molecules. In human tissue, ferritin and its breakdown product (hemosiderin) are the only known physiologic sources of such particles [66,118,156–158]. The FDRI measure is therefore interchangeably referred to as ferritin iron in this chapter [13,20,21]. The specificity of the FDRI measure is most easily demonstrated when examining white matter data, which has a relatively low FDRI, consistent with lower iron levels than basal ganglia structures (Figure 2), yet the white matter R2 values are higher than basal ganglia values (not shown) which have twice the iron levels of the frontal white matter (Figure 2). This disagreement between simple R2 measures and iron levels occurs because the presence of myelin (as well as multiple other possible factors such as calcifications) increases R2 in a non field-dependent fashion and all non field-dependent effects on R2 are eliminated by the FDRI method through its use of two R2 measures obtained at two different field strengths [59,118]. Since only the iron in ferritin has a field-dependent effect, FDRI is a specific measure of ferritin iron.

5.2 Measuring Ferritin Iron in Neurodegenerative Diseases In some brain regions such as caudate, putamen, and globus pallidus, normal age-related ferritin iron accumulations continue into old age [8,57,59,61]. These increases occur as ferritin iron decreases in white matter regions [61]. In disease states associated with increased gray matter iron levels such as AD and HD, myelin breakdown may release iron from white matter regions (Figure 2) and contribute to subsequent iron accumulation in gray matter regions (see Sections 3.1.1 and 3.1.2). In brain, age-related increases in gray matter iron may underlie the age-related risk of toxicity and manifest as degenerative diseases ([3], for review see [26,61]). Several lines of circumstantial evidence support the possibility that brain ferritin iron levels may be a modifiable risk factor for age-related degenerative brain diseases (for review see [3,26]). For example, men who are diagnosed with AD and PD at younger ages have increased brain ferritin iron [20,110] and compared to women, men also have an earlier age at onset of PD and DLB (reviewed in [110,137]).

5.3 Ferritin Iron as a Biomarker The advent of in vivo neuroimaging methods that can assess tissue ferritin iron as well as myelin breakdown on a regional basis provides the means to prospectively Met. Ions Life Sci. 1, 151–177 (2006)

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examine the impact of age-related changes in iron and myelin breakdown on neurodegenerative disease processes (see Sections 2.2 and 2.3). The FDRI measure of ferritin iron has already shown itself to be a useful biomarker. It is a highly reproducible measure and has been validated in several studies of normal individuals that used different MRI instruments and produced the same high correlations r > 095 with post mortem measures of regional brain iron [59,61,75,118]. The method was further validated by examining ferritin iron levels in three neurodegenerative diseases (AD, PD, and HD) and confirmed postmortem evidence of increased iron levels in all three diseases [13,20,22,74] including striking iron increases in specific regions of HD that could not be detected with other imaging methods [13] (Figure 2).

6 NOVEL TREATMENT CONSIDERATIONS Since in many degenerative brain diseases myelin breakdown and iron accumulation begin before the first appearance of pathological changes (reviewed in [3,26]), there is a decades-long period in which therapeutic interventions could alter the course of these disorders, before clinical evidence such as cognitive decrements appears [3,25,26,29]. Thus, it may be possible that medication development could be carried out in very early stages of disease using noninvasive in vivo neuroimaging markers of myelin breakdown and iron levels [3,25,26,29]. Imaging biomarker methods could thus be used to target emerging therapeutic interventions ([161–164], for review see [165,166]) years before clinical manifestations of disease [30]. This approach may be especially rewarding in diseases such as HD and AD where genetic tests can identify individuals that will definitely develop the disease or subgroups that are at high risk. Early intervention may make it possible to increase effectiveness of such treatments, decrease the need for later more aggressive approaches, and ultimately may represent a heretofore unexplored opportunity for primary prevention of degenerative brain diseases (reviewed in [3,28,30]).

7 CONCLUSIONS Myelin plays an essential role in brain structure and function and the human brain is uniquely dependent on the elaboration of this late evolutionary invention. Our brain has the most extensive and protracted process of myelination. The unique vulnerabilities of myelin and the oligodendrocytes that produce it are directly pertinent to many uniquely human neuropsychiatric diseases including many degenerative disorders such as AD, PD, and HD and the striking patterns of spread of their pathognomonic lesions across the brain in predictable symmetric bilateral patterns. Met. Ions Life Sci. 1, 151–177 (2006)

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Age-related myelin breakdown releases considerable stores of iron and can promote tissue oxidative damage to which the brain is especially vulnerable. In brain, iron levels increase with age and are inexorably intertwined with the myelination and myelin breakdown processes. Increased iron levels may be directly toxic by promoting free radical reactions and indirectly contribute to pathologic changes through excitotoxicity or by promoting the development of proteinopathies (abnormal deposits of proteins) associated with several prevalent neurodegenerative diseases such as AD, PD, DLB, and possibly HD. Iron levels are abnormally elevated in these age-related degenerative brain diseases, suggesting that increased iron levels may contribute to the striking age-related manifestation of such diseases as well as influencing their age at onset and other phenotypic facets. Magnetic resonance imaging technology permits the assessment of myelin breakdown as well as iron levels in vivo. There is close agreement between neuropsychological, neuropathologic, and imaging measures, suggesting that the process of myelin breakdown normally begins in early adulthood, accelerates as aging progresses, and underlies both age-related cognitive declines and the subsequent development of dementia-causing disorders. This myelin-centered model and the ability to measure the lifelong trajectory of myelin development, breakdown, and its impact on brain iron levels provides a framework for developing novel treatments as well as assessing the efficacy of currently available treatments. Although many issues remain to be resolved regarding both normal aging and age-related disease states, the ability to track myelin breakdown and iron accumulation with MRI provides the opportunity to assess these processes directly in humans. MRI biomarkers can also provide a means to assess the efficacy of treatments aimed at mitigating myelin degeneration and iron toxicity in clinically healthy as well as symptomatic populations. Such treatments may have a wide spectrum of efficacy and potentially could delay or prevent brain aging and some of the uniquely human disorders associated with the aging process of the human brain.

ACKNOWLEDGMENTS This work was supported in part by NIMH grants (MH51928; MH6357-01A1; and MH066029-01A2); an NIA Alzheimer’s Disease Center Grant (AG 16570); funds received from the State of California, Department of Health Services, contract #013608-001; the Sidell–Kagan Foundation; and a Merit Review Grant from the Department of Veterans Affairs.

ABBREVIATIONS A AD

amyloid  Alzheimer’s disease Met. Ions Life Sci. 1, 151–177 (2006)

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APP Syn CNS BDNF DLB FDRI Fwm GABA HD MRI N NMDA PD R2

amyloid precursor protein -synuclein central nervous system brain-derived neurotrophic factor dementia with Lewy bodies field-dependent R2 increase (an in vivo MRI measure of ferritin iron) frontal lobe white matter -aminobutyric acid Huntington’s disease magnetic resonance imaging normal control N -methyl-d-aspartate Parkinson’s disease transverse relaxation rate (an in vivo MRI measure of myelin breakdown)

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8 Copper-Zinc Superoxide Dismutase and Familial Amyotrophic Lateral Sclerosis Lisa J. Whitson and P. John Hart Department of Biochemistry and the X-ray Crystallography Core Laboratory, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio TX, 78229-3900, USA <[email protected]>

1 INTRODUCTION 1.1 The Connection of Amyotrophic Lateral Sclerosis to Copper-Zinc Superoxide Dismutase (SOD1) 1.2 SOD1 Enzymatic Activity 2 MOLECULAR MECHANISMS OF fALS SOD1 PATHOGENESIS 2.1 Pathogenic SOD1 Proteins and Oxidative Chemistry 2.2 Pathogenic SOD1 Proteins and Aggregation 2.3 The Link Between Oxidative Chemistry and Aggregation 3 STRUCTURAL FEATURES OF HUMAN SOD1 3.1 Metal Ions and SOD1 Structure 3.2 Negative Design of SOD1 3.3 Classification of fALS Mutations Based on Structure 4 ‘WILD-TYPE-LIKE’ fALS MUTANTS 5 ‘METAL BINDING REGION’ fALS MUTANTS 5.1 Linear, Amyloid-like Filamentous Arrays 5.2 Helical, Pore-like Filamentous Arrays 6 MONOMERIC SOD1 AND PATHOGENESIS 6.1 Metal-deficient SOD1 6.2 SOD1 Dissociation 6.3 Aggregation of Monomeric SOD1

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Edited by Astrid Sigel, Helmut Sigel and Roland K. O. Sigel

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1 INTRODUCTION Copper-zinc superoxide dismutase (SOD1) has been studied extensively over the past four decades. Initial interest in the protein came from its unique metal binding and spectroscopic properties. More recently, the enzyme has received considerable attention because of its link to the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS). SOD1 normally functions as an antioxidant by detoxifying the potentially damaging superoxide radical anion, a natural byproduct of respiration. Distinct amino acid substitutions convert the SOD1 protein into a species that is selectively toxic to motor neurons. Although the pathogenic mechanism remains elusive, evidence is accumulating that ALS mutant SOD1 proteins have a propensity to oligomerize into higher order structures. This tendency to self-associate is not a property of the wild-type enzyme. Soluble oligomers or insoluble aggregates of pathogenic SOD1 may function as toxic elements by compromising neuronal cellular machinery in a way analogous to that observed in other neurodegenerative disorders such as Alzheimer’s, Huntington’s and Parkinson’s diseases.

1.1 The Connection of Amyotrophic Lateral Sclerosis to Copper-Zinc Superoxide Dismutase (SOD1) First reported as Charcot’s sclerosis in 1869 [1], ALS is characterized by progressive degeneration of both upper and lower motor neurons. Symptoms generally arise later in life, with an average age of onset of ∼48 years [2]. There currently exists no effective treatment, and the disorder leads to progressive loss of muscle function and death, generally within 3–5 years after the patient is diagnosed [3]. Approximately 90% of cases are termed sporadic (sALS) with no identifiable inheritable links [4]. The remaining ∼10% of cases have an inherited component, and are termed familial (fALS). Approximately 20% of fALS cases are caused by dominantly inherited mutations in SOD1 [5,6]. One mutant, D90A, has been reported to be recessive in Met. Ions Life Sci. 1, 179–205 (2006)

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Scandinavian populations [7]. The vast majority of mutations are single amino acid substitutions, although a few result in single residue deletions or protein truncations. The discovery of a connection between mutations in SOD1 and fALS generated enormous excitement in the research community because it provided the first concrete opportunity for researchers to study the role of such a well characterized protein in disease etiology. Because sALS and SOD1-associated fALS are clinically quite similar, it is possible that both arise through common molecular mechanisms. Researchers in the field are hopeful that information gleaned from work on pathogenic SOD1 and its relation to fALS can be applied to all cases of the disease.

1.2 SOD1 Enzymatic Activity SOD1 is a ubiquitously expressed metalloprotein located in the cytosol [8] and in the mitochondrial intermembrane space [9,10]. It is one of the most abundant polypeptides in the cell [11,12], particularly in motor neurons of the spinal cord and brainstem [13]. SOD1 is a relatively small homodimeric enzyme consisting of 153 amino acid residues per monomer. Each subunit has an eight-stranded Greek key -barrel fold, binds one zinc ion and one copper ion, and contains one intrasubunit disulfide bond (Figure 1A). The superoxide disproportionation reaction is catalyzed by the bound copper ion through alternating cycles of reduction (Reaction 1) and oxidation (Reaction 2) [14]: 1+ + O2 SOD1-Cu2+ + O− 2 → SOD1-Cu

SOD1-Cu

1+

+ + O− 2 + 2H

→ SOD1-Cu

2+

(1) + H2 O2

(2)

The reaction kinetics are near the diffusion limit [15,16] in part due to a series of specially placed charged residues that help to guide the negatively charged substrate into the active site [17–19]. Figure 1B shows that over 100 distinct mutations are now associated with fALS (a complete list can be found at www.alsod.org). These mutations affect 66 residues out of the 153 residues in the SOD1 monomer, and they are scattered throughout the protein architecture. A significant question is whether the various pathogenic mutations all elicit their toxic effects via a common molecular mechanism.

2 MOLECULAR MECHANISMS OF fALS SOD1 PATHOGENESIS Although ALS was first described a century and a half ago, researchers are just beginning to learn about the pathogenic mechanism(s) leading to motor neuron death. Efforts aimed at unraveling this mystery are concentrated on four main Met. Ions Life Sci. 1, 179–205 (2006)

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Figure 1. The human SOD1 dimeric structure and location of fALS mutations. (A) Each SOD1 monomer displays an eight-stranded Greek key -barrel fold. The zinc and electrostatic loop elements and the disulfide loop and -strand 8 that form the bulk of the dimer interface are labeled. The disulfide bond between Cys57 and Cys146 holds the disulfide loop against the -barrel and stabilizes the dimer interface. The copper and zinc ions are represented as dark and light spheres, respectively. The molecular two-fold axis is indicated by the 180 symbol. (B) Human SOD1 showing the location of fALS mutations. Those fALS mutations predicted to directly affect SOD1 dimerization are shown as black spheres, those that are predicted to affect dimerization indirectly through loss of metal binding are shown as light spheres, and those that fall in the -barrel are shown as medium spheres. (Images taken from [81] and reproduced by permission).

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areas: abnormal glutamate transport [20], neurofilament dysfunction [21], SOD1 copper-mediated oxidative damage [22,23], and SOD1 aggregation [24]. Because they relate directly to SOD1, only the latter two aspects will be discussed in this chapter. When the link between fALS and mutations within SOD1 was first discovered, it was believed that a loss of enzymatic function was the likely cause of the disease [5,6]. However, a subsequent series of transgenic mouse studies strongly suggest that this initial hypothesis is incorrect. Mice that are knockout for SOD1 (and hence, completely lacking the SOD1 protein) do not develop paralysis akin to ALS, suggesting that it is not a loss of superoxide dismutase activity that causes motor neuron loss [25]. In contrast, transgenic mice expressing their own normal SOD1 as well as a pathogenic human polypeptide develop motor neuron dysfunction and paralysis, despite possessing normal levels of enzymatic activity [26–28]. That these mice are able to handle superoxide anion production with their own endogenous SOD1 yet develop symptoms of paralysis in the presence of the human pathogenic polypeptide strongly suggests that these proteins possess a motor neuron specific ‘toxic gain-of-function’. Explanatory hypotheses as to the nature of this toxic property range from aberrant coppermediated oxidative chemistry to nonproductive self-association and aggregation of the mutant proteins. These two views of SOD1-mediated toxicity are examined in more detail below.

2.1 Pathogenic SOD1 Proteins and Oxidative Chemistry SOD1 has an elegantly designed active site channel that provides electrostatic guidance of the negatively charged substrate to the catalytic copper ion while simultaneously preventing larger anionic molecules from entering. It has been hypothesized that mutations within SOD1 could cause a ‘loosening’ of its structure, thereby compromising the selectivity provided by the active site channel [29]. It has also been suggested that the deleterious gain-of-function property comes from an altered reactivity of the catalytic copper resulting in the production of damaging oxidants [30,31]. Possible aberrant oxidative activities of pathogenic SOD1 proteins include enhanced peroxidase activity, elevated peroxynitrate production, or other oxidative reactions that may arise from any mishandled copper ion that may become released from the protein. Several pathogenic mutants of SOD1 have been demonstrated to have a significantly enhanced peroxidase activity relative to the wild-type enzyme [23,32–36]. The normal SOD1 disproportionation reaction produces hydrogen peroxide from superoxide anion, but when hydrogen peroxide levels rise, it can also act as a substrate, resulting in the production of a powerful oxidant (hydroxyl radical) as shown in Reactions (3) and (4). Met. Ions Life Sci. 1, 179–205 (2006)

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SOD1-Cu2+ + H2 O2 → SOD1-Cu1+ + O− 2 SOD1-Cu

1+

(3)

+ H2 O2 → SOD1-Cu OH  + OH •

2+

−

(4)

This chemistry is known to be dramatically enhanced in the presence of bicarbonate anion, the concentrations of which are substantial ∼25 mM in vivo [36]. The presence of bicarbonate also permits the oxidation of exogenous substrates too large to enter the active site channel, although there is debate as to the exact molecular/chemical basis for this property [36–41]. In this context, it is possible that certain proteins (such as signaling molecules) or other factors critical for motor neuron viability become oxidized in the presence of pathogenic SOD1 proteins, which could eventually lead to motor neuron dysfunction. An alternative oxidative pathway involves the production of peroxynitrite ONOO− . Peroxynitrite is produced spontaneously from the reaction of nitric oxide and superoxide (Reaction 5). − • O− 2 + NO → OONO

(5)

It has been postulated that when peroxynitrite levels rise, pathogenic SOD1 proteins could catalyze the nitration of tyrosine residues as shown in Reactions (6) and (7) [22,42–44]. SOD1-Cu2+ + ONOO− → SOD1-CuO-NO+ 2 SOD1-CuO-NO+ 2 + H-Tyr

→ SOD1-Cu

2+

(6) −

+ OH + NO2 -Tyr

(7)

In support of this idea, increased protein nitration in various fALS SOD1 backgrounds has been documented [42,44,45]. However, studies in transgenic mice engineered such that they have substantially decreased nitric oxide production did not affect the disease onset or progression [46]. A prerequisite for fALS SOD1 proteins to engage in oxidative reactions is that these proteins must bind copper (or another redox active metal ion) in the active site. However, many fALS-associated SOD1 mutants are known to be metal-deficient, particularly with respect to copper ion [47,48]. Moreover, a study of transgenic mice that lack the copper chaperone for superoxide dismutase (CCS) and therefore express copper-deficient pathogenic SOD1 proteins did not demonstrate slowed progression of motor neuron disease [49]. Similarly, SOD1 mutants with an abrogated copper binding site were still able to cause the disease when expressed in transgenic animals [50,51]. The observations described above seem to suggest that fALS SOD1-catalyzed oxidative chemistry may not be directly involved in disease etiology. However, oxidative stress from other sources may in general be involved in ALS pathogenesis [52] and in particular, self-oxidation may play a role in pathogenic SOD1 Met. Ions Life Sci. 1, 179–205 (2006)

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aggregation (see Section 2.3). Of interest is the late onset of the disorder, a characteristic common to other neurodegenerative diseases. It is well established that oxidative damage increases with age [53] and that copper and iron levels increase in concentration in the brain [54,55]. In addition, there exists evidence of oxidative stress in certain tissues of ALS patients [56–58]. One possibility is that the observed oxidative damage could represent a downstream effect of motor neuron dysfunction, particularly since mitochondrial abnormalities are among the earliest signs of pathology in the ALS transgenic mice [27]. In vitro studies have shown that mild oxidative damage to SOD1 results in an increased propensity for the molecule to be subjected to proteasomal digestion [59] and/or aggregation [60].

2.2 Pathogenic SOD1 Proteins and Aggregation ALS is similar to other neurodegenerative disorders such as Alzheimer’s, Huntington’s and Parkinson’s diseases in that it has an age-related component and is characterized by neuron-specific degeneration. Moreover, protein misfolding and aggregation appear to play a key role in the etiology of these diseases [61]. A hallmark of both sALS and fALS is the presence of intracytoplasmic proteinaceous inclusions within the affected neuronal tissue [62–64]. Some inclusions in fALS transgenic mice have been found to contain SOD1 along with ubiquitinylation machinery, heat shock proteins, metallothioneins, and CCS [65–67]. Transgenic mice expressing various pathogenic human SOD1 mutant proteins form aggregates containing pathogenic SOD1 [24,67–69] that become more and more prominent as the disease progresses and paralytic symptoms manifest. SOD1 misfolding and aggregation is hypothesized to be harmful to motor neurons by slowing axonal transport [70], overloading proteasomal machinery [68], sequestering chaperonins [71–73], and disrupting mitochondrial function [27,74]. Prior to symptom onset and the formation of the visible proteinaceous inclusions, higher molecular weight, soluble SOD1 oligomers can be detected concomitant with the onset of slight defects in axonal transport [28,63,68,69], suggesting that it may be these soluble species that are toxic and that the larger insoluble inclusions may come from a cytoprotective mechanism that arises when the burden of these soluble oligomers exceeds the capacity of the protein degradation machinery to handle them [75]. Over the last two years, in vitro aggregation studies of wild type and fALSassociation SOD1 mutants have revealed two tendencies: (1) pathogenic SOD1 proteins aggregate more readily than does the wild-type [76,77]; and (2) point mutations can cause destabilization of SOD1, but it is metal deficiency of these proteins that is likely the key factor enhancing the propensity of these proteins to aggregate [78–81]. Recent structural studies of various SOD1 mutants demonstrate the tendency of these proteins to form filamentous arrays when they are metal deficient [82]. Met. Ions Life Sci. 1, 179–205 (2006)

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2.3 The Link Between Oxidative Chemistry and Aggregation Figure 2A shows that in the presence of H2 O2 , wild-type human SOD1 selfinactivates as a function of time. Figure 2B represents a schematic version of what can occur after Reaction (4), where the copper-bound oxidant (hydroxyl radical) can attack one of the histidine copper ligands to form 2-oxo-histidine. Over time, oxidative modification of metal ligands could compromise the ability of these mutants to bind metal ions and may convert them to forms that are much more prone to aggregation, as metal deficiency can lead to filamentous

kcalc (m–1 s–1) Dismutation of Superoxide

A wt t1/2 = 380 s 8 mM H2O2, 100 mM NaCl No bicarbonate, 0.5 mM Tris

1e + 9

pH 8.0 37 °C

1e + 8

wt t1/2 = 128 s 8 mM H2O2,100 mM NaCl 25 mM bicarbonate, 0.5 mM Tris 1e + 7 0

200

400

600

800

B Arg143

H N

Thr137

O N H C O O 2-oxo-histidine

N H

•OH

His120 His48

Cu2+

His63

His71 Zn2+ Asp83

His46

His80

Figure 2. Self-oxidation of human SOD1 results in inactivation and metal ion loss. (A) Effect of bicarbonate on hydrogen peroxide-mediated SOD1 self-inactivation. (B) The attack of an oxygen radical species on one of the histidine copper ligands in SOD1 leads for the formation of 2-oxohistidine, leading to cofactor loss and enzyme inactivation and aggregation (see text). (Images taken from [36] and reproduced by permission). Met. Ions Life Sci. 1, 179–205 (2006)

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assembly of SOD1 dimers as described in Sections 5.1. and 5.2 and can promote monomerization followed by aggregation as described in Section 6.3.

3 STRUCTURAL FEATURES OF HUMAN SOD1 3.1 Metal Ions and SOD1 Structure First characterized in 1969 [83], SOD1 is a homodimeric molecule characterized by an eight-stranded Greek key -barrel fold in each subunit [84]. The two polypeptides are related by a two-fold axis of rotation at the dimeric interface (Figure 1A), which buries approximately 660 Å2 of solvent accessible surface area per polypeptide and is formed by a series of reciprocal hydrogen bonding, hydrophobic, and water-mediated interactions [81]. The wild-type SOD1 holoenzyme is unusually stable, retaining substantial activity in 4% SDS and 8 M urea [85] and having a melting point of ∼95  C [86]. The dimeric quaternary structure is important for catalytic function, as demonstrated by an engineered SOD1 monomer that retains only 10% of normal SOD1 activity [87]. Each subunit contains a copper- and zinc-binding site. The metal binding arrangement is unique in that one ligand, His63, binds simultaneously to both copper and zinc metals when the enzyme is in the cupric form (Figure 3). This ligand arrangement is unique to SOD1 and His63 is termed the ‘imidazolate bridge’ [84]. The copper(II) ion is further coordinated by three additional histidine residues at positions 46, 48, and 120 in a distorted square planar arrangement [84,88–90]. Structural studies have revealed a fifth ligand to Cu(II), a water molecule approximately 22–25 Å from copper [84,88–93]. The coordination geometry changes to a pseudotrigonal planar arrangement during the catalytic cycle when the imidazolate bridge is lost as the copper ion becomes reduced (Reaction 1). The breaking and reforming of the imidazolate bridge to the copper ion during the catalytic cycle has been verified both spectroscopically and structurally [91,93–97]. Figure 3 shows that the zinc ion is bound by one aspartate residue, Asp83, the bridging His63 and two other histidine residues, His71 and His80, in a tetrahedral coordination geometry. Zinc does not have catalytic function, but instead functions to structurally stabilize SOD1 [86,98]. Each -barrel of the dimer is flanked by two major loop elements termed the ‘electrostatic’ and ‘zinc’ loops (Figure 1A). The electrostatic loop contains charged residues Glu132, Glu133, and Lys136 essential for electrostatic guidance of the substrate into the active site channel [17,19,99–104]. The active site channel is formed on one side by residues from the electrostatic loop and on the other side by residues of the zinc loop. The channel opening is wide at the entrance, approximately 24 Å across, and narrows to about 4 Å immediately above the copper ion [84]. The narrowing of the channel allows access to small anionic molecules such as azide, cyanide, and fluoride [93,105]. One of the most critical Met. Ions Life Sci. 1, 179–205 (2006)

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Gly 85 N H

Conserved water O

Asp 124

H

H

O

O

H

His 46

H

N

N

His 71

H H

H

N

O

N

Asp 83

N

Cu

N

N

N

Zn

O O

His 120

N

H

His 63

N

His 80

N

His 48

N

Figure 3. Schematic diagram of the copper- and zinc-binding sites in SOD1. The ligand to the copper and zinc ions are labeled. His63 is the ‘primary bridge’ or the ‘bridging imidazolate’. Asp124 is the ‘secondary bridge’ (see text). (Image taken from [150] and reproduced by permission).

residues for enzymatic activity, Arg143, sits approximately 6 Å away from the copper ion and functions to correctly position the substrate relative to the active site in what is termed the ‘anion binding site’ [106]. Mutation of this residue results in an approximate 90% decrease in enzymatic activity [107,108]. Thr137 is positioned at the bottom of the active site channel and together with Arg143, sterically excludes larger nonsubstrate anions from reacting with the copper ion. The zinc and electrostatic loop elements are connected by the ‘secondary bridge’ of Asp124 (the bridging imidazolate is the ‘primary’ bridge). Asp124 links the two loop elements and the metal binding sites by making hydrogen bonds simultaneously to the zinc ligand His71 and to the copper ligand His46 (Figures 3 and 4). Thus, disruption of one loop element through mutation directly affects the mobility and the conformation of the other. The intrasubunit disulfide bond between residues C57 and C146 is a feature conserved among SOD1 proteins across many species [109]. As shown in Figure 1A, it is an important element of SOD1 stability and function where it helps to hold firmly the structural elements that make up the homodimeric interface. Recent evidence suggests that transport of yeast SOD1 into the intermembrane space of mitochondria requires the loss of metal ions and reduction Met. Ions Life Sci. 1, 179–205 (2006)

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Figure 4. Negative design of human SOD1 and the locations of the wild-type-like (WTL) and metal binding region (MBR) fALS mutations. In the left subunit, the edge strands of the -sheets and the gain-of-interaction (GOI) interface are boxed. In the wild-type protein, the zinc loop projects from the plane of the paper toward the viewer, preventing SOD1-SOD1 protein–protein interactions from occurring at the edge strands. The copper and zinc ions are shown as light and dark gray spheres, respectively. In the right subunit, the positions of the MBR mutations are shown as black spheres and the WTL mutations (not shown for clarity) are scattered throughout the -barrel. Metal binding stabilizes the conformation of the zinc and electrostatic loop elements and therefore plays an intimate role in the negative design of the molecule (see text).

of the disulfide bond, and that these are prerequisites for monomerization and/or unfolding of SOD1 [110]. Functionally, the reduced disulfide bond has been suggested to be recognized by the copper chaperone for superoxide dismutase during copper transfer from CCS to SOD1 [111]. Notably, all forms of SOD1 isolated through standard purification methods have its intrasubunit disulfide bond intact, presumably due to their exposure to air during the purification process. However, given the highly reducing environment of the cytosol [112,113], the status of this disulfide bond in vivo is uncertain.

3.2 Negative Design of SOD1 Amyloid fibers, associated with many diseases [75], are extremely stable entities made up of many -strands aligned perpendicular to the fiber axis [114]. Due to the fact that they already contain -sheets, -proteins are constantly Met. Ions Life Sci. 1, 179–205 (2006)

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under evolutionary pressure to avoid the formation of amyloid fibers. Nature has addressed this issue by integrating structural elements into -proteins that protect the edge strands of -sheets from nonproductive self-associative interactions. This protection has been termed ‘negative design’ [115]. -barrel formation is one example of how proteins have incorporated negative design elements to prevent self-association at edge strands. Other ways  proteins protect their -sheet edges include covering them with loops or other structural elements, dimerization, and the introduction of -bulges, proline residues, and strategically placed charged amino acids [115]. Loss of this protection through mutation can result in edge-strand interactions leading to oligomerization. As shown in Figure 4, although SOD1 is classified as a -barrel, each monomer is actually more like a -sandwich that contains two sets of edge strands. The negative design of SOD1 comes both from its dimerization, protecting edge strands 1 and 8 (Figure 1A), and from well-ordered zinc and electrostatic loop elements that protect strands 5 and 6 (boxed in the left subunit of Figure 4). Dissociation of SOD1 or conformational mobility of the zinc and electrostatic loop elements could render the molecule vulnerable to self-association and/or aggregation with other proteins (see below).

3.3 Classification of fALS Mutations Based on Structure The ∼114 mutations spread throughout the three-dimensional protein architecture of SOD1 are grouped into categories based on their location in the structure (Figure 4) and on the metal content of the purified proteins [47]. fALS mutations located throughout the -barrel generally bind metals normally and have characteristics similar to wild-type SOD1 proteins isolated from the same expression system. Such mutants are said to be in the ‘wild-type-like’ (WTL) class of pathogenic SOD1 proteins. In contrast, fALS mutations located at positions of the metal ligands or in the zinc and electrostatic loop elements are said to be in the ‘metal binding region’ (MBR) class of pathogenic SOD1 proteins due to the tendency for these molecules to be metal deficient relative to the wild-type enzyme [47,82].

4 ‘WILD-TYPE-LIKE’ fALS MUTANTS fALS-associated WTL mutations in SOD1 are scattered throughout the -barrel of the protein, including -strands, loops connecting the -strands, and the dimeric interface. The specific activities and spectroscopic properties of this group of mutants are nearly indistinguishable from the wild-type [47]. The thermal stabilities of the WTL mutants in their metal bound forms are decreased by 1–7  C relative to the wild-type protein [116]. However, the metal-free forms of these mutants are substantially destabilized compared to metal-free wild-type Met. Ions Life Sci. 1, 179–205 (2006)

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SOD1 [117]. The least stable apo-mutant, A4V, causes the most severe form of SOD1-associated fALS, and patients harboring this mutation have a mean survival period of little over one year [118]. This hints at the likely importance of the metal-deficient forms of pathogenic SOD1 proteins to the disease state. Three-dimensional structures of several metal bound versions of the WTL class of pathogenic SOD1 mutants are known and these structures do not differ considerably from that of the wild-type [29,77,119–121]. Structures of G37R and G93A demonstrate increased mobility in the electrostatic loop and -plug regions, respectively [29,119]. The A4V and I113T mutations are located within a hydrophobic pocket at the dimer interface and because of the molecular twofold, exert their effects reciprocally in each subunit. Although structurally similar to wild-type, the A4V and I113T mutant proteins display a slight shift of the two monomers relative to each other, indicative of an alteration of interactions across the dimer interface [121]. Although the structural data of metal bound WTL mutants do give some clues as to how they differ from the wild-type protein, it is not immediately obvious how they might exert toxicity through aggregation.

5 ‘METAL BINDING REGION’ fALS MUTANTS In contrast to the WTL class, fALS-associated MBR mutations in SOD1 are located at the metal binding sites themselves or in the lengthy zinc and electrostatic loop elements. Metal analyses of a number of these MBR mutants purified from insect and yeast cells using inductively coupled plasma mass spectrometry demonstrate that these proteins have a significantly decreased metal content relative to the wild-type enzyme [47,122]. MBR mutants display decreased thermal stabilities of their metal bound forms when compared to the metal bound forms of the wild type and WTL mutant proteins [47,116,122]. Lowered stability of these mutants is likely to be directly due to the decrease in metal binding. In contrast, the metal free forms of these mutants have equivalent, and in some cases, increased thermal stabilities in comparison to metal free wild-type protein [117]. As shown in Figure 1A and Figure 4, loss of metal ions is likely to affect the conformations of the zinc and electrostatic loop elements while leaving the -barrel and dimer interface unaltered relative to the wild-type protein. Interestingly, the metal free form of MBR mutant H46R has the highest thermal stability of all of the mutants tested and patients harboring this mutation have one of the longest time courses for the disease, over 20 years [123]. While it is tempting to correlate disease progression with relative stabilities of the various SOD1 mutants, most of the fALS SOD1 patient pools are quite small in number, and it is currently difficult to generate reliable statistics or to quantify Met. Ions Life Sci. 1, 179–205 (2006)

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the effects of other factors (such as diet and genetic background) on disease progression. Crystallographic studies of two MBR mutants, H46R and S134N, were the first metal-deficient SOD1 structures to be determined [82]. As discussed below, it is precisely the metal deficiency of these proteins that led to the discovery of their propensity to form higher-order assemblies.

5.1 Linear, Amyloid-like Filamentous Arrays Structures of the metal deficient SOD1 MBR mutants S134N and H46R reveal novel interactions between SOD1 dimers [82] (Figure 5). The -barrel elements of these protein structures do not differ from wild-type SOD1. However, the electrostatic and zinc loop elements are disordered such that some of the residues of these loops were unable to be modeled. The S134N substitution occurs in the electrostatic loop (Figure 4, right subunit), and has both copper sites occupied, but one zinc site empty per dimer. The H46R mutation occurs at the position of a copper ligand, and the protein purified from its yeast expression system is completely devoid of metal ions.

Figure 5. Linear, amyloid-like filaments formed by pathogenic SOD1 mutants H46R, H80R, and S134N. The SOD1 filaments are represented by three dimers from top to bottom in green, gold, and blue. In (i), the GOI interfaces are represented as red patches and in (ii) the GOI is boxed. -strands 1,2,3 and 6 are shown in red in (iv). The long axes of the -strands run perpendicular to the long axis of the filament, an architecture similar to the ‘cross-’ structure observed in amyloid fibers as shown schematically in (v). (Images taken from [82] and reproduced by permission). Met. Ions Life Sci. 1, 179–205 (2006)

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In both the H46R and S134N structures, metal deficiency causes disorder and conformational changes in the electrostatic and zinc loop elements that generate an unusually tight SOD1–SOD1 interaction between dimers. As shown in Figure 5, residues of the electrostatic loop from adjacent dimers ‘swap’ in a reciprocal fashion to form an extra -strand with the exposed SOD1 edge strands. These edge strands become accessible for protein–protein interactions through conformational mobility of the zinc and electrostatic loop elements due to metal deficiency. We term this type of nonnative interaction between pathogenic SOD1 dimers to be a ‘gain-of-interaction’ (GOI). Figure 5 shows that the SOD1 dimers are arranged within the crystal such that the -sheets of each dimer are oriented with their -strands perpendicular to the long axis of the linear arrangement of dimers. This arrangement has been termed ‘amyloid-like’ as is shown schematically in Figure 5(v) [114]. We have found that metal deficiency is a requirement for ‘true’ amyloid formation (that is, the formation of fibers that bind congo red) of pathogenic SOD1 proteins in solution [149]. In addition, crystallographic studies of the pathogenic SOD1 zinc ligand substitution H80R reveal that the protein is capable of binding metal in its copper-binding site but is unable to bind metal in the zinc-binding site. The H80R SOD1 mutant assembles into the same amyloid-like arrangement shown in Figure 5 in two distinct crystal systems [149]. This finding highlights the potential importance of metal deficiency on SOD1 structure and its propensity towards aggregation.

5.2 Helical, Pore-like Filamentous Arrays A second, distinct structure of H46R that binds zinc but not copper (Zn-H46R) forms a higher-order filamentous structure resembling a pore (Figure 6) [82]. As in the metal deficient SOD1 structures that form the amyloid-like filaments, the zinc-loaded form of H46R has deprotected edge strands of a -strand due to the mobility of the electrostatic and zinc loop elements. This deprotection allows residues of the zinc loop (blue in Figure 6A(iii)) to change conformation and add a strand to the exposed edge strand of a -sheet in a neighboring dimer within the crystal (red in Figure 6A(iii)). Propagation of this reciprocal -sheet addition between dimers results in a helical arrangement of SOD1 molecules. Each turn of the helical filamentous array consists of four Zn-H46R dimers (Figure 6B) with an overall outer diameter of approximately 95 Å. The inside ‘pore’ is water-filled with a diameter of approximately 30 Å. Intriguingly, annular pores of similar dimensions are found to be formed by proteins involved in Parkinson’s and Alzheimer’s diseases [124] (Figure 7). These pores are hypothesized to permeabilize membranes by generating unregulated channels [125]. Recent evidence suggests that metal-free SOD1 mutants, A4V and G85R can also form pore-like structures following copper-induced oxidation [126] and that these pores may play a pathological role in ALS. Met. Ions Life Sci. 1, 179–205 (2006)

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Figure 6. Helical, ‘pore-like’ filaments. (A) (i) Two Zn-H46R dimers in green and gold that form one-half turn of helical filament. The GOI is boxed. (ii) and (iii) Ribbon diagrams of what is depicted in (i). Zn ions are shown as magenta spheres in (ii) and (iii). The GOI interface between Zn-H46R molecules is formed by reciprocal interactions between residues of the zinc loop (blue in (iii)) with the exposed edges of -strands 5 and 6 of adjacent molecules in the crystal lattice (boxed in (iii)). The GOI in Zn-H46R forms a hollow, pore-like structure. (B) Divergent stereo view of the helical pore-like filamentous array with four Zn-H46R dimers per turn shown in green, gold, blue, and magenta, respectively (see text). (Images taken from [82] and reproduced by permission).

6 MONOMERIC SOD1 AND PATHOGENESIS 6.1 Metal-deficient SOD1 As described previously in this chapter, mounting evidence suggests that metal deficiency of pathogenic SOD1 mutants likely plays a role in fALS etiology, at least for a subset of the pathogenic SOD1 mutations. Although a small Met. Ions Life Sci. 1, 179–205 (2006)

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Figure 7. Annular, pore-like structures observed in proteins causing Parkinson’s (synuclein) and Alzheimer’s A-1-40ARC  diseases are similar to the pore-like structure observed for Zn-loaded H46R SOD1 (Figure 6). The inner and outer dimensions of the pores are given. (Images taken from [124] and reproduced by permission).

destabilizing effect is often observed with the fALS mutations in the metalbound protein, the destabilization tends to be enhanced when the protein becomes metal deficient. However, it remains unclear to what degree SOD1 molecules are metallated in vivo (in the motor neuron) and whether the degree of metal loading changes over time. SOD1 metal-deficient pools are observed in anaerobic yeast [127] and human K562 cells [128], and an estimated 35% of SOD1 in lymphoblasts exists in the apo-form [129]. SOD1 molecules have an enhanced risk for incurring oxidative damage followed by metal ion loss due to their high concentration and extended half-life within motor neurons. Protein synthesis (and presumably metal loading) occurs in the cell body and the nascent proteins travel to the nerve termini through the slow component of anterograde axonal transport at a maximum rate of 2–6 mm per day [130]. Thus for a meter long motor neuron, it can take between 200–500 days for a SOD1 protein to travel to the synapse, ample time to incur oxidative damage and perhaps lose metal ions as described in Section 2.3. Indeed, SOD1 self-oxidation has been documented in transgenic mice expressing certain pathogenic SOD1 proteins [131]. Given these factors, a pool of aggregation-prone pathogenic metal deficient SOD1 may expand with age as overall oxidative stress increases. Met. Ions Life Sci. 1, 179–205 (2006)

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6.2 SOD1 Dissociation The metal loaded SOD1 protein is an enormously stable dimer. However, it has recently been shown that dissociation of SOD1 can occur under quite mild conditions through modification of two of its structural elements. Disulfide bond reduction between residues C57 and C146 in the metal-free form of SOD1 results in complete dissociation of the protein to its monomeric subunits at physiologically relevant pH [81,111,132]. However, the disulfide-reduced, metalbound protein remains a stable homodimer. The binding of a single zinc ion shifts monomeric SOD1 back to its dimeric form [133]. Taking into account that approximately one-third of SOD1 may be apo for metal ions and that the environment of the cell is reducing, there could exist a significant pool of SOD1 monomer in vivo. Monomeric SOD1 also likely plays a functional role, particularly in the context of copper insertion by CCS, where the heterodimer model of copper incorporation into SOD1 requires one monomer of apo SOD1 to form a complex with one monomer of CCS [134]. There is some evidence that copper transfer from yeast CCS to yeast SOD1 may be mediated through an intermolecular disulfide involving Cys57 of SOD1. Thus, a reduced disulfide bond for SOD1 recognition and metal loading by CCS is required [111]. The disulfide bond is proposed to be formed concomitant with copper ion transfer from yeast CCS to yeast SOD1 resulting in the mature form of the enzyme [135]. How might the fALS mutations affect the quaternary structure of SOD1? fALS mutants of the MBR class (Figure 4, right subunit) have decreased affinity for metals in comparison to the wild-type protein and are thus more prone to dissociate, depending on the status of the intrasubunit disulfide bond [81]. In contrast, some WTL mutations (black spheres in Figure 1B) may directly destabilize dimeric SOD1 by perturbing the dimer interface. These same mutations may indirectly destablize the protein through metal deficiency if they interfere with CCS/SOD1 complex formation and metal transfer to SOD1. Other WTL mutations in the -barrel (medium gray spheres in Figure 1B) may destabilize the -barrel fold, slowing the maturation process. We recently discovered, using analytical ultracentrifugation, that purified fALS SOD1 mutants A4V and G93A in their metal-free form are partially monomeric, even without reduction of the disulfide bond [149]. These mutants also demonstrate an increased susceptibility for reduction of their disulfide bond in comparison to the wild-type protein [136]. Finally, the immature form of SOD1 is more prone to oxidative damage, underscoring the role that this form may play in the disease state [111].

6.3 Aggregation of Monomeric SOD1 Based on the presence of inclusion bodies containing SOD1 in ALS patients and transgenic mice, the toxic property of SOD1 mutants likely lies in their Met. Ions Life Sci. 1, 179–205 (2006)

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propensity for aggregation into insoluble complexes. However, finding meaningful conditions to replicate SOD1 aggregation in vitro has proven to be difficult. Recent studies have demonstrated aggregation of SOD1, particularly when the protein is devoid of metals [76,77]. However these studies employ extreme conditions of low pH, high temperature, and exposure to organic solvents to generate the aggregates, conditions that are not easily translated into a cellular environment. With the recent discovery of physiologically relevant conditions under which SOD1 is monomeric [81] and the likelihood that the monomeric form is a part of the in vivo SOD1 pool, the question follows: is the SOD1 monomer the toxic species responsible for the aggregation associated with ALS? Accumulating evidence suggests that this may indeed be the case [78,111,137]. SOD1 mutant samples that are partially monomeric (such as metal-free A4V and G93A) aggregate at neutral pH, whereas metal-loaded, dimeric samples do not aggregate under the same mild conditions. Furthermore, the aggregation propensity of these proteins is enhanced by driving the monomer/dimer equilibrium toward the monomer form through reduction of the disulfide bond. This also holds true for wild-type SOD1 [149]. Given that ALS appears to be related to the aging process, it is possible that conditions within neuronal tissue change over time, and that these changes shift the equilibrium toward the SOD1 monomer species and initiating aggregation and/or 20 S proteasome overload (see below). Age-related factors that could be involved are an increase in oxidative damage [53], a change in cellular reduction potential [138,139], a decrease in proteasomal activity [140], and an increase in translational errors [141]. These factors may affect the status of SOD1 (and likely, other proteins) and enhance their propensity for aggregation. An important recent study strongly suggests that mildly oxidized pathogenic SOD1 proteins aggregate through a monomeric intermediate [142]. In this context, Lansbury and colleagues have identified a series of FDA-approved compounds that appear to bind at the SOD1 dimer interface, stabilizing the pathogenic, metal-deficient proteins against dissociation and aggregation [143]. The research community eagerly awaits the testing of these compounds in transgenic mice overexpressing pathogenic SOD1 to determine their efficacy and the validity of the monomer theory of aggregation.

6.4 fALS SOD1 and the 20 S Proteasome As a result of mutation, some fALS SOD1 proteins may be partially unfolded or mimic oxidatively modified SOD1 and thus adversely affect proteasome function in vivo. SOD1 has been used as a model system and it has been demonstrated that mildly oxidized SOD1 is one of the proteins that is specifically targeted for proteolysis by the 20 S proteasome as a consequence of the damage. The pathway for degradation of oxidized SOD1 requires no ubiquitination or ATP Met. Ions Life Sci. 1, 179–205 (2006)

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and recognition of the mildly oxidized SOD1 protein is likely due to partial unfolding leading to exposure of hydrophobic portions of the protein [59]. Recent studies have strongly suggested that proteasome levels and/or activities are adversely affected by the presence of the ALS-mutant SOD1 proteins [144–147]. The level of 20 S proteasome activity was substantially reduced in lumbar spinal neurons relative to the surrounding neuropil in the G93A SOD1 transgenic mouse and impairment of the proteasome is an early event and contributes to ALS pathogenesis [144]. We hypothesize that some of the fALS-mutant SOD1 proteins are likely to monomerize or be partially unfolded in vivo as a consequence of their loss of metal ion binding ability, destabilization of their apo-proteins, or because of enhanced oxidative damage to the mutant SOD1 that may or may not be mediated by bound copper ions. In any of these cases, the partially unfolded fALS-mutant SOD1 proteins are likely to expose hydrophobic portions of their structures and they may be abnormally targeted to the proteasome even without oxidation. If this is true, it could help explain the inhibition of proteasome function observed in the disease models. To test directly the susceptibility of SOD1 to be digested by the 20 S proteasome, a number of in vitro proteasomal digestion assays were performed using wild-type and pathogenic SOD1 proteins [148]. Contrary to the in vivo studies, none of the SOD1 proteins, metal-bound or metal-free, are found to be inhibitors of proteasomal activity in vitro. Furthermore, all the metal-bound, dimeric forms of SOD1 (wild-type and mutant) are not substrates for degradation. However, the metal-free forms of both wild type and mutant are substrates to varying degrees, with the disulfide-reduced, monomeric form of SOD1 being the best substrate by far. If the burden of misfolded SOD1 proteins exceeds the capacity of the protein degradation system in vivo, these monomeric proteins may accumulate and self-associate. All of the SOD1 proteins digested, including wild-type, are cleaved into four distinct fragments ranging from 1.7 to 7.8 kDa in size. It is possible that it is the products of this proteolytic digestion that may be toxic to motor neurons, either through direct aggregation of the fragments or through other as yet unknown toxic effects of these peptides.

7 CONCLUSIONS In the twelve years since the link between mutations in SOD1 and ALS was first identified, enormous amounts of research effort have been invested to understand the molecular basis for this deadly disease. It is clear that there are many factors involved in the pathogenesis of ALS, and it is becoming increasingly evident that researchers are unlikely to find the answer in one factor alone. Based on the data reviewed here, metal deficiency likely plays a substantial role in the propensity for protein aggregation, either through dimeric or monomeric forms. As our understanding of the molecular basis for pathogenic SOD1 expands, the Met. Ions Life Sci. 1, 179–205 (2006)

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research community looks forward to the application of this knowledge into prospective therapies for the disease.

ACKNOWLEDGMENTS This work was supported by the NIH NINDS (NS39112), the ALS Association, and the Robert A. Welch Foundation (AQ-1399). LJW was supported in part by a predoctoral fellowship from the American Federation for Aging Research and in part by a Ford Foundation Predoctoral Fellowship for Minorities. Collaborative efforts on the Zn-H46R structure by R. Strange, M. Hough, S. Antonyuk, and S. Hasnain (CLRC Lab, Daresbury, UK) and on 20 S proteasome digestion studies by R. Levine and L. Di Noto (NIH) are gratefully acknowledged. We thank Alex Taylor and Stephen Holloway for help with the figures, and members of the J. S. Valentine lab for helpful discussions.

ABBREVIATIONS ALS CCS CNS fALS FDA GOI MBR sALS SDS SOD1 WTL

amyotrophic lateral sclerosis copper chaperone for superoxide dismutase central nervous system familial amyotrophic lateral sclerosis Food and Drug Administration gain of interaction metal binding region sporadic amyotrophic lateral sclerosis sodium dodecyl sulfate copper-zinc superoxide dismutase 1 wild-type-like

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9 The Malfunctioning of Copper Transport in Wilson and Menkes Diseases Bibudhendra Sarkar The Research Institute, The Hospital for Sick Children, Toronto and The Department of Biochemistry, University of Toronto, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada

1 INTRODUCTION 1.1 Biological Transport of Copper 2 CLINICAL AND BIOCHEMICAL FEATURES OF COPPER TRANSPORT DISORDERS 2.1 Wilson Disease 2.2 Menkes Disease 3 GENES IDENTIFIED IN COPPER TRANSPORT DISORDERS 3.1 Wilson Disease Gene 3.2 Menkes Disease Gene 4 STRUCTURE AND FUNCTION OF COPPER-TRANSPORTING ATPases 4.1 Wilson Disease ATPase (ATP7B) 4.1.1 Structural Studies of ATP7B 4.1.2 Functional Studies of ATP7B 4.2 Menkes Disease ATPase (ATP7A) 4.2.1 Structural Studies of ATP7A 4.2.2 Functional Studies of ATP7A 5 TREATMENT OF COPPER TRANSPORT DISORDERS 5.1 Treatment of Wilson Disease 5.1.1 BAL (2,3-Dimercaptopropanol) 5.1.2 d-Penicillamine 5.1.3 Trientine

Metal Ions in Life Sciences, Volume 1 © 2006 John Wiley & Sons, Ltd

Edited by Astrid Sigel, Helmut Sigel and Roland K. O. Sigel

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5.1.4 Zinc 5.1.5 Tetrathiomolybdate 5.2 Treatment of Menkes Disease 5.2.1 Copper-Histidine 6 CONCLUSIONS ACKNOWLEDGMENTS ABBREVIATIONS REFERENCES

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1 INTRODUCTION 1.1 Biological Transport of Copper Copper is an essential trace element which forms an integral component of many enzymes [1,2]. While trace amounts of copper are needed to sustain life, excess copper is extremely toxic. Copper enters circulation by absorption from the gastrointestinal tract which is dependent on the action of the Menkes ATPase, ATP7A (Figure 1). The word ‘copper’ refers to both Cu(I) and Cu(II). Once in circulation it joins an exchangeable pool consisting of copper bound to albumin and amino acids in the Cu(II) form [1,3]. Prior to cellular uptake, Cu(II) is reduced to Cu(I) by a membrane-bound reductase and enters the cell via a passive transporter hCTR1 [4]. In the cell, copper may be complexed to various ligands and copper chaperones, which shuttle copper to various copper-dependent proteins distributed in the cytoplasm and all cellular organelles including the nucleus and mitochondria [5]. The majority of cytoplasmic copper is complexed to glutathione (GSH) as Cu(I). The Cu(I)-GSH complex donates copper to various intracellular proteins such as metallothionein [6]. The copper chaperones COX17 and CCS1 have been suggested to deliver copper to cytochrome oxidase [7] and superoxide dismutase [8], respectively. Atox1 is a copper chaperone, which delivers copper to Wilson ATPase, ATP7B, in trans-Golgi network (TGN) [9]. In the hepatocyte, ATP7B is responsible for providing copper to proteins such as ceruloplasmin. ATP7B is also involved in excreting copper into bile at the bile canalicular membrane. Mutation of the ATP7B gene disrupts the biliary excretion of copper and impairs copper transport across the trans-Golgi membrane to copper-requiring proteins, specifically ceruloplasmin. Thus the intracellular trafficking of copper is vital to all aspects of cellular function. Met. Ions Life Sci. 1, 207–225 (2006)

Figure 1.

2+

Intestine

Cu+

GSH

CCS1

Reductase

MT

hCTR1

COX17

ATOX1

Portal circulation

CCO

ApoCp

HoloCp

Biliary excretion of Low-MW complexes

Trans-Golgi Network

Secretion to Plasma

ATP7B

Golgi

Hepatocyte

Biological copper transport pathways. (Reproduced with permission from [2]).

Menkes CuATPase

Cu Exchangeable pool

SOD1

Nucleus

Mitochondria

Canalicular membrane

Trafficking in response to ↑ [Cu]

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2 CLINICAL AND BIOCHEMICAL FEATURES OF COPPER TRANSPORT DISORDERS 2.1 Wilson Disease Progressive lenticular degeneration was described by S. A. K. Wilson in 1912 [10]. It was eventually established that copper plays a central role in the pathogenesis of Wilson disease. The disease is inherited in an autosomal recessive manner. Wilson disease occurs worldwide, with an average incidence of ∼30 affected individuals per million population. Wilson disease can lead to liver disease, progressive neurological disorder, and psychiatric illness. There are three phases in the progression of the disease. In the first phase copper accumulates in the cytoplasm of hepatocytes. As more copper is absorbed, in the second phase the increased concentration of cytoplasmic copper results in the induction of large amounts of metallothionein. It causes storage of copper in the hepatocytes, leading to necrosis and release of copper into the blood stream [11]. In the third phase, copper begins to accumulate in other organs such as brain, kidney and cornea. Sudden release of copper in the blood stream due to oxidative damage to erythrocyte membrane leads to hemolytic anemia [12]. Wilson disease may present as fulminant hepatic failure [13] due to hepatocellular apoptosis [14], along with acute intravascular hemolysis and rapidly progressive renal failure. Wilson disease is fatal if not treated early. One of the important diagnostic features of Wilson disease is the Kayser– Fleisher ring caused by deposition of copper in the cornea. Patients with early onset of the disease usually display predominantly hepatic symptoms, whereas those with a late onset mostly display neurologic symptoms [15]. Liver dysfunction symptoms which include jaundice and fatigue and other conditions include asymptomatic cirrhosis, subacute hepatitis and hepatitis resembling autoimmune hepatitis [16]. The mechanism of liver damage in Wilson disease is thought to be oxidant stress [17]. Levels of antioxidants such as vitamin E are low [18]. Accumulation of copper is most likely the source of activated oxygen species (superoxide • O− 2 , hydrogen peroxide, and hydroxyl radical). Studies using primary cultures of rat hepatocytes show that copper generates more activated oxygen species and causes more lipid peroxidation than cadmium [19]. Generation of peroxides and hydroxyl radicals leads to nuclear damage. Wilson disease causes disturbance of copper excretion through bile. This is consistent with the biochemical findings in patients with Wilson disease (Table 1). Due to total body overload of copper, urinary copper excretion is increased. Incorporation of copper in ceruloplasmin is impaired, resulting in greater proportion of copper bound to albumin and amino acid complexes in the serum. But the serum copper concentration remains low. The low ceruloplasmin level in Wilson disease is the result of a disturbance in the transfer of copper to the apoprotein during its synthesis. Met. Ions Life Sci. 1, 207–225 (2006)

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2.2 Menkes Disease Menkes disease was first characterized by John Menkes and co-workers in 1962 during the course of a study of five patients from the same family who died before the age of three [20]. In their original study they noticed that the disease seemed to be confined to males and was inherited in a sex-linked recessive manner. The disease is caused by an impairment in the absorption of dietary copper and severe disturbance in the intracellular transport of copper [21]. Menkes disease patients are not able to absorb copper and hence suffer from the effect of copper deficiency (Table 1). The disease has a very early age of onset and affected children suffer from neurodegeneration and die before the age of three years. All patients gain very little weight following birth. Several gross abnormalities are noted with hairs that are coarse and brittle and either twisted or fractured at various intervals. This abnormality of hair in Menkes disease led to the disorder to be referred to as kinky hair syndrome. The clinical features of the disease include hypothermia, arterial rupture and bone changes. Patients are found to have extremely low levels of copper in the liver, brain and serum as well as having very low ceruloplasmin levels. All of these are due to the basic biochemical defect of a severely reduced ability to absorb copper from the intestine and a disturbance in the intracellular transport of copper [21,22]. The clinical symptoms of classical Menkes disease can be traced back to the deficiency of developmentally important copper enzymes such as lysyl oxidase, tyrosinase, cytochrome oxidase, dopamine -hydroxylase, superoxide dismutase, and amine oxidase [23]. Lysyl oxidase is needed for the crosslinking of connective tissue and deficiency in this enzyme causes weakened connective tissue and connective tissue disorder such as arterial ruptures as observed in these patients. Low levels of cytochrome c oxidase cause temperature instability and the absence of tyrosinase explains the hair depigmentation observed in affected individuals [24]. Table 1. Clinical and biochemical features of Wilson and Menkes diseases. Laboratory findings

Defect

Treatment Genes

Wilson disease

Menkes disease

↓Serum copper levels ↑Liver copper levels ↑Urinary copper levels Decreased billiary excretion of copper Incorporation of copper into ceruloplasmin Chelation therapy Chromosome 13/recessive

↓Serum copper levels ↓Liver copper levels ↑Intestinal copper levels Intestinal copper absorption Deficiency of cuproenzymes Cu-histidine administration X chromosome/recessive

Reproduced with permission from [2].

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3 GENES IDENTIFIED IN COPPER TRANSPORT DISORDERS 3.1 Wilson Disease Gene The Wilson disease gene was cloned independently by two groups and designated as ATP7B [25,26]. The gene consists of 22 exons out of which 21 are expressed in the liver. Exon 22 is expressed in the kidney. The size of the exons ranges from 77 bp to 2355 bp. There is a high level of expression of the gene transcript in the liver and kidney with a lower level in the lung and placenta. One finds a good correlation of the pattern of expression and the observed clinical and biochemical features of the disease. There are more than 250 mutations reported for Wilson disease [27]. Several mutations in the Wilson disease gene, with small insertion/deletions, non-sense, frameshift, and splice-site mutations have been found. Most common mis-sense mutation in many populations is a change from histidine in position 1069 to glutamine. This mutation is found in about 38% of homozygous Wilson disease patients of North European descent. It occurs in a conserved loop motif (SEHPL) which is adjacent to the conserved phosphorylation motif DKTG. It is not clear what specific function is performed by this motif, but it seems to be necessary for the function of the heavy metal ATPases, as this motif is seen conserved in the Wilson disease, Menkes disease and Cop A copper-transporting ATPases [26,28]. It was found that patients homozygous for the His 1069 Glu mutation has later age of onset as compared to the heterozygous patients, i.e., 20 years versus 15.4 years [29]. This His 1069 Glu mutation was found at high frequency in Mediterranean populations. The data from several studies suggest that the His 1069 Glu mutation is the most common molecular defect in the Wilson disease protein and it probably arose from an ancient mutational event [30].

3.2 Menkes Disease Gene The Menkes disease gene (ATP7A) was isolated and identified independently by three groups using positional cloning [31–33]. The gene has 23 exons and spans a genomic region of approximately 150 kb [34,35]. After the exon coding for the fifth metal-binding domain (exon five in ATP7A and exon 3 in ATP7B), the coding region for both the Wilson disease gene (ATP7B) and Menkes disease gene (ATP7A) are virtually identical. Approximately 20% of the mutations in the Menkes disease are insertion/ deletion mutations of varying sizes. Exon 8 codes for the region between metalbinding 6 and the first transmembrane segment of the protein. The exact role of this region in the function of the ATPase remains unknown but it could play a role in the precise localization of the metal-binding domain relative to the ATPase core. The importance of this region of the protein is evident from the Met. Ions Life Sci. 1, 207–225 (2006)

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finding that individuals carrying mutations in exon 8 present the most severe form of Menkes disease [36]. In the Wilson disease protein only two mutations have been found in the corresponding region [30]. These findings may indicate that these domains play somewhat different roles in the Wilson and Menkes ATPases, despite their high degree of sequence homology.

4 STRUCTURE AND FUNCTION OF COPPER-TRANSPORTING ATPases 4.1 Wilson Disease ATPase (ATP7B) 4.1.1 Structural Studies of ATP7B The Wilson disease gene is predicted to encode a copper transporting P-type ATPase having many features common to cation-transporting P-type ATPases (Figure 2). There are six metal-binding motifs, GMTCXXC at the N-terminus, the TGEA motif (actuator domain), the CPC motif in the sixth transmembrane, the DKTGT and TGDN motifs (phosphorylation domain) and SEHPL, a conserved motif found in all heavy-metal transporting ATPases. A complete structural description of ATP7B remains unsolved. However, significant advances have been made to characterize the N-terminal copperbinding segment of ATP7B. It can bind a total of six Cu(I) in a cooperative manner and undergoes specific conformational transitions [37–39]. Attempts to crystallize this domain have not been successful to date. However, X-ray absorption spectroscopy (XAS) experiments provided crucial information regarding

Lumen C P C

Golgi Membrane

Cytoplasm Cu6 KT

D

EA

G

TG

Cu5 Cu4

SEHPL

COOH

Cu3 Phosphatase

Phosphorylation/ATP binding

Cu2 Cu1 H 2N

Metal binding

Figure 2. Schematic representation of Wilson copper-transporting ATPase showing domains which are common in other cation-transporting P-type ATPases. Met. Ions Life Sci. 1, 207–225 (2006)

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copper-binding to ATP7B. The N-terminal region binds Cu(I) with two cysteine side chains in a distorted linear geometry with a Cu-S distance of 216–219 Å. The metal ion specificity of ATP7B is emphasized by XAS and circular dichroism (CD) results demonstrating non-cooperative zinc-binding [38,39]. Further studies by XAS have shown the role of exogenous ligands in structural as well as conformational changes of the N-terminal region [40]. The exogenous ligand induces formation of a Cu–Cu interaction, which may signal conformational changes important for copper transport. Mutational and recombinant experiments for ATP7B showed that domains 4–6 are sufficient for copper transport and other domains cannot replace this for copper transport activity [41–43]. A more specific function has also been assigned to domains 5 and 6 as the ones responsible for the cooperative effect of copper on the catalytic phosphorylation activity of ATP7B [44]. Subsequent studies have found that either domain 5 or domain 6 alone is enough for copper transport, but domain 6 appears to be critical for both copper transport and copper-dependent cellular trafficking of ATP7B [45]. Recent studies with N-terminal metal-binding domains of rat ATPase lacking a CXXC motif in domain 4, demonstrate similar copper-binding affinity and pattern to that of ATP7B, indicating that certain domains that are not crucial for direct copper binding might be involved in protein–protein interactions [46]. Thermodynamic studies have shown that individual domains bind copper with slightly different affinities [47]. The experimental determination of the 3D structure of the entire ATP7B poses many technical challenges. However, studies have been carried out by homology modeling using gapped-BLAST analysis [83]. A low resolution 3D model of the ATP7B [48,49], depicting the rough spatial organization of the various domains and conserved sequence motifs with respect to each other was obtained based on a 26 Å crystal structure of the homologous P-type calcium pump of sarcoplasmic reticulum SERCA1a [50] (Figure 3a). Mammalian copper-transporting ATPase has a conserved CXXCPC motif in the sixth transmembrane (TM6) domain. Copper-binding affinity of synthetic peptides representing the native sequence of TM6 and their various cysteine mutated analogues was determined by homology modeling, CD and NMR spectroscopy [51]. The residues Cys 980, 983 and 985 in the unwound part of TM6 are essential for copper transport [52]. The homology model generated as shown in Figure 3(b) could provide a suitable coordination geometry for Cu(I) binding. Experimental results indeed showed that the three cysteine thiolates present in the peptide sequence act as bridging ligands for Cu(I) binding in the TM6 peptide [51].

4.1.2 Functional Studies of ATP7B The unique metal-binding cytosolic N-terminal segment of ATP7B has been a major focus of research. Among all the copper-binding domains of ATP7B, the closest to the transmembrane region appears to be functionally more important Met. Ions Life Sci. 1, 207–225 (2006)

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Figure 3. (a) Homology model of ATP7B based on the known X-ray structure of CaATPase. The core of ATP7B encompassing the regions between transmembrane helix 3 and 7 is very similar to Ca-ATPase. The actuator domain (A); nucleotide-binding domain (N); phosphorylation domain (P) contain important and highly conserved residues. The R-loop is a potential regulatory region. This disordered proline-rich region of 57 amino acids is between domain 4 and 5 and contains several putative phosphorylation and myristoylation sites. The six copper-binding domains in the N-terminal metal-binding region (M) of ATP7B each adopt a heavy-metal associated fold found in other metal-binding proteins. The arrangement of these domains is arbitrary since the overall organization of the N-terminal region is not known. (b) Modeling of the Cu-binding site with cysteine residues in the CXXCXC motif of TM6 of ATP7B using the arrangement of residues in M4, which contribute to one of the two high affinity Ca 2+ ion-binding sites in Ca-ATPase. (Part (a) reproduced with permission from [48]).

than the ones closest to the amino terminus [41]. It is possible that the role of the N-terminal copper-binding domains may be to increase the overall catalytic rate of the transporter. It has been shown that the N-terminal domain undergoes secondary and tertiary conformational changes in response to copper-binding [38]. Although it is not known how conformational changes in the N-terminal copperbinding domain affect the function of ATP7B, it has been suggested that conformational changes in the N-terminal region of ATP7B may signal the protein to traffic between the trans-Golgi network and the plasma membrane. Met. Ions Life Sci. 1, 207–225 (2006)

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Further, in addition to its role in trafficking, a regulatory role has also been suggested for the N-terminal domain. It has been proposed that the nucleotide/phosphorylation domains interact with the N-terminus and the copper-bound N-terminal domain induces a conformational change in the nucleotide/phosphorylation domain, altering the protein–protein interaction [53]. The copper chaperone Atox1 has been shown to interact with some, but not all the N-terminal copper-binding repeats [54,55]. Homology modeling of the six metalbinding repeats of ATP7B reveals that their electrostatic surfaces differ from one another and thus not all are equally capable of interacting with the chaperone [56]. It has been suggested that copper-binding to the N-terminus weakens its interaction with the nucleotide binding domain, thereby causing this to dissociate. The dissociation of the N-terminus from the nucleotide-binding domain restores the nucleotide-binding affinity of this domain and thus allows the binding of ATP to the nucleotide-binding site. The successive conformational changes are transmitted to the translocation domain. The transmembrane domains are associated with the transduction channel and contain residues critical to cation binding. There is a conserved proline residue in the ATPase transmembrane domain 6 which is flanked by two cysteine residues to form the highly conserved CPC motif, which is predicted to be a copper-binding site within the channel [50,52,55–58] and subsequent model peptide studies as discussed above, showed it to bind copper [51]. The hydrolysis of the phosphoenzyme intermediate requires the release of copper from the channel to the lumen. The release of cation may trigger the movement of the activator domain, which would allow water to access and hydrolyze the phosphoenzyme.

4.2 Menkes Disease ATPase (ATP7A) 4.2.1 Structural Studies of ATP7A The Menkes disease protein has a high degree of homology with Wilson disease ATPase (Figure 4) [33]. The overall identity between these two proteins is 57%. The identity rises to 79% and higher in the phosphatase transduction– phosphorylation and ATP-binding domain. These two proteins have a significant similarity, but there are some significant differences as well, most notably a 78-amino acid insertion between metalbinding domain 1 and 2 in the ATP7A which is not the case in ATP7B. ATP7A is a 1500 amino acid protein. The copper-binding properties of the N-terminal metal-binding domains are similar to that in Wilson disease ATPase. This protein also binds copper in a 1:6 protein to copper ratio [59]. The geometry of copperbinding site is digonal similar to what has been reported in the case of Wilson protein [60]. Binding of Ag to domain 4 of the metal binding segment has been carried out by NMR studies [61]. Met. Ions Life Sci. 1, 207–225 (2006)

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ATP7Ap

Metal Binding

1

ATP7Bp

217

2

3 4

5

6

Td

Ch/Ph

ATP

Metal Binding

1

2

3

78 aa 18 aa

8 aa

4

5

3 aa

6

Td

17 aa

Ch/Ph

ATP

17 aa

18 aa

Figure 4. Comparison of Menkes (ATP7A) and Wilson (ATP7B) ATPases. – Transmembrane segments, ATP – ATP binding, Td – Transduction, Ch – Channel, Ph – phosphorylation, ∧, Deletions relative to ATP7A, , Insertions relative to ATP7A. (Reproduced with permission from [2]).

4.2.2 Functional Studies of ATP7A A recent review has summarized the mechanism of catalysis and copper dependent trafficking of the ATP7A [62]. Biochemical studies have shown some interesting results. Mutation of six metal-binding sites did not abolish the catalytic activity of ATP7A. Thus, it was considered that these motifs were not essential for copper translocation. However, the binding of copper to these sites increased the affinity of the protein for translocation, which would involve the interaction between the binding sites and the cytosolic ATP-binding domain of ATP7A. Further work is necessary to understand the mechanism of this process.

5 TREATMENT OF COPPER TRANSPORT DISORDERS 5.1 Treatment of Wilson Disease The damage to vital organs such as liver, brain and kidney in Wilson disease is caused by the toxic action exerted by large amounts of copper deposited in these organs. However, there are several treatment modalities available for the management of Wilson disease patients (Table 2).

5.1.1 BAL (2,3-Dimercaptopropanol) It was the first agent used to treat Wilson disease patients [63,64]. The drug has to be administered by intramuscular injection, but its use was abandoned in favor of orally available chelating agents. Met. Ions Life Sci. 1, 207–225 (2006)

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Table 2. Agents for the treatment of Wilson disease. Agent

Mechanism of action

Daily adult dosage

d-Penicillamine

Reduction and chelation of copper. Urinary excretion of copper by mobilizing copper from organs Copper chelator and urinary excretion Inhibits intestinal absorption of copper by induction of intestinal cell metallothionein; may also induce hepatic metallothionein Copper chelator

1–2 g orally in divided doses

Triethylenetetramine (trientine) Zinc salts

British anti-Lewisite (BAL) (2,3-dimercaptopropanol) Tetrathiomolybdate

Blocking the intestinal absorption of copper and a copper chelator

0.75–1.5 g orally in divided doses 150–200 mg of elemental zinc orally in divided doses

3 mL of 10% BAL in peanut oil IM Up to 2 mg/kg orally in divided doses

Reproduced with permission from [1].

5.1.2 d-Penicillamine It was first introduced to treat Wilson disease patients in 1956 [65,66]. The recommended adult dose is 1 g/day. d-penicillamine reduces copper that is bound to protein, which in turn causes the reduction of affinity of the protein for copper and allows d-penicillamine to bind copper. There is a marked increase of urinary copper excretion. Removal of copper from the liver is slow and copper level in the liver may remain elevated even after years of therapy. The high level of toxicity is the major problem with d-penicillamine. A significant number of patients with neurological aspect of the disease may become worse after the use of this drug. The immune system and connective tissues suffer from long-term side effects. It could be the result of reaction with d-penicillamine or could be the result of its interaction with the enzyme lysyl oxidase, which is a copper-enzyme and responsible for the cross-linking of collagen.

5.1.3 Trientine Trientine (triethylenetetramine) has been an alternative treatment for Wilson disease [67]. It chelates copper and causes increased urinary excretion of copper. The initial effect of this treatment is the excretion of large amounts of Met. Ions Life Sci. 1, 207–225 (2006)

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copper but it diminishes rapidly, compared to d-penicillamine. But this treatment has been very helpful when d-penicillamine has to be stopped because of its adverse side effects. Trientine helps the removal of copper from copper bound to proteins in serum, but it is not very effective to remove copper from the liver [68].

5.1.4 Zinc Zinc has been known to produce copper deficiency in experimental animals. The first report of zinc treatment for Wilson disease was published in 1979 [69]. The mechanism of action of zinc involves the induction of metallothionein in the intestinal cells which binds copper with a high affinity and holds it until the intestinal cells are sloughed off. Thus, zinc inhibits absorption of copper from the intestine and increases the fecal excretion of copper. Zinc not only inhibits the absorption of copper from the food, it also blocks the reabsorption of endogenously secreted copper from saliva and gastric juice. Advantage of zinc treatment is in its low toxicity.

5.1.5 Tetrathiomolybdate It has been used to treat patients who were intolerant to d-penicillamine, trientine, and zinc sulfate [70]. These patients were in better health upon treatment than at any time since their disease was diagnosed. Tetrathiomolybdate seems to act both by blocking the intestinal absorption of copper and keeping the absorbed copper in a metabolically inert chelated form which is not taken up by the liver. However, there are reports of toxic effects on the skeletal system of growing animals [71]. In cases where patients have been intolerant of conventional therapy, it could be considered as an alternate choice for the management of Wilson disease [72].

5.2 Treatment of Menkes Disease 5.2.1 Copper-Histidine Efforts to treat Menkes disease patients have been concentrated on restoring normal copper levels in the body by administering copper. Since intestinal copper absorption is extremely low in Menkes patients, copper must be administered parenterally. There is another problem in delivering copper into cells. Albumin is known to bind copper with a very high affinity in human blood and causes inhibition of copper uptake into cell [23,73,74]. The administration of copper salts induces the formation of a copper-albumin complex and therefore copper in this form is not bioavailable to the cells [75]. Copper has been administered Met. Ions Life Sci. 1, 207–225 (2006)

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Figure 5. X-ray structure of copper-histidine shows a neutral five coordinate distorted square-pyramidal complex. One of the histidine ligands act as a monoanionic bidentate form through Nam and Ocarboxyl atoms, while the other binds as a monoanionic tridentate ligand towards copper center through Nam  Nim , and Ocarboxyl atoms. The Ocarboxyl atom lies in an axial position. (Reproduced with permission from [79]).

in various forms including copper chloride, copper sulfate and copper-EDTA, and copper-albumin, but none of these copper compounds is able to produce significant clinical improvement [76]. The only currently available treatment for Menkes patients is subcutaneous injections of copper-histidine. The complex was originally discovered in human blood and was shown to transport copper across the cell membrane [77,78]. The structure of the copper-histidine complex has recently been solved by X-ray crystallography (Fig. 5) [79]. Patients treated with copper-histidine have shown significant clinical and biochemical improvements. They all show relatively good neurological outcome and the serum copper, and ceruloplasmin levels have been normalized [80]. Genetic diagnosis of copper-histidine treated patients revealed that they had fatal mutations in Menkes disease gene [81]. Despite these significant improvements by the copper-histidine treatment of these patients, connective tissue disorders continue to persist, indicating that lysyl oxidase levels are not restored by copper-histidine treatment. A long-term clinical follow-up of copper-histidine treated patients over 10–20 years has been reported [82]. Subcutaneous injections of copper-histidine when initiated very early in life, have been tolerated best and seem to be the most effective treatment at this time in preventing some of the serious complications of Menkes disease. Met. Ions Life Sci. 1, 207–225 (2006)

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6 CONCLUSIONS The discovery of Wilson and Menkes disease genes and their respective coppertransporting ATPases ushered in a new era in the studies of copper transport mechanism. It has opened the door for in-depth studies of copper-transport to determine the specific role played by these proteins in the homeostatic control of cellular copper. The knowledge of the intracellular localization of transporter proteins and the understanding of their functions will be key to our understanding of how the defective ATPase gene interferes with the normal transport of copper. The role played by copper chaperone and regulatory protein partners in the functioning of copper-transporting ATPases has added yet another piece to the copper-transport puzzle. Future research will be critically important for developing a broader understanding of normal copper transport and pathophysiology of Wilson and Menkes diseases and other genetic copper toxicosis disorders.

ACKNOWLEDGMENTS I would like to thank Drs Prasad P. Kulkarni, Patrick Deschamps, and Eve Roberts for many helpful discussions. Research in the author’s laboratory was supported by grants from the Canadian Institutes for Health Research and the Canadian Liver Foundation.

ABBREVIATIONS ATP7A ATP7B BAL gapped-BLAST CD GSH IM TGN trientine XAS

Menkes disease ATPase Wilson disease ATPase British anti-Lewisite = 2,3-dimercaptopropanol gapped-basic local alignment search tool circular dichroism glutathione intramuscular trans-Golgi notwork triethylenetetramine X-ray absorption spectroscopy

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10 Iron and Its Role in Neurodegenerative Diseases Roberta J. Ward and Robert R. Crichton Unité de Biochimie, Université de Louvain, Place Louis Pasteur 1, B-1348 Louvain-la-Neuve, Belgium <[email protected]>

1 INTRODUCTION 2 THE INORGANIC CHEMISTRY OF IRON AND ITS ROLE IN HUMAN BIOLOGY 3 IRON METABOLISM 3.1 Absorption from the Gastrointestinal Tract 3.2 Transport and Delivery to Cells 3.3 Storage and Intracellular Utilization 3.4 Iron Homeostasis 4 THE ROLE OF THE ‘LABILE IRON POOL’ IN FREE RADICAL PRODUCTION 5 THE IMPORTANCE OF IRON IN BRAIN 5.1 Iron in Brain Development and Function 5.2 Brain Iron as a Function of Age 6 THE INVOLVEMENT OF IRON IN NEURODEGENERATIVE DISEASES 6.1 Parkinson’s Disease 6.2 Alzheimer’s Disease 6.3 Hallervorden–Spatz Syndrome 6.4 Neuroferritinopathy 6.5 Aceruloplasminemia 6.6 Friedreich’s Ataxia 7 EXPERIMENTAL APPROACHES TO BRAIN IRON LOADING 8 CONCLUSIONS

Metal Ions in Life Sciences, Volume 1 © 2006 John Wiley & Sons, Ltd

Edited by Astrid Sigel, Helmut Sigel and Roland K. O. Sigel

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ACKNOWLEDGMENTS ABBREVIATIONS REFERENCES

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1 INTRODUCTION Many of the key proteins which are involved in brain function, for example neurotransmitter synthesis, involve metalloenzymes, notably iron and copper. An obvious example is the Fe2+ enzyme, tyrosine hydroxylase which synthesizes dihydroxyphenylalanine (DOPA) from tyrosine (Figure 1), for dopamine formation. Despite this need for iron, it has become increasingly clear that a small elevation in brain iron concentration will cause a great number of disorders of the central nervous system, which are often age-related, since iron progressively accumulates in brain with age. However, iron is a Damocles sword [1], since, in addition to being catalytically essential for many enzymes, it can also catalyze the formation of reactive oxygen and nitrogen species (ROS and RNS), resulting in oxidative stress, which

H CH2

HO

COO–

C +

NH3 Tyrosine

tyrosine hydroxylase

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Tetrahydrobiopterin + O2 Dihydrobiopterin + H2O

HO H HO

CH2

C

COO

–

Melanin

+ NH3

Dihydroxyphenylalanine (L-DOPA)

Figure 1. The synthesis of l-DOPA from tyrosine by tyrosine hydroxylase. l-DOPA is then transformed into adrenaline, and also into the skin pigment, melanin. (Reproduced with permission from [169]). Met. Ions Life Sci. 1, 227–279 (2006)

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can cause neurodegeneration. Two classes of iron-related neurodegenerative disorders can be distinguished; the first due to iron accumulation in specific brain regions, and the second resulting from defective iron metabolism and perturbation of iron homeostasis [2]. As life expectancy increases and, as a consequence, brain iron accumulates, we would expect the occurrence of iron-related neurodegenerative diseases to inexorably increase [3].

2 THE INORGANIC CHEMISTRY OF IRON AND ITS ROLE IN HUMAN BIOLOGY Iron is the second most abundant metal and the fourth most abundant element of the earth’s crust. Iron, in the middle of the first transition series, has the possibility of various oxidation states (from −II to +VI), the principal being II d6  and III d5 , although higher valent Fe(IV) or Fe(V) reactive intermediates are generated during the catalytic cycle of a number of enzymes such as catalases, peroxidases, and cytochrome P450s [4]. Fe2+ is extremely water soluble, whereas Fe3+ is quite insoluble in water Ksp = 10−39 M and at pH 7.0, Fe3+  = 10−18 M). Significant concentrations of water-soluble Fe3+ species can be attained only by strong complex formation. Fe(III), with an ionic radius of 67 pm and a charge of 3+ is a ‘hard’ acid and prefers oxygen ligands like phenolate and carboxylate to imidazole or thiolate. Fe(II) with an ionic radius of 83 pm and a charge of 2+ is on the borderline between ‘hard’ and ‘soft’ favoring nitrogen (imidazole and pyrrole) and sulfur ligands (thiolate and methionine) over oxygen ligands. The coordination number of 6 is the most frequently found for both Fe(II) and Fe(III) giving octahedral stereochemistry although four- (tetrahedral) and particularly five-coordinate complexes (trigonal bipyramidal or square pyrimidal) are also present. For octahedral complexes, two different spin states can be observed. Both oxidation states are Lewis acids, particularly the ferric state. It is doubtful that life on earth would be possible in the absence of iron. The unique suitability of iron comes from the extreme variability of the Fe2+ /Fe3+ redox potential, which can be fine-tuned by well-chosen ligands, so that iron sites can encompass almost the entire biologically significant range of redox potentials, from about −05 V to about +06 V. Iron is a constituent of numerous proteins, which can be classified [1] according to the coordination chemistry of their iron: heme proteins, iron–sulfur (Fe–S) proteins, and nonheme, non-iron–sulfur proteins. This latter group includes proteins that contain single Fe atoms and di-iron -oxygen-bridged centers as well as proteins involved in iron transport and storage [1]. Five-coordinate heme containing enzymes, including cytochrome oxidase, peroxidases, catalases, and cytochrome P450s, are involved in the activation of O2 or Met. Ions Life Sci. 1, 227–279 (2006)

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in the metabolism of peroxides, whereas hemoglobin and myoglobin are involved in oxygen transport and storage, respectively. In contrast, most cytochromes (e.g., cytochromes a, b, c) do not bind oxygen, the 6th coordinate position of the Fe2+ is occupied by an amino acid residue of the protein (often histidyl or methionyl), such that they only transfer electrons with the iron in the heme alternating between Fe2+ and Fe3+ . Iron sulfur proteins in man mostly contain 4Fe–4S clusters, involved in electron transfer reactions, in the mitochondrial electron transport chain as well as in various other electron transport systems. However, a number of Fe–S proteins have functions other than electron transfer. These include catalysis, exemplified by the mitochondrial enzyme aconitase, whose 4Fe–4S center acts as a Lewis acid in the dehydration reaction by which citrate is converted to isocitrate, and which, in its apo-form acts as a biological sensor for iron (in the iron regulatory proteins (IRPs) described below). A number of enzymes contain a single Fe atom, which is coordinately bound to imidazole (histidine) or carboxylate (glutamate and aspartate) ligands in the protein. These Fe2+ enzymes are involved in reactions that use O2 as substrate, and include prolyl and lysyl hydroxylases, involved in the maturation of collagen, which require ascorbate as cofactor, and aromatic amino acid hydroxylases, that require tetrahydrobiopterin as cofactor. Some other Fe2+ -containing enzymes add both oxygen atoms from O2 to the substrate; these dioxygenases include the 5-lipoxygenase involved in eicosanoid synthesis. Yet another family of proteins containing carboxylate-bridged dinuclear iron sites are involved in functions as diverse as mediating reversible oxygen binding (hemerythrin), the oxidation of Fe2+ to Fe3+ (ferritins), the catalysis of hydroxylation, epoxidation, and desaturation reactions, and the conversion of ribonucleotides into deoxyribonucleotides [1]. Finally, there are the proteins involved in iron assimilation and storage, more of which later.

3 IRON METABOLISM 3.1 Absorption from the Gastrointestinal Tract The mechanisms by which iron crosses the membrane of the enterocyte and is then transferred to the circulation have become much more clearly understood in the last few years. A number of new proteins have been identified which are involved in mucosal iron absorption (Figure 2). Heme iron is the most effective source of dietary iron in man, and after its uptake across the apical (brush-border) membrane of the intestine, probably via a specific receptor, its iron is released by the action of heme oxygenase as Fe2+ , with concomitant production of CO and the bile pigment, biliverdin [1]. Met. Ions Life Sci. 1, 227–279 (2006)

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Uptake

DMT1

Transfer

Fe(II)

Fe(II)

NTBI IREG

Iron pool

Fe(II) hephaestin

Fe(II) Dietary Iron

Haem

CP

DcytB

Fe(III)

Fe(III) Haem Ox Fe(II)

Ferritin ApoTf

Tf

Figure 2. Schematic representation of iron absorption in normal subjects. Iron is taken up from the gastrointestinal tract either as heme or nonheme iron. The former is degraded to release Fe(II) by heme oxygenase, whereas the latter is reduced by DcytB and transported across the apical membrane by DMT1. Within the enterocyte the iron pool can equilibrate with the intracellular storage protein ferritin. At the basolateral membrane, iron is transported out of the cell by IREG 1: its incorporation into apotransferrin may be aided by the ferroxidase activity of hephaestin. DcytB, duodenal cytochrome b; DMT1, divalent cation transporter protein 1; IREG 1, iron-regulated gene; Haem Ox, heme oxygenase; Cp, ceruloplasmin; Tf, transferrin; NTBI, non-transferrin-bound iron. (Reproduced with permission from [1]).

Nonheme dietary iron seems to cross the brush border membrane of the enterocyte, after reduction by a duodenal ferric reductase, Dcytb (for duodenal cytochrome b), a plasma membrane di-heme protein [5]. The amino acid sequence of Dcytb has around 50% similarity to the cytochrome b561 family of plasma membrane reductases, and it is highly expressed in the brush border membrane of duodenal enterocytes. The ferrous iron is then transported into the intestinal cell by the divalent metal transporter 1 (DMT-1). (DMT-1 is also involved, as mentioned below, in the transport of Fe2+ in the transferrin to cell iron cycle). DMT-1 is not specific for Fe2+ and transports other divalent metal ions, including cobalt, copper, manganese, nickel, and zinc [6]. The metal ion transport is coupled to co-transport of a proton and depends on the cell membrane potential. The DMT1 protein has 12 putative membrane-spanning domains, is expressed at the highest levels in the proximal duodenum, and is up-regulated by dietary iron deficiency [6]. Within the intestinal cell, Fe2+ derived from both heme and nonheme Fe2+ enters a low molecular weight pool. This iron can either be stored in ferritin within the mucosal cell (and lost from the body when the mucosal cell exfoliates), or it can be transported across to the basolateral membrane. There, the trans-membrane transporter protein IREG 1 can allow the Fe2+ to reach the interstitial fluid/plasma. This basolateral iron transporter, also known as ferroportin or MTP1 was found Met. Ions Life Sci. 1, 227–279 (2006)

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almost simultaneously by three groups [7–9]. It was isolated from duodenal mucosa of homozygous atransferrinemic mice, which have high levels of iron absorption [7]. IREG-1 is a transmembrane protein, with 10 potential membrane spanning regions, that localizes to the basolateral membrane of polarized epithelial cells. The incorporation of iron into the apotransferrin of plasma may be facilitated by the oxidation of Fe2+ to Fe3+ , either by hephaestin, a transmembranebound multicopper ferroxidase, which is highly expressed in intestine [10], or by ceruloplasmin, the principal copper-containing protein of serum.

3.2 Transport and Delivery to Cells Iron is normally transported in plasma and other extracellular fluids bound to transferrin, a bilobal protein which can bind two Fe3+ ions, in octahedral coordination, with four protein ligands and a bidentate carbonate anion [1]. It is taken up by cells via the transferrin-to-cell cycle (Figure 3), which begins with the binding of the holo(diferric)-transferrin molecule (HOLO-TF) to transferrin receptors at the cell surface. The complexes localize to clathrin-coated pits, which pinch off from the membrane to form coated vesicles, initiating the process of endocytosis. After budding is complete, the clathrin coat is removed, and smoothsurfaced endosomes are formed. The pH of the endosome is acidified by the action of an ATP-dependent proton pump. At the acidic pH, the holotransferrin undergoes a conformational change, releasing iron from transferrin as Fe3+ , presumably accompanied by protonation of the bound carbonate. Acidification facilitates proton-coupled transport of iron transport out of the endosomes through the action of the divalent metal transporter, DMT1, a member of the SLC11 family of H+ -coupled metal ion transporters [11]. However, the ferric reductase, which is assumed to reduce the iron, prior to its transport out of the endosome by DMT1, has not yet been identified. Apotransferrin (APO-TF) bound to its receptor, returns to the plasma membrane, where, at neutral pH, the complex dissociates. The two proteins can then participate in further rounds of iron delivery. This transferrin-to-cell cycle ensures iron uptake by cells that have transferrin receptors. Certain specialized cells obtain additional iron by other pathways. The cells of the reticuloendothelial system acquire significant amounts of iron from the phagocytosis and catabolism of red blood cells. And, when there is iron overload, and transferrin is iron-saturated, non-transferrin bound iron, attached to a variety of ligands, is taken up mostly by liver hepatocytes.

3.3 Storage and Intracellular Utilization Most of the iron delivered to or acquired by cells is believed to cross the cell membrane in ferrous form and to enter a poorly characterized intracellular Met. Ions Life Sci. 1, 227–279 (2006)

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Clathrincoated pit DMT1 HOLO-TF

APO-TF TFR

Fe2+

Mitochondria Ferritin H+ Non-erythroid cells

Proton pump H+

Haemosiderin Acidified endosome

TFR

Figure 3. The transferrin cycle. Holotransferrin (HOLO-TF) binds to transferrin receptors (TFR) at the cell surface. The complexes localize to clathrin-coated pits, which invaginate to initiate endocytosis. Specialized endosomes form, and are acidified by a proton pump. At the acidic pH, iron is released from transferrin and is co-transported with protons out of the endosomes by the divalent metal ion transporter DMT1. Apotransferrin (APO-TF) bound to TFR is returned to the cell membrane, where, at neutral pH they dissociate to participate in further rounds of iron delivery. In nonerythroid cells, iron is stored as ferritin and hemosiderin. (Reproduced with permission from [2]).

transit pool, usually referred to as the labile iron pool (LIP), which has been described as ‘somewhat like the Loch Ness monster’, only to disappear from view before its presence, or indeed its nature, can be confirmed [12]. The LIP (reviewed in [13]), represents iron in transit between extracellular supplies and intracellular demands, both functional as well as storage, and is discussed below. To prevent unwanted effects of iron-catalyzed free radical generation or precipitation of ‘free iron’, it is imperative that cells store excess iron safely. This Met. Ions Life Sci. 1, 227–279 (2006)

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‘housekeeping’ function in all cells is carried out by the iron storage proteins ferritin which can safely sequester 4500 atoms/molecule, or its degradation product hemosiderin. Other specialized cells, such as macrophages of the reticuloendothelial system and parenchymal cells of the liver will also store excess iron. Body iron pools can then be replenished as iron is utilized or lost. In normal subjects ferritin is the principal storage form, mostly in liver, spleen, and skeletal muscle, with hemosiderin representing a very small fraction of normal body iron stores, mostly in macrophages and spleen, which increase their iron content dramatically in iron overloading syndromes. The ferritin molecule is made up of 24 subunits which form a hollow protein shell, delimiting a cavity within which up to 4500 atoms of iron are stored in a nontoxic, water-soluble, yet bioavailable form, with a structure similar to the mineral ferrihydrite. Mammalian ferritins are heteropolymers of two biochemically distinct types of subunit, H and L, which play different roles in storing, detoxifying, and maintaining Fe3+ in a soluble form [1,14]. In the first step of iron incorporation, Fe2+ penetrates the apoferritin shell through channels at the surface of the protein and is oxidized at dinuclear iron centers (ferroxidase centers) localized in the H subunits. Fe3+ then migrates to nucleation centers within L subunits, in the interior of the protein shell. Once a nucleus of iron core has formed at the nucleation sites, deposition of iron on this inorganic core, essentially in the form of the mineral ferrihydrite, drives the further incorporation of iron. The mechanism of iron release from ferritin is not at all well understood, although it appears to be facilitated by reducing conditions and by a more acidic pH. Hemosiderin, the second storage form of iron, found predominantly in conditions of iron overload is thought to be the product of lysosomal degradation of ferritin [15,16]. The intracellular labile iron pool (described above) is not only in equilibrium with the storage iron compartment, but also serves as a source of iron for intracellular iron-protein synthesis. Recently, considerable progress has been made in our understanding of the way in which iron is used within the mitochondria for incorporation into both Fe/S protein clusters, and into protoporphyrin IX to form heme (see Section 6.6).

3.4 Iron Homeostasis It has been known for a long time that ferritin biosynthesis can be regulated in response to changes in cellular iron content even when transcription has been inhibited [17], and it was subsequently shown that this involves a stem loop of approximately 30 nucleotides in the 5 untranslated region of the ferritin mRNA. Met. Ions Life Sci. 1, 227–279 (2006)

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This sequence is highly conserved in all ferritin mRNAs. Biosynthesis of the transferrin receptor is also regulated at the level of translation, but in this case the regulation involves up to 5 corresponding tandem nucleotide sequences in the 3 untranslated portion of the transferrin receptor mRNA. These sequences all show significant homology and have been termed iron-responsive elements or IREs [18,19]. The consensus sequence of these IREs, shown in Figure 4 consists of a lower stem of variable length made up of complementary pairs of RNA bases; between this lower stem and an upper stem of five complementary base pairs, is an unpaired cytosine base, which produces a characteristic bulge in the stem structure; on top of the upper stem is a loop in which 5 of the 6 bases are almost always CAGUG. In addition to the ferritin H-chain, ferritin L-chain, and transferrin receptor mRNAs, IREs or IRE-like sequences have been found in the mRNAs for several other proteins, including the erythroid form of -aminolevulinate synthase (the first enzyme in the heme biosynthetic pathway), the mitochondrial form of aconitase, and the iron transporters IREG-1 and DMT-1. IREG-1 transports iron across the basolateral membrane of the small intestine and across the plasma membrane of macrophages, while the divalent cation transporter protein, DMT-1, transports Fe2+ across the apical membrane of the small intestine and is also involved in the transport of iron out of the endosome during the transferrin uptake cycle. Two cytosolic proteins, designated iron regulatory proteins (IRPs) interact directly with the IREs to modulate mRNA translation (Figure 5). IRP-1 and IRP-2 have MWs of 90 and 105 kD, respectively, the difference being a 73 residue inclusion. When iron is in adequate amounts, IRP-1 exists in a 4Fe–4S form, has aconitase activity, and lacks IRE-binding activity. When the cellular iron concentration is low, the protein lacks its 4Fe–4S cluster, loses its aconitase G U A C N N N N N C N N N N N N 5′

G N N N N N N

6 Base loop

5 Base pair upper stem Unpaired cytosine

N N N N N N 3′

Variable length lower stem

Figure 4. Consensus sequence for iron-responsive elements. Met. Ions Life Sci. 1, 227–279 (2006)

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3′-mRNA Transferrin-R

IREs

Transferrin-R

IRPs

Ferritin

Ferritin

5′-mRNA

Figure 5. Production of transferrin receptor and of ferritin are regulated at the level of mRNA. Iron responsive elements (IREs) in mRNA of the transferrin receptor are localized in the 3 -mRNA region, and in mRNA of ferritin in the 5 -mRNA promotor region. The system is designed to allow the cells to procure iron from the plasma, by expressing more transferrin receptors, if they need iron for production of proteins, and to protect cells against potentially toxic iron by expressing ferritin molecules, able to hide Fe(III) within its core. The system is regulated by iron-responsive protein 1 (IRP). In iron deficiency IRP inhibits RNase attack of TFR mRNA, and inhibits production of ferritin while sitting on an IRE in the 5 -mRNA region. If the LIP contains abundant amounts of iron, modified IRP has no affinity for IRE, resulting in destruction of TFR mRNA. At the same time free IRE on ferritin mRNA allows sufficient expression of ferritin. (Reproduced with permission from [1]).

activity but acquires IRE-binding activity (Figure 6). IRP-2 has around 80% homology with IRP-1, is less abundant than IRP-1 in most cells, but is highly expressed in brain. Although the cysteine residues that coordinate the iron–sulfur cluster in IRP-1 are conserved in IRP-2, it has no detectable aconitase activity. The apo form of IRP-2 binds to IREs when iron is in short supply, but, in iron replete cells, IRP-2 is ubiquitinated within its 73 residue inclusion, and subsequently degraded by the proteosome (Figure 6). In conditions of iron depletion, the IRPs bind to the IREs, preventing ferritin synthesis, but allowing the synthesis of transferrin receptor, by protecting its mRNA from nuclease degradation (Figure 5). When iron is in excess of requirements, the IRPs no longer bind to the IREs, allowing ferritin to be synthesized, whereas the transferrin receptor mRNA, no longer protected by the IRPs, is degraded, and transferrin receptor is no longer produced. Met. Ions Life Sci. 1, 227–279 (2006)

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ACTIVE

IRP-1

INACTIVE de novo synthesis

7

mG

4 C

2+

Fe (2–4h)

CAG N U G

4

Dos (–15h) NO (–15h)

G AU

C506 C437 C503 1–3

C506 C437 C503 1–3

H2O2 (<1h)

Apo-IRP-1: no [4Fe-4S] cluster IRE-binding site accessible

Cytoplasmic aconitase with [4Fe-4S] cluster IRE-binding site not accessible

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IRP-2 7

mG

4 C

CAG N U G

G AU

C581 C512 C578 1–3

Apo-IRP-2 IRE-binding site accessible

UNSTABLE de novo synthesis

4

2+

Fe (2–4h) –

Dos NO

C581 C512 C578 1–3

IRP-2 modified (proteasome-mediated) degradation

Figure 6. Regulation of IRP-1 and IRP-2. The two IRPs are shown as homologous four domain proteins that bind to IREs. Left: In iron-replete cells, IRP-1 assembles a cubane Fe-S cluster that is liganded via cysteines 437, 503 and 506. Similar cysteines are conserved in IRP-2 (Cys512, 578, and 581), but it is unresolved whether they also coordinate an Fe-S cluster. Right: In iron-replete cells, IRP-2 is targeted for destruction via a specific region (shaded in black), whereas IRP-1, with a 4Fe-4S cluster, is stable and active as a cytoplasmic aconitase. Multiple signals induce IRE-binding by IRP-1 with distinct kinetics. Whether or not NO and H2 O2 induce IRP-1 by apoprotein formation remains to be addressed directly. Dos: desferrioxamine. (Reproduced with permission from [18]).

The regulation of the transcription of ferritin, transferrin receptor, or transferrin genes may also be modulated by a number of nuclear transcription factors. Several DNA-binding proteins have been identified that interact with the transferrin promoter region. Tissue differences in the expression of H and L ferritins exist and are in part due to differences in transcriptional activity. Changes in expression of ferritin in response to cellular iron content also differ for the H- and L-ferritin subunits. Transcriptional control of ferritin expression has been documented in a number of human cells and cell lines. Factors such as Met. Ions Life Sci. 1, 227–279 (2006)

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cellular differentiation and inflammatory cytokines (tumor necrosis factor and interleukin-1) may bring about differential subunit expression [14]. Changes in expression of the transferrin receptor have been observed in response to different phases of the cell cycle, to cellular differentiation, and to cytokines, including - and -interferons. These responses appear to be predominantly mediated by changes in transcription.

4 THE ROLE OF THE ‘LABILE IRON POOL’ IN FREE RADICAL PRODUCTION The labile iron pool within cells is defined as a pool of redox-active, mostly low molecular weight iron complexes, originally proposed to be a pool of iron in transit between extracellular and intracellular forms of iron. Operationally, it is a pool of chelatable iron, both Fe2+ and Fe3+ , associated with a diverse population of ligands such as organic anions (phosphates and carboxylates), polypeptides, and surface components of membranes (e.g., phospholipid head groups) [13]. This means that the LIP can participate in redox-cycling, with clear implications for production of reactive oxygen species, notably hydroxyl radical, and that it can be scavenged by chelators which are able to penetrate the cell. The levels of LIP in cultured cells are maintained within a relatively narrow range of concentrations [20,21], in the interests of cellular iron homeostasis, allowing the cells to meet their metabolic requirements for iron while maintaining their potential for ROS formation at an appropriate level. Logically, cytosolic LIP levels will be sensed by the IRPs [19] and, subsequently, readjusted; when LIP levels are low, the IRPs will up-regulate iron uptake via the transferrin receptor and the endosomal transmembrane cation transporter DMT 1 while downregulating the synthesis of the intracellular iron storage protein, ferritin; when LIP levels are high in iron repletion, IRPs will be inactivated, leading to increased translation of ferritin and down-regulation of the iron importer proteins. The LIP reflects the readily available iron within the cell and is believed to be the key regulator of internal and external iron exchange, and its labile nature is underlined by its capacity to promote the formation of reactive oxygen species, levels of LIP and ROS following similar ‘rises and falls’ as a function of cellular iron homeostasis. For example, it was shown that accompanying rising LIP levels there was a demonstrable rise in ROS production, lipid peroxidation, and eventual cell death [22]. Using repression of ferritin synthesis it was shown that the upregulation of steady state LIP significantly increased protein oxidation [23]. The production of both ROS and RNS (including nitric oxide and peroxynitrite) results in covalent modification of proteins by products of lipid peroxidation including acrolein and 4-hydroxy-2 nonenal (HNE), involving Michael addition (Figure 7) to lysine, cysteine and histidine residues[24]. The ROS/RNS induced Met. Ions Life Sci. 1, 227–279 (2006)

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OH ∗

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OH

O

∗

O

X

XH

Protein

Protein ∗

O

∗

OH

∗

X Protein

Figure 7. The Michael-type addition of 4-hydroxy-2 nonenal to proteins. X: the sulfhydryl group of cysteine, the imidazole group of histidine or the amino group of lysine. (Reproduced with permission from [24]).

protein misfolding finally results in the formation of protein aggregates. On account of ineffective functioning of the cytoplasmic protein elimination system, involving ubiquitination and digestion by the proteasome (Figure 8), this leads to the formation of the typical intracellular inclusion bodies which are the postmortem hallmark of many neurodegenerative diseases.

5 THE IMPORTANCE OF IRON IN BRAIN 5.1 Iron in Brain Development and Function The brain has several characteristics that make it unique among organs with regard to iron metabolism. It resides behind a vascular barrier, which limits its access to plasma iron. A transport mechanism at the blood–brain barrier (BBB) moves iron across the endothelial cells and into the brain [25]. However, little is known about the mechanism of iron release into the brain or the regulation of the transport mechanism. The concentration of iron in various regions of the brain varies greatly. Regions of the brain that are associated with motor functions (extrapyramidal regions) tend to have more iron than non-motor-related regions [26] which might explain why movement disorders are commonly associated with iron imbalance. It is known that at birth these regions have little iron, however, with ageing they accumulate significant amount of iron, probably through axonal transport. The amounts of iron present is in excess of what is required by the various iron dependent processes (calculated to be about 5–10% of brain iron). The rest of the iron must have some other function. In the brain, the most common cell type to stain for iron under normal conditions is the oligodendrocyte [27] which expresses both H- and L-ferritin. Neurons and microglia, have much lower amounts of ferritin present [27], neurons mostly Met. Ions Life Sci. 1, 227–279 (2006)

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O Ubiquitin

COO–

+

E1

— SH

ATP 1 AMP + PPi O Ubiquitin

C—S

— E1 E2 — SH

2 E1 — SH O Ubiquitin

C—S

— E2 Condemned protein

3 E3

E2 — SH

O Ubiquitin

C — NH

— Lys

Condemned protein

Isopeptide bond

Figure 8. The reactions involved in the attachment of ubiquitin to a protein. In the first part the carboxyl group of ubiquitin is coupled to E1 by a thioether linkage in a reaction driven by ATP hydrolysis. The activated ubiquitin is subsequently transferred to a sulfhydryl group of E2 and then in a reaction catalyzed by E3 to the -amino group of a Lys residue on a condemned protein, thereby flagging the protein for proteolytic degradation by the 26S proteosome. (Reproduced with permission from [169]).

express H-ferritin while microglia appear to express L-ferritin [28,29]. Very little ferritin expression is seen in astrocytes. In the dopaminergic neurons of the substantia nigra and the noradrenergic neurons of the locus coeruleus, large amounts of iron are sequestered in neuromelanin granules [30]. Met. Ions Life Sci. 1, 227–279 (2006)

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Neuromelanin is synthesized by oxidation of excess cytosolic catechols that are not accumulated in synaptic vesicles by vesicular monoamine transporter-2 [31]. Neuromelanin binds iron avidly, forming stable octahedral complexes that contain high-spin oxyhydroxide iron(III) clusters [32]. Neuromelanin is a complex molecule whose structure is arranged as a multilayer system where each layer is a polymer composed of melanic groups bound to aliphatic and peptide chains. This melanic group contains benzothiazine and dihydroxyindole units (ratio 1:3) and the latter is responsible for the strong chelating ability for iron and other metals [31,33]). The iron transport protein transferrin (Tf) was originally thought to be synthesized only by oligodendrocytes in the brain [34], although evidence for Tf mRNA has recently been found in neurons in the human substantia nigra [33]. Choroid plexus epithelial cells and other cells outside the brain that make Tf secrete transferrin, but it does not seem to be secreted by oligodendrocytes [35]. There are two receptors in the brain for iron uptake – TFRs are expressed exclusively in the gray matter, whereas ferritin receptors are present only in white matter [36]. This dichotomous expression pattern is unique to the brain. In summary, the brain is unique among organs because of the nonuniformity of iron distribution, both regionally and cellularly, and because of the apparent selectivity in cellular responsibilities for iron storage and mobilization, and presumably usage and methods of uptake.

5.2 Brain Iron as a Function of Age The iron content of some parts of the brain is very high – significantly greater iron concentrations, as high as 150–200 g/g protein, are found in the substantia nigra and the globus palidus than in liver [37], and other brain regions with high concentrations are the dentate gyrus, interpeduncular nucleus, thalamus, ventral pallidus, nucleus basilis and red nucleus (Figure 9). There is a consensus that iron accumulates in the brain as a function of age. However, this process is quite specific and involves the accumulation of iron-containing molecules in specific cells, particularly in brain regions that are preferentially targeted in neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). Levels of nonheme iron (mostly ferritin) increase in the putamen, motor cortex, prefrontal cortex, sensory cortex and thalamus during the first 30–35 years of life and variable changes are observed in older individuals [38]. Some studies have shown that levels of H-ferritin in older subjects (67–88 years of age) were higher than in younger controls (27–66 years) in the frontal cortex, nucleus caudatus, putamen, substantia nigra and globus pallidus. In the case of L-ferritin, this increase is observed only in the substantia nigra and globus pallidus [39,40]. Met. Ions Life Sci. 1, 227–279 (2006)

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250

Iron μg / g protein

200 150 100 50

R LI VE

FC

ER C

IP P H

C C

N R

G D

TH

IP N

SN

G P

0

Figure 9. Distribution of iron in human brain. GP, globus pallidus; SN, substantia nigra; IPN, interpeduncular nucleus; TH, thalamus; DG, dentate gyrus; RN red nucleus; CC, cerebral cortex; HIPP, hippocampus; CER, cerebellum; FC, frontal cortex. (Reproduced with permission from [37]).

In the parieto-occipital lobe of the cortex, the expression of heme oxygenase-1 and ferritin increases with age, whereas in the hippocampus, only heme oxygenase-1 expression increases. Heme oxygenase-1 is found in both glia and neurons, whereas ferritin is only found in glia. The increase in heme oxygenase-1 might contribute to an increased susceptibility to oxidative stress in the elderly [41]. Several lines of evidence indicate that in the brains of older individuals, iron homeostasis is accomplished more efficiently in the locus coeruleus than in the substantia nigra. A linear increase in iron concentration, both H and L ferritin, occurs with age in the substantia nigra, whereas in the locus coeruleus, the iron concentration is lower and remains constant throughout life [29,40]. In subjects over 80 years of age, numerous extraneuronal iron deposits are present in the substantia nigra, especially in oligodendrocytes, but very few iron deposits are observed in the locus coeruleus. Iron deposits are found in neurons of the substantia nigra that do not contain neuromelanin, but no iron deposits are seen in neuromelanin-containing neurons in either the substantia nigra or the locus coeruleus (Figure 10). The concentration of the neuromelanin–iron complex in neurons of the substantia nigra and locus coeruleus increases linearly during life, but the slope of the accumulation curve in the substantia nigra is steeper than in the locus coeruleus [29]. In older subjects (60–90 years of age), as compared with younger subjects (28–49 years), more iron staining is observed in microglia and astrocytes of the cortex, cerebellum, hippocampus, basal ganglia and amygdala, and ferritin immunoreactivity is also stronger in these cells. Oligodendrocytes contain the Met. Ions Life Sci. 1, 227–279 (2006)

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Figure 10. Iron deposits in the ageing brain. Iron histochemistry with modified Perls’ staining of: (a) human substantia nigra (SN); and (b) locus coeruleus (LC), from a normal male subject aged 88 years. Neuromelanin in dopaminergic neurons of SN and noradrenaline neurons of LC are seen as brown granules and iron deposits stain blue. (Reproduced with permission from [2]).

largest amount of iron, ferritin and Tf, but their content remains constant during ageing [42]. Iron accumulation in microglia might stimulate the activation of these cells in the neuroinflammatory processes that contribute to AD and PD. All the above data are from postmortem tissue, but iron levels can also be assessed by non invasive methods, such as magnetic resonance imaging (MRI) which has revealed an age-related increase in nonheme iron concentration in the nucleus caudatus, putamen, and globus pallidus [43–45]. The increased iron levels in certain brain regions could result from the altered vascularization that is observed during ageing and in neurodegenerative diseases [46–48]. The role of vascular factors in AD and the common features shared by AD and vascular dementia has been recently discussed [49]. The increase in iron levels in neurons, astrocytes and microglia, which normally have low iron contents up to middle age, is typically present in regions such as the cortex, hippocampus, and substantia nigra, which are particularly susceptible to the neuropathological changes that characterize AD and PD [50]. This iron ‘invasion’ and ‘perennial occupation’ might directly damage these cells or perturb the cellular milieu, making it more susceptible to toxins and activation processes of a pathogenic type (for example, inflammation, factor release, morphological changes or apoptosis). However, in brain aging iron is partially converted from its stable and soluble form (ferritin) into other oxyhydroxide derivatives such as hemosiderin, which might reduce its reactivity. Despite this, the prelude of the pathogenic role of iron in brain ageing can be summarized in this triad: iron accumulation, invasion, and increased reactivity. Met. Ions Life Sci. 1, 227–279 (2006)

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6 THE INVOLVEMENT OF IRON IN NEURODEGENERATIVE DISEASES 6.1 Parkinson’s Disease Parkinson’s disease (PD) (see also Chapter 6) was initially described as Shaking Palsy in 1817 by the English surgeon James Parkinson. The second most common form of motor system degeneration, it is characterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta in the ventral midbrain. The etiology of the disease is unknown. It is possibly multifactorial, with input from a variety of genetic, environmental, and endotoxin factors, as well as iron, which increases in specific brain regions with ageing. Oxidative stress together with abnormal protein turnover and degradation, are likely to be key factors in the development and progression of this disease. There is strong evidence implicating the abnormal processing of a number of cellular proteins (see Sections 3.1.1–3.1.4) via the ubiquitin/26S proteosomal system in PD. Such proteins are present in the cytosol as insoluble, unfolded, ubiquitinated polypeptides. The characteristic inclusion bodies of PD, Lewy bodies (LB), present in affected neurons in the brain (Figure 11), are eosinophilic intracytoplasmic inclusions, with a core of granular and filamentous material surrounded by radiating filaments 10–15 nm in diameter. Wild-type -synuclein is found as a major component of Lewy bodies, suggestive that -synuclein is involved in the pathophysiology of PD. Three human synuclein proteins , and are known, and they are encoded by separate genes on three different chromosomes, 4q21.3-q22, 5q23 and

Figure 11. A Lewy body contains a mass of proteins including -synuclein and is a characteristic feature of Parkinson’s disease. Met. Ions Life Sci. 1, 227–279 (2006)

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10q23.2-q23.3, respectively. The synucleins, in common with the microtubuleassociated protein tau, as well as mutants of -synuclein and tau, are associated with inherited forms of neurodegenerative disease and have a very similar type of structure. They are classed as natively unfolded proteins with practically no organized secondary structure in solution. - and -synucleins are found primarily in brain tissue, predominantly in the neocortex, hippocampus, striatum, thalamus, and cerebellum: immunoreactive proteins are enriched in presynaptic terminals. -synuclein is found primarily in the peripheral nervous system (in primary sensory neurons, sympathetic neurons, and motor neurons) and in retina: its expression in breast tumors is a marker of tumor progression [51]. It has been proposed that -synuclein may play an important biological role in the modulation of dopamine release, as a regulatory protein, which binds and inhibits tyrosine hydroxylase, the rate-limiting enzyme in dopamine biosynthesis [52]. -synuclein is predominantly expressed in the central nervous system within the substantia nigra and localized within the cytosol, in close proximity to synaptic vesicles at presynaptic termini. It shares both physical and functional homology with 14-3-3 proteins, a family of ubiquitous cytoplasmic chaperones, which are, like -synuclein, particularly abundant in brain. In idiopathic Parkinson’s disease, aggregates of wild-type -synuclein, which are heavily ubiquitinated, are found in Lewy bodies within dopaminergic neurons, axons, and synapses of the substania nigra. The dopaminergic neurons seem to be particularly and selectively vulnerable to the toxic effects of -synuclein [53]. The family, known as the Iowan kindred described recently [54], exhibit a triplication of the -synuclein gene, which results in autosomal dominant PD giving four copies of the gene rather than the usual two. This clearly implies that overproduction of wild type -synuclein is the cause of this form of PD rather than simply the production of mutant protein. The presence of Lewy bodies in affected regions of PD brains indicates improper handling of this protein. -synuclein may be the actual building block of the fibrillary components of LBs, binding tubulin amongst a host of other ubiquitinated proteins, such as, transglutaminase [55], synphilin-1, and parkin [56], and acts as the focal point for the aggregations. Its polymerization is associated with concomitant changes in secondary structure which can range from the natively unfolded state in solution, to -helical in the presence of lipid containing vesicles to anti-parallel -pleated sheet structures or amyloid structure in fibrils. Such products will initiate disturbances in the cytosolic cellular compartment interacting with vesicles, dopamine transporters, and intraneuronal mitochondria, which may lead to cellular death. A link between iron and -synuclein has been identified in vitro. Iron enhances intracellular aggregation of -synuclein [57–59], leading to the formation of advanced glycation end products [60,61] while -synuclein liberated hydroxy radicals when incubated with Fe2+ [62]. Extracellular -synuclein will generate ROS [63]. Pretreatment of cells with cell-permeable iron chelators, Met. Ions Life Sci. 1, 227–279 (2006)

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transferrin-receptor antibodies or transfection with glutathione peroxidase, inhibited intracellular oxidant generation, -synuclein expression/aggregation as well as apoptosis [64]. Parkin is located on chromosome 6, 6q25-27, and has a distinct domain structure consisting of a ubiquitin-like (UBL) domain at its amino terminus and two carboxy-terminal cysteine-rich RING (really interesting new gene) fingers flanking an in-between RING (IBR) domain [65].1 Wild-type parkin has been shown to have E3 ubiquitin-protein ligase activity, whereas autosomal recessive juvenile parkinsonian-related mutant parkin proteins do not have this activity [66–68]. Parkin is thus one of the large number of E3 ubiquitin-protein ligases whose cellular role is to add the polyubiquitin tail to targeted proteins for their degradations, through the binding of their Ubl to proteosomes. The Ubl of parkin has a typical ubiquitin fold and binds to the Rpn10 subunit (Rnt6 and C3) of the 26S proteasome complex [69] while the extreme C-terminal region interacts with at least one membrane protein, CASK [70], and co-localizes with it in lipid rafts. This means that parkin is tethered to membranes where it can recruit proteasomes. Mutations affecting Ubl function have been identified in both human (R42P) and Drosophila (A46T) which cause neurodegeneration in humans and mitochondrial dysfunction in Drosophila. Parkin can also recruit other proteins, such as the heat-shock chaperone Hsp70 [71], another E3 ubiquitin ligase, CHIP [71], and a component of modular E3 ligase systems, hse110 [72]. Putative substrates that may be ubiquitinated by parkin include the O-glycosylated form of -synuclein, synphilin 1, and PaelR [73]. The substrates of parkin associate with the RING1 domain, whereas the E2 enzyme carrying the activated ubiquitin binds to the IBR and RING2 domains. Most of the mutations in parkin are loss-of-function mutations whereby parkin does not ubiquitate and remove substrates which subsequently accumulate with toxic consequences. One of these is an endoplasmic reticulum-resident substrate, a putative G-protein-coupled receptor (Pael-R), which is abundantly expressed in dopaminergic neurons in the substantia nigra, and has the tendency to unfold, even under physiological conditions. Accumulation of insoluble PaelR, could lead to neuronal death as a result of unfolded protein stress: indeed, insoluble Pael-R accumulates in the brains of patients with autosomal recessive juvenile parkinsonism (AR-JP), suggesting that accumulation of unfolded Pael-R could lead to selective death of dopaminergic neurons in AR-JP. Hsp70 and CHIP enhance the ability of parkin to inhibit cell death induced by Pael-R [74]. Pael-R is selectively toxic to dopaminergic neurons in Drosophila which overexpress it [75]. Therefore, an important role of parkin is to protect against the 1 A RING finger is a cysteine-rich zinc-binding motif, which is found in a wide variety of proteins. The domain mediates binding to other proteins, either via their RING domains or other motifs. In several proteins, RING domains are found in combination with other cysteine-rich binding motifs and some proteins, like parkin, contain two RING domains. Recent evidence suggests that RING finger proteins function in the ubiquitin pathway as E3 ligases.

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toxicity induced by mutant forms of -synuclein and Pael-R, by promoting their ubiquitination and proteasomal degradation. Since the cysteine residues of parkin are integral to its E3 activity, their modification by reactive oxygen species (possibly catalyzed by the increasing iron accumulation) may impair its function. Two recent reports indicate that S-nitrosylation may alter parkin’s ability to ubiquitate target proteins, although they report opposite effects [76,77]. Peroxide will induce mis-folded parkin which can be prevented if heat shock chaperones, Hsp70 and its co-chaperone Hsp40, are induced. The three carboxy-terminal amino acids of parkin are necessary for proper folding as well as function. Impairment of proteosomal activity by stable expression of parkin mutants leads to accumulation of oxidized proteins and lipids, thereby sensitizing the neurons to various forms of stress, leading to neuronal death. Caspase 1 and 8 can directly cleave parkin, leading to loss of ubiquitin ligase activity, causing accumulation of toxic parkin substrates and triggering dopaminergic cell death [78]. Lymphocytes from patients with AR-JP, homozygous for the Cys212Tyr parkin mutation, show increased sensitivity to dopamine, iron and hydrogen peroxide [79]. Studies of parkin-deficient mice suggest that the function of parkin is to maintain synaptic function in dopaminergic neurons. A decrease in several subunits of mitochondrial complexes I and IV in the ventral midbrain was identified in parkin-deficient mice, while functional assays showed reduced respiratory capacity. Decreased levels of cytoprotective enzymes were also identified which resulted in increased protein and lipid peroxidation [80]. Such results reveal an essential role for parkin in the regulation of mitochondrial function. Overexpression of parkin is beneficial to dopaminergic neurons protecting them from mutant forms of -synuclein and from Pael-R. Parkin may also protect against aberrant accumulation of cyclin E [72], facilitate the clearance of proteins which have polyglutamine expansions [81], proteasomal inhibition, kainin acid excitotoxicity and ceramide-induced mitochondrial apoptosis (reviewed in [73]). Synphilin-1 is an -synuclein-binding protein that is ubiquitinated by parkin, and promotes the formation of cytosolic inclusions. It also interacts with the E3 ubiquitin-ligases SIAH-1 and SIAH-2 [82]. SIAH proteins ubiquitinate synphilin1 in vivo, and promote its degradation by the ubiquitin-proteasome system. An inability of the proteasome to degrade the synphilin-1/SIAH complex will lead to the formation of ubiquitylated cytosolic inclusions. The importance of this protein in Parkinson’s disease awaits further clarification although SIAH immuno-reactivity has been shown in Lewy bodies. Other genes have been identified which may play a role in Parkinson’s disease; these include Park 3 and Park 4, located respectively on chromosome 2 and 4, Park 5, an ubiquitin carboxy-terminal hydrolase (UCHL 1), Park 6, a putative protein kinase, which locates primarily to the mitochondria [83], Park 7, Park 8-11, and NR4A2 which encodes a member of a nuclear receptor super family and is essential for the differentiation of nigral dopaminergic neurons. Further clarification of their roles at the protein level is required. Met. Ions Life Sci. 1, 227–279 (2006)

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In the brains of Parkinson’s patients there is a specific two-fold elevation of iron in two brain regions, the substantia nigra (SN), and the lateral globus pallidus (LGP), in comparison to age-matched controls [37,84]. It is remarkable that, in contrast to other iron storage diseases, where 10–20-fold increases in iron stores are required before clinical abnormalities occur, e.g., untreated genetic hemochromatosis (GH), and thalassemia patients (THAL), only a relatively small elevation of this redox active metal is needed within the SN of PD brains to produce extensive pathological consequences. Although hemosiderin (the product of lysosomal degradation of L-ferritin) is the predominant iron storage protein in GH and THAL, the predominant iron protein present is H-ferritin which is not an iron storage protein. This may explain why excess iron associates with neuromelanin within the dopaminergic neurons of SN and LGP. As these neurons degenerate, the iron–neuromelanin complex is released into the extraneuronal environment to stimulate microglia proliferation [37], triggering the inflammatory process, which in turn will induce degeneration and the death of more neurons, thereby consolidating the process. The importance of H-ferritin in brain function is exemplified by the fact that homozygous knockout mice for H-ferritin die in utero, whereas heterogenous mice are viable [85] and show brain iron levels comparable to controls although H-ferritin content is decreased by approximately 50%. Transferrin, transferrin receptors, L-ferritin, DMT1, and ceruloplasmin content are increased. Oxidative stress was increased in these mice, as indicated by the presence of oxidatively modified proteins and decreased activities of cytoprotective enzymes, e.g., superoxide dismutase [86]. Cells which overexpressed human H-chain ferritin, accumulated the protein at levels 14-16-fold over background, and show an iron-deficient phenotype manifested by a five-fold increase of IRP activity, 2-2.5-fold increases in both transferrin receptor, and iron-transferrin iron uptake, and approximately 50% reduction of the labile iron pool [87]. Overexpression of the H-ferritin strongly reduced cell growth and increased resistance to H2 O2 toxicity; such effects were reverted by prolonged incubation in iron-supplemented medium. Neuromelanin (Figure 10), a granular dark brown pigment, is produced in catecholaminergic neurons of the SN and LGP and is possibly the product of reactions between oxidized catechols with a variety of nucleophiles, including thiols from glutathione and proteins [84]. The function of neuromelanin in the pigmented neurons is unknown, but it could play a protective role by binding transition metals such as iron to attenuate the induction of oxidative stress via Fenton chemistry. Whether the ability of the neurons to synthesize neuromelanin is impaired in PD patients is unknown, it is reported that the absolute concentration of nigral neuromelanin is less than 50% in PD with respect to age-matched controls. In vitro, melanin can bind a significant amount of iron at two sites [88], although the pigment appears to be only 50% saturated with iron in PD. EPR studies of the SN show that ferric iron is bound to neuromelanin as a high spin complex with an octahedral configuration [31,89]. Met. Ions Life Sci. 1, 227–279 (2006)

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The explanation for the dysregulation of iron in Parkinson’s disease remains unknown, changes in iron release mechanisms across the blood–brain barrier, or in the regulation of iron transport across the cellular membranes of SN and LGP may be relevant [90]. Divalent metal transporter 1, DMT1, which facilitates the release of iron from the endosomes after internalization of the transferrin– transferrin receptor complex shows increased expression in the SN dopaminergic neurons of Parkinson’s patients [91] which may reflect up-regulation of transferrin receptors on such neurons. Perturbation of iron homeostasis via the IRP-IRE system has also been implicated. Iron regulatory proteins, IRP-1 and IRP-2, act as iron sensors and regulate ferritin and transferrin receptor synthesis. Although no studies of IRP-2 binding activity in SN of Parkinson’s patients have been reported, IRP-1 binding activity in the SN of Parkinson’s patients was comparable to age matched controls [92]. This result is of interest, since it might have been expected that the IRP activity would have been altered by the increasing iron content. No IRP-2 polymorphisms are reported in subjects with sporadic PD which might have played an important role in the development of the disease. Genetically engineered mice which lack IRP-2, but have the normal complement of IRP-1, develop adult onset neurodegenerative disease associated with inappropriately high expression of ferritin in degenerating neurons, while mice that are homozygous for a targeted deletion of IRP-2 and heterozygous for a targeted deletion of IRP-1 developed severe neurodegeneration with severe axonopathy, with increased levels of ferric iron and ferritin expression as well as neuronal cell bodies degenerating in the substantia nigra. The increased iron burden may contribute to mitochondrial dysfunction, a defect in mitochondrial oxidative phosphorylation, as a consequence of reduction in the activity of NADH CoQ reductase (complex 1) [93]. Such derangement in complex I causes -synuclein aggregation, contributing to the demise of dopaminergic neurons. Aggregates of -synuclein may enhance such dysfunction by specific protein-protein interaction of -synuclein with cytochrome c oxidase, the terminal complex of the mitochondrial electron transport chain [94] while dopamine oxidized metabolites may interact with complex I of the chain. Protein misfolding and aggregation in vivo can be suppressed and promoted by several factors such as molecular chaperones, protein degradation systems and free radicals, many of which are under the control of normal mitochondrial function. Substantial amounts of parkin are present on the mitochondrial outer membrane which has led to suggestions that this protein may have an important role in maintaining mitochondrial function, possibly protecting the cell from oxidative stress. Indeed, overexpression of parkin has been shown to attenuate C2-ceramidemediated mitochondrial swelling. Pretreatment of cells with cell-permeable iron chelators, transferrin-receptor antibodies or transfection with glutathione peroxidase inhibited intracellular oxidant generation, -synuclein expression/aggregation as well as apoptosis [95], indicating that removal of iron would be beneficial. Met. Ions Life Sci. 1, 227–279 (2006)

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The use of chelation therapy to remove the excess iron has received considerable interest over the last decade. Initially, the only iron chelator available was deferrioxamine, which required subcutaneous administration with overnight infusion, because of its rapid clearance. A large number of new chelators have therefore been synthesized, which are orally active, and not so rapidly metabolized, and have been proposed as useful therapeutic agents for the removal of iron from the brain. So far, the majority of these have only been tested in animal models of iron overload and not been evaluated in clinical trials. Only one orally active drug is in current clinical use for the treatment of iron overload in thalassemia patients, deferipone, L1, but it is unsuitable for PD patients since adverse effects on dopamine metabolism were identified [96]. Therefore, since this time there has been an urgent requirement for the development of new orally bioactive chelating agents. Previously, a tridentate, desferrithiocein was also tested [97] which successfully removed excess brain iron without adversely altering dopamine metabolism. Unfortunately, further development of this compound was not possible because of nephrotoxicity. Some interest has been directed towards the increased expression of lactoferrin receptors on SN neurons and microvessels of PD patients which may be secondary to iron accumulation [49]. The explanation for their up-regulation is unknown but may be related to lactoferrin’s protective properties — it is released in response to inflammation and infection, removing iron from the circulation and allowing it to be sequestered within macrophages. Cumulative evidence supports an ‘oxidative stress hypothesis’ for initiation of nigral dopamine neuron loss. Substantia nigra has a relatively high metabolic rate, with a high content of dopamine, neuromelanin, polyunsaturated fatty acids, and iron but low antioxidant protection, e.g., glutathione. Therefore the imposition of oxidative stress will readily overwhelm the cytoprotective and antioxidant capacity, leading to the initiation, propagation and programmed cell death (apoptosis) of the dopaminergic neurons.

6.2 Alzheimer’s Disease Alzheimer’s disease (AD) (see also Chapter 5) is one of the most common neurodegenative maladies in Western societies. Clinical symptoms occur between the ages of 60–70 years. This disease, for which no effective treatment is currently available, initially presents with symptoms of memory loss, after which a progressive decline of both cognitive and motor function occurs. Both genetic and environmental factors are implicated in its development. The amyloidosis which occurs in AD involves abnormal protein processing; a structural transition of a polypeptide chain from a natively folded protein to one with an improperly folded conformation which leads to inefficient protein degradation. Truncated proteins are formed, and the resulting peptides tend to aggregate. There is considerable evidence that dyshomeostasis of redox-active metals, i.e., Met. Ions Life Sci. 1, 227–279 (2006)

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iron and copper, together with oxidative stress, contribute to the neuropathology of AD. The characteristic histology of AD is the deposition of both the amyloid peptide, A , as neuritic plaques (Figure 12a), and of the protein tau, as neurofibrillary tangles (Figure 12b), predominantly in the cerebral cortex and hippocampus. Amyloid precursor protein (APP), a type I membrane protein, resembles a cell surface receptor and is physiologically processed by site specific proteolysis (Figure 13). APP is cleaved by -secretase (within the A domain between Lys687

(a)

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Figure 12. Histopathology showing (left) plaques and (right) neurofibrillary tangles in Alzheimer’s disease.

Figure 13. Hypothetical mechanisms of conformational transitions for (A) A peptides and (B) tau. (A) The peptides are proteolytically cleaved from APP, precipitate as B structures in equilibrium with oligomers which may eventually redissolve into the membrane. (B) A schematic picture of the architecture of tau. The four repeats are indicated as R1-R4. The sites identified as calpain and caspase-3. (Reproduced with permission from [170]). Met. Ions Life Sci. 1, 227–279 (2006)

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Figure 14. Proteolytic processing of amyloid precursor protein (APP) by the secretases. APP is a type 1 transmembrane glycoprotein. The majority of APP is processed in the Met. Ions Life Sci. 1, 227–279 (2006)

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and Leu688 ), to yield APPs and the C-terminal fragment containing p3 peptide. The production of the amyloid peptide, A , is thus precluded. The presence of increasing amounts of iron may alter -secretase activity; one hypothesis suggested that iron might be required as a cofactor or be an allosteric modifier of -secretase activity. Iron may also decrease -secretase cleavage rates, since 24–48 h stimulation with ferric ammonium citrate (500 mM) reduced APP levels, but at the same time intracellular APP levels were enhanced significantly [98]. The membrane anchored -carboxy terminal fragment, -CTF, is then cleaved by

-secretase within the membrane, releasing p3 peptide and the APP intracellular domain (AICD) (Figure 14) [99]. AICD may function in nuclear signaling; it can fuse to the DNA binding domain of the Gal4 transcription factor to induce the expression of an upstream activating sequence-dependent reporter in combination with the adaptor protein Fe65 and the histone acetyltransferase Tip60. The fate of p3 is unknown. A can exist in three biochemical fractions in the brain: membrane-associated, aggregated, and soluble; in healthy individuals, most of the A will be membraneassociated, with small amounts of the soluble monomers detectable in cerebrospinal fluid (CSF) and blood. A possibly exists as an -helical conformation when it is part of the transmembrane protein, APP, with the hydrophobic C terminus embedded in the membrane lipid. In amyloidogenesis, the APP is cleaved sequentially by the proteolytic enzymes -secretase (aspartyl protease) and then by -secretase. -secretase has a C-terminal transmembrane domain and two active site motifs located in the luminal domain. -secretase cleaves APP between Met 671 and Asp672 and yields APP s and C99 fragments. The enzyme -secretase, a multi-subunit complex (containing presenilins 1 and 2 (PS1 and PS2)), will then cleave APP s to produce -amyloid peptide, A A 42 and A 40 , and AICD. Some recent studies indicate that there may be other factors involved in the action of PSs on the intramembranous proteolysis of APP [100]. Mutations in PS1 or PS2 genes will increase the production of the toxic A 42 . In individuals with AD, the aggregated and soluble fractions of A are markedly increased [100], the two -amyloid peptides, A 42 and A 40 , migrate from the cell to form aggregates, fibrils, and eventually neuritic plaques. The structure of A 40 consists of two helices spanning residues 15–23 and 31–35, Figure 14. (Continued) nonamyloidogenic pathway (thick arrow); APP is first cleaved by -secretase within the amyloid protein A  domain leading to APPs secretion and precluding A generation. Membrane-anchored -carboxy terminal fragment ( -CTF) is then cleaved by -secretase within the membrane releasing the p3 peptide and the APP intracellular domain (AICD). Alternatively, amyloidogenesis (thin arrow) takes place when APP is first cleaved by

-secreatase, producing APPs . A and AICD are generated upon cleavage by -secretase of the -CTF fragment retained in the membrane. (Reproduced with permission from [99]). Met. Ions Life Sci. 1, 227–279 (2006)

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which are separated by a disordered region. The A 42 peptide adopts a regular type I -turn, yielding a well defined tertiary structure which shows similarities with the fusion domain of the hemagglutinin of the influenza virus. A accumulation and aggregation is considered to be the initiating factor in AD pathogenesis, although it is known that such deposition occurs over many years, if not decades, prior to the clinical cognitive impairment. A may be oxidized within the membrane, perhaps as a result of the increased Cu and Fe levels in the brain [101] (Figure 15), from where it is ultimately liberated in a soluble form, to precipitate in the amyloid plaques. A peptides will increase calcium influx through voltage gated calcium channels (N and L types) by reducing magnesium blockade of N -methyl-d-aspartate (NMDA) receptors, as well as forming cation-selective ion channels after A peptide incorporation into the cellular membrane, thereby increasing excitotoxicity. A peptides may interfere with long-term hippocampal potentiation and cause synaptic dysfunction in Alzheimer’s disease [102]. The balance between the - and -secretase processing of APP is partially regulated by subcellular transport. -secretase typically cleaves the protein during the transport route to and at the cell surface. -secretase apparently meets APP at the cell surface and both are internalized together into early endosomes. Therefore the bulk of -secretase cleavage of APP occurs in the endosomes which is related to the acid pH optimum for -secretase. Cleavage of APP may also be regulated by ligands although no such functionally active ligands have yet been identified. F-spondin will bind to the central APP domain (CAPPD) and inhibit the initial - and -cleavage of APP, thereby regulating cleavage. This may indicate a potential pharmaceutical target to inhibit APP cleavage by synthesizing new molecules which bind to this CAPPD motif [103]. Trafficking and processing of APP can also be regulated by many adaptor proteins, e.g., members of the XII and the Fe65 families, which can bind to the APP cytosolic tail. Fe65 is involved in gene transcription regulation and appears to promote A secretion [99]. It is unknown whether A , which is continuously secreted under normal physiological conditions, may have a physiological role, possibly functioning as an antioxidant. There is an inverse relationship between A content and in vivo oxidative damage, suggesting that A might be a modulator of ROS generation [104,105]. The precipitation of A into plaques, associated with increased levels of metal ions, may be an efficient means of presentation to phagocytic cells for its removal from the cell. Other physiological functions assigned to A are as a superoxide scavenger (SOD activity), a cholesterolbinding molecule, and an acute phase reactant (reviewed in [106]). Many forms of stress, including head injury and trauma, may be regulated via -amyloid. Tau proteins are highly hydrophilic microtubule-associated proteins (Figure 13). There are six isoforms of tau, ranging between 352 and 441 amino acids in length, which are produced as a result of alternative mRNA slicing from a single gene on chromosome 17 [107] and are differentially expressed in neurons. In the normal brain, the functions of tau include stabilization of axonal Met. Ions Life Sci. 1, 227–279 (2006)

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Figure 15. Hypothetical model for the metallobiology of A in Alzheimer’s disease. The proposed sequence of events: (1) Concentration of iron and copper increase in the cortex with aging. There is an overproduction of APP and A in an attempt to suppress cellular metal-ion levels. (2) Hypermetallation of A occurs which may facilitate H2 O2 production. (3) Hypermetallated A reacts with H2 O2 to generate oxidized and crosslinked forms which are liberated from the membrane. (4) Soluble A is released from the membrane and is precipitated by zinc which is released from the synaptic vesicles. Oxidized A are the major components of the plaque deposits. (5) Oxidized A initiate microglia activation. (6) H2 O2 crosses cellular membranes to react with Cu and Fe to generate hydroxyl radicals which oxidize a variety of proteins and lipids. (Reproduced with permission from [101]). Met. Ions Life Sci. 1, 227–279 (2006)

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microtubules, modulation of signal transduction and regulation of vesicle transport (reviewed in [108]). Tau protein monomers can polymerize to form fibrils known as paired helical filaments (aggregated hyperphosphorylated tau protein), which become the building blocks for the neurofibrillary tangles (NFT), neuropil threads, and dystrophic neuritis that accumulate in AD. Such tau hyperphosphorylation may be associated with an increase in kinase activity or a decrease in phosphatase activity. PS1 mutations also modulate neurofilament-H protein phosphorylation [109], which in turn can modulate tau phosphorylation, leading to disruption of neurofilament-microtubule binding. This will disrupt axonal transport of APP and other proteins increasing accumulation and subsequent degradation of APP. Tau is a natively unfolded protein, containing neither -helix nor -sheet, which regulates microtubule function in the axon. It shows an even distribution of hydrophobic fragments along its sequence with up to four repeats of approximately 30 residues in its central region. Peptides from tau are more soluble than A . Short synthetic peptides spanning tau repeat sequences are able to aggregate as -structures. Neurofibrillary tau lesions are not restricted to AD; they are also the defining feature of other neurodegenerative diseases collectively referred to as tauopathies and include frontotemporal dementias including Pick’s disease, progressive supranuclear palsy and corticobasal degeneration. When such protein aggregates occur, their normal cellular function is lost and neurotoxicity occurs. NFTs contain redox active iron. Accumulation of tau in neurofibrillary tangles is associated with the induction of heme oxygenase 1, a potent antioxidant which plays an important role in metabolizing heme released from damaged mitochondria. Although this will reduce oxidative damage [110], Fe2+ will be released which may participate in Fenton chemistry to produce hydroxyl radicals. Tau within the neurofibrillary tangles is oxidatively damaged. High levels of Zn, Cu, and Fe are constitutively found in the neocortical regions which are most prone to AD pathology. Enriched amounts of copper, iron, and zinc are also present in the insoluble amyloid plaques in post mortem AD brain [111]. Alternatively, it has been suggested that the formation of the

-sheet configuration of A may actually be a protective mechanism. It may be that the increased synthesis of APP and A is an attempt by the brain cells to detoxify the elevated levels of redox active metals, copper and iron; other studies suggest that zinc and copper are inhibitory and prevent -sheet formation [112]. Membrane-bound A may be damaged by metal-induced reactive oxygen species prior to their liberation from the membrane, and consequently precipitated by zinc which is released from synaptic vesicles. In vitro, zinc rapidly accelerates A aggregation, the zinc being associated with the N-terminal region of A which has an autonomous zinc binding domain. Zinc induces conformational change of the 1–16 N-terminal region of A [113]. In adult rat brain, pools of zinc are detectable within glutamatergic synaptic vesicles in the neocortex which represents 30% of total brain zinc (Figure 16). Zinc is released during synaptic transmission, possibly Met. Ions Life Sci. 1, 227–279 (2006)

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Figure 16. Brain Zn2+ localization in normal neocortex and in an amyloid plaque from AD brain. (a) Timm’s stain for pools of dissociable ionic Zn2+ or low affinity-bound Zn2+ in adult rat brain. The stain detects Zn2+ within the glutamatergic synaptic vesicles in the neocortex. Basal ganglia: H, hippocamous; N, neocortex. (b) Zinc staining of amyloid plaques in AD. The plaques are stained with TSQ fluorescent stain for zinc, top panel and anti-A antibody. (Reproduced with permission from [101]).

in conjunction with glutamate, and induces cerebral A deposition in a transgenic mouse model for AD. The transporter ZnT3 carries zinc into vesicles such that its genetic ablation will inhibit A deposition in Tg2576 mice [114]. Iron may modulate APP processing, by virtue of the presence of a putative iron response element in APP mRNA (based on sequence homology). The IRE Met. Ions Life Sci. 1, 227–279 (2006)

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was mapped within the 5 -untranslated regions (5 -UTR) of the APP transcript +51 to + 94 from the 5 cap site (Figure 17). The APP mRNA IRE is located immediately upstream of an interleukin-1 responsive acute box domain +101 to + 146. In response to intracellular iron chelation, translation of APP was selectively down regulated [99], thereby causing a striking decrease in the production of APPsol . Iron influx reversed this inhibition, by a pathway similar to iron control of the translation of the ferritin-L mRNAs by iron responsive elements in its 5 -UTRs. IRP binding to the APP 5 -UTR is also increased after interleukin-1 stimulation. When the APP RNA probe is mutated in the core IRE

CAA 90 G G A G G A A C C–G G–C 80 G G G C–G G–C C–G G 100 A G–U G–C C G–C G–C 70 U–A G–C U G–C 110 C–G 5′.(66)...G–C...(35)...AUG

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Figure 17. Evidence for an iron-responsive element in the 5 -UTR of APP mRNA. APP 5 -UTR sequences were computer-folded to generate the predicted RNA stem loop. (Reproduced with permission from [98]). Met. Ions Life Sci. 1, 227–279 (2006)

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domain, IRP binding is abolished. In addition, binding of the IRP to the IRE might interfere with APP translation and translocation across the endoplasmic reticulum membrane. This interference could be significant since -secretase activity has been shown to require membrane bound APP. The role played by IRP-2 in Alzheimer’s disease remains undefined, but may be more important than was previously thought. A has a very effective binding domain for copper in its N-terminal domain and can bind copper in nmol amounts (Figure 18). It is unclear whether APP

Figure 18. Binding domain for copper in APP. A: The initial coordination site of Cu2+ on A in solution (His6 His13 His14 , and Tyr 10 ) as determined by NMR and EPR spectroscopy. B: A proposed model explaining the aggregation, cooperative binding, and redox properties of metal-bound A peptides. The imidazole ring of His6 is shown forming a bridge between copper atoms to form a dimeric species; this residue is used as an example, and other histidine residues could form similar bridges and therefore lead to aggregation. (Reproduced with permission from [171]). Met. Ions Life Sci. 1, 227–279 (2006)

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or A , when associated with copper, are in fact neuronal metallochaperones. Knockout and knockin mice for APP show that in the former, cerebral cortex copper levels are increased, whereas in the latter reduced copper levels were assayed. Copper was also influential in APP processing in the cell, copper will reduce levels of A and cause an increase in the secretion of the APP ectodomain.

6.3 Hallervorden–Spatz Syndrome Hallervorden–Spatz syndrome is an autosomal recessive trait which is relatively uncommon, characterized by progressive Parkinson-like rigidity and mental and emotional retardation [115]. Onset is in late childhood, with death occurring usually within 10 years. It can be considered as a form of iron storage disease, since both the globus pallidus and the substantia nigra have large accumulation of iron; mostly in microglia and macrophages, but occasionally in scattered neurons. Many patients with Hallervorden–Spatz syndrome have mutations in the gene encoding pantothenate kinase 2 (PANK2) [116]. Pantothenate kinase is the first enzyme in the biosynthesis of the key metabolic intermediate, coenzyme A from its vitamin precursor, pantothenate (Figure 19). PANK2 is the only one of the four human pantothenate kinase genes predicted to be targeted to, and localized within, mitochondria [117]. It is proposed that the disease may result in tissue-specific coenzyme A deficiency. This could result in accumulation of cysteine in the basal ganglia, chelation of iron and its accumulation [118,119]; the cysteine–iron complex might also promote oxidative stress, resulting in neurodegenerative damage.

6.4 Neuroferritinopathy A previously unknown, dominantly inherited late-onset basal ganglia disease, presenting with features similar to those of Huntington’s disease or Parkinson’s disease was reported in a large family from Cumbria in north west England [120]. The disease was shown to be due to the insertion of an adenine residue at position 460–461 of the gene for the ferritin light chain, which was predicted to alter the carboxy terminal sequence of the protein. The same mutation was reported in a French family [121], and a similar ferritinopathy accompanied by intranuclear and intracytoplasmic bodies in glia and neurons in the central nervous system (CNS) has been reported [122,123]. The main constituents of these bodies were ubiquinated ferritin light and heavy chains, which stained by the Perl’s reagent for ferric iron [123]. Molecular genetic studies showed the presence of a two-base pair insertion mutation in exon 4 of the ferritin light chain gene. Met. Ions Life Sci. 1, 227–279 (2006)

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O O

O– Adenine

P O O–

CH2

H3C

OH

O

C

CH

C

O NH

CH2

CH2

C

H3C

O H

H

O

OH

H

H PO32–

Coenzyme A (CoA)

Figure 19. The biosynthetic pathway of coenzyme A (CoA) from the essential vitamin pantothenate. CoA is vitally important for metabolism; acetyl-CoA is the link between the metabolism of amino acids, fats, and sugars and the citric acid cycle. (Reproduced with permission from [169]). Met. Ions Life Sci. 1, 227–279 (2006)

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6.5 Aceruloplasminemia As was pointed out earlier, ceruloplasmin is thought to play an important role as a ferroxidase, in the incorporation of iron into apotransferrin. The role of ceruloplasmin in iron metabolism was established unequivocally with the identification of aceruloplasminemia [124,125]. Ceruloplasmin is a member of the multicopper oxidase family of enzymes, mostly synthesized in liver, the major source of serum ceruloplasmin, but also in other tissues, including brain. Within the CNS, ceruloplasmin is expressed in glial cells surrounding dopaminergic melanin-containing neurons in the substantia nigra and within the inner cortex of the retina [126]. In astrocytes it is synthesized as a glycophosphatidylinositol (GPI)-anchored protein, suggestive of a role in oxidation and mobilisation of iron at the blood–brain barrier [127]. Patients with aceruloplasminemia usually present in their 40s or 50s with neurologic symptoms, a history of insulin-dependent diabetes, and retinal degeneration, all reflecting iron accumulation in most parenchymal tissues,

Cp Fe3+ Fe2+

Tf

Fe3+

Fe3+ Tf

Fp

Fe2+ macrophage

RBC

Figure 20. In the reticuloendothelial system (macrophage), the red blood cell (RBC) is phagocytosed, iron derived from heme is returned to the plasma via the membrane transporter ferroportin (Fp). Ceruloplasmin (Cp) plays an essential role in determining the rate of iron efflux via oxidation, which is required for binding to transferrin (Tf). (Reproduced with permission from [127]). Met. Ions Life Sci. 1, 227–279 (2006)

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including the basal ganglia of the brain (a 10-fold increase, with iron accumulation in both neurons and microglia). While serum ferritin is elevated, reflecting significant hepatic iron accumulation, liver function tests are normal. In macrophages of the reticuloendothelial system, effete red blood cells are captured by phagocytosis, and the iron derived from heme degradation is returned to the plasma via the membrane transporter ferroportin (Fp, also known as IREG1). Ceruloplasmin plays an essential role in determining the rate of iron efflux from the macrophages through its ferroxidase activity, oxidizing Fe2+ prior to its incorporation into apotransferrin (Figure 20). This is consistent with the clinical findings in aceruloplasminemia, with iron accumulation in parenchymal tissues because ceruloplasmin is necessary for iron efflux. Administration of ceruloplasmin to patients with aceruloplasminemia results in a rapid increase in serum iron [128,129], and the administration of the yeast homologue of ceruloplasmin restores iron homeostasis in aceruloplasminemic mice [130]. The neurologic symptoms associated with the pathogenesis of aceruloplasminemia are improved by therapy with the iron chelator desferrioxamine [131].

6.6 Friedreich’s Ataxia Friedreich’s ataxia (FRDA) is an adolescent-onset ataxia, associated with clumsiness in walking, which is accompanied by sensory loss, curvature of the spine, foot deformity and heart disease [132]. It is one of 15 neurological diseases in man which are known to be caused by the anomalous expansion of unstable trinucleotide repeats. However, in FRDA, the trinucleotide expansion occurs in a non-coding region of the gene (Figure 21). FRDA was found to be due to an expanded trinucleotide repeat (GAA) within the first intron of the gene for a 210 residue mitochondrial protein named frataxin [133]. Frataxin protein levels are severely decreased in FRDA patients. In FRDA patients, the first intron of the frataxin gene contains between 90 and 1700 GAA units. Most normal alleles carry six to nine GAA repeats, and never more than 34 repeats [133–135]. The expansion of this GAA trinucleotide repeat severely decreases the expression of the frataxin protein, reducing the quantity of frataxin mRNA produced. It has been suggested that the GAA-rich sequence is ‘sticky’ DNA, which forms a triple-helical structure, and impedes the transcription of the gene by RNA polymerase [136]. The three-dimensional structure of human frataxin consists of a stable seven-stranded antiparallel -sheet packed against a pair of parallel -helices (Figure 22) [137]. The recently determined solution structure of the yeast protein confirms this [138]. There has been considerable debate on the role of frataxin in iron metabolism, but, by far the most promising hypothesis is that frataxin is required for Met. Ions Life Sci. 1, 227–279 (2006)

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Truncating mutations [GAA] 90–1300 1A 2T 3G

C∗ C∗ T∗∗∗

L106X∗ 317deIT∗ 340deI13∗

157insC∗ 157delC∗ 202GTCA

1

TTG∗

2

IVS3 + 1G IVS362A

W155X∗

3

4

D122Y∗ L106S∗

Mis-sense mutations

IVS4 + 3 del A∗ IVS4 + 2T G∗

A∗ G∗

G130V∗∗∗∗∗∗ l154F∗∗∗ L156P∗

5a R165C∗ W173G∗∗ L182H∗ L182F∗ H183R∗

Figure 21. Frataxin mutations. The commonest mutation is the GAA expansion in the first intron of the frataxin gene (98% of cases). Boxes represent exons and bars represent introns of the frataxin gene. Asterisks indicate the number of families reported for each of the other mutations. (Reproduced with permission from [172]).

β1

α1

β2

N β3 β4 C β5

β6 β7

α2

Figure 22. Structure of frataxin. Ribbon diagram showing the fold of frataxin, a compact sandwich with -helices and -strands. Strands 1− 5 form a large flat antiparallel

-sheet, which interacts with the two helices 1 and 2. The two helices are almost parallel to one another and to the plane of the large -sheet. A second smaller -sheet is formed by the C-terminus of 5 and strands 6 and 7. (Reproduced with permission from [137]). Met. Ions Life Sci. 1, 227–279 (2006)

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iron–sulfur cluster biosynthesis, and also for heme biosynthesis. A role for frataxin in iron–sulfur cluster assembly was supported by the finding of reduced levels of iron–sulfur proteins in FRDA patients as well as in yeast and mouse frataxin-deficient models [139–142]. Frataxin appears to play an important role in this process in yeast. It is necessary for assembly of the iron-sulfur cluster into yeast ferredoxin [143] and it interacts both in vivo [144] and in vitro [145,146] with the iron–sulfur cluster scaffold proteins Isu1. Isu1 which, together with the cysteine desulfurase Nfs1, constitute the central Fe–S assembly complex. It seems reasonable to assume that frataxin supplies iron to this complex for Fe–S cluster assembly, as has been shown by in vitro experiments [146,147]. Yeast cells which lack frataxin show low content of cytochromes, and it has been suggested from genetic studies [148] and from in vitro studies with iron-containing frataxin and ferrochelatase, the enzyme which inserts iron into protoporphyrin IX [138,149,150] that frataxin may also be the donor of iron for heme synthesis.

7 EXPERIMENTAL APPROACHES TO BRAIN IRON LOADING The neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA), induce many of the pathological changes that occur in PD and are widely used as animal models of this disease. MPTP induces nigral degeneration in several species including rat, mouse, dog, cat, and monkey (reviewed in [151]). Essentially, MPTP will cross the BBB to be converted by monoamine oxidase B in glial cells to the active form, 1-methyl-4phenylpyridinium MPP+  (Figure 23). MPP+ will accumulate in dopaminergic cells prior to its entry into mitochondria, via an energy-dependent mechanism. MPP+ will inhibit mitochondrial activity leading to decreases of ATP levels, thereby inducing cell death. MPTP induces changes in iron metabolism, up-regulating the expression of both transferrin and lactoferrin receptors, as well as increasing free iron content in substantia nigra. Oxidative stress occurs which is associated with cellular changes of both GSH-GSSG ratio and cytoprotective enzymes. The loss of GSH may reflect the inactivation of glutathione peroxidase [152]. Primates treated with MPTP develop motor disturbances resembling those seen in idiopathic PD, including bradykinesia, rigidity and postural abnormalities. 6-Hydroxydopamine, a hydroxylated analogue of dopamine, is unable to cross the BBB, such that intracerebral administration is necessary to destroy nigral dopaminergic neurons in the striatum, the SN or the ascending medial forebrain Met. Ions Life Sci. 1, 227–279 (2006)

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OH• •

MPP

Oxidative Stress

Xanthine oxidase

Glial cell MPP+

Increase in free Fe2+

DAT

MPP+ MAO MPTP

• Lipid peroxidation • Protein peroxidation • DNA damages • Complex I inhibition • α-ketoglutarate dehydrogenase inhibition

MPTP

Periphery of nervous system ATP decrease

Do

Peroxinitrite increase Activation of kinases, proteases endonucleases

Blood–brain barrier

pa m ne ine uro rg n ic

Cell death

Increase in calcium cytoplasmic levels (indirect excitotoxicity)

Figure 23. Hypothetical mechanism of MPTP toxicity. MPTP injected peripherally, crosses the BBB and is transformed by glial monoamine oxidase (MAO) into the active compound MPP+ . Once inside the cells, MPP+ will inhibit the respiratory chain, cause oxidative stress, both triggering cell death. DAT = dopamine transporter. (Reproduced with permission from [151]).

bundle (reviewed in [151]) (Figure 24). The iron content of SN and striatum is increased, which will participate in Fenton chemistry to generate reactive oxygen species to exacerbate the lesion. The importance of iron in the exacerbation of this lesion can be verified by the facts that iron-deficient rats are resistant to 6-OHDA-induced damage [152,153], and iron chelating agents can prevent 6-OHDA-induced deleterious effects [154]. It has been difficult to engender an animal model which shows selective accumulation of iron in specific brain regions. In normal rats the blood–brain barrier will normally restrict iron uptake. However, the orally administered compound 3,5,5-trimethyl hexanoyl ferrocene, will rapidly traverse the BBB, its iron moiety being concealed between the two aromatic rings, releasing and increasing iron content of various brain regions, including SN, cerebellum and cerebral cortex, by approximately 50%, after four weeks of administration. This animal model has been utilized to test the ability of a range of chelators to reduce brain iron content [96]. Met. Ions Life Sci. 1, 227–279 (2006)

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Dopamine

Ferritin-Fe3+ Melanine

6-OHDA

6-OHDA

Auto-oxidation

Ferritin-Fe3+ Fe2+ Quinones

Fe2+

•

OH

H2O2

Oxidative Stress

H2O2 MAO

H2O2 6-OHDA

•DNA •Lipid

defects peroxidation disorganization

•Cytoskeleton

•Complex I inhibition •Oxidation phosphorylation

uncoupling •Mitochondrial membrane potential collapse

DAT 6-OHDA

Cell death

ATP decrease

Figure 24. Hypothetical mechanism of 6-OHDA toxicity. This is induced by (a) reactive oxygen species generated by intra- or extracellular auto-oxidation, (b) hydrogen peroxide formation induced by MAO activity, or (c) direct inhibition of the mitochondrial respiratory chain. (Reproduced with permission from [151]).

In our early study [96] intraperitoneal injection of either bidendate (e.g., deferiprone), or the hexadendate (desferrioxamine = DFO) iron chelators (Figure 25) to ferrocene-loaded rats, reduced excess iron from a variety of brain regions, with no apparent adverse effect on brain iron metabolism. Dopamine metabolism was adversely affected by both groups of chelators in this study. Tridendates (e.g., desferrithiocein) were also efficient at removing iron from various brain regions in the ferrocene-loaded brain and most importantly did not interfere with dopamine metabolism [97]. Both 6-OHDA and MPTP increase the iron content of the substantia nigra pars compacta (SNPC) in rats and monkeys [155–159] and in mice [160,161]. Various factors are implicated in iron accumulation, which include up-regulation of DMT1 [162], down regulation of transferrin receptors, loss of IRP-2 activity, and iron release from ferritin [163]. Many compounds have been evaluated in these two animal models for their iron-chelating properties and antiinflammatory action. In the MPTP model, desferal [161,162] and green tea catechin ((-)-epigallactocatechin-3-gallate) all show iron chelating properties and, as such, are potent neuroprotective agents against Met. Ions Life Sci. 1, 227–279 (2006)

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O OH N OH O (a) Deferiprone

S N

N N OH

N

N

O OH

OH

HO

(b) Desferrithiocin and ICL670

O H N

O

N O

O

N OH

OH

OH

O

N

NH2

N H

(c) Desferrioxamine

Figure 25. Iron chelators, past and future therapeutic agents for the removal of iron from neurodegenerative diseases where excessive iron accumulation occurs. (a) Bidendates: deferiprone; (b) tridendates: desferrithiocein and ICL670; (c) hexadendates: desferrioxamine.

MPTP. DFO administration either by intracranial pretreatment or intraperitoneally prevented 6-OHDA-induced changes in dopaminagic neurons [162]. Another iron chelator, the lipophilic VK-28, (5-[4-hydroxyethyl) piperazine1-ylmethyl]-quiniline-8-ol) (Figure 26), traverses the blood–brain barrier to prevent 6-OHDA-induced malondialdehyde (MDA) production, without altering dopamine metabolism [163]. IRP-1 knockout mice show misregulation of iron metabolism in the kidney and brown fat, the two tissues in which the endogenous expression of IRP-1 exceeds that of IRP-2 [164]. IRP-2 knockout mice, have dysregulated iron metabolism associated with cytosolic iron accumulation (possibly as ferritin) in neuronal axons, particularly striatum, which in normal circumstances Met. Ions Life Sci. 1, 227–279 (2006)

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CH2

269

N

N

CH2CH2OH

N OH

VK-28

OH OH O H

HO

OH

H OH

O OH

O

OH OH Epigallactocatechin-3-gallate

Figure 26. Iron chelators of possible therapeutic use: (-)-epigallactocatechin-3-gallate and VK-28.

has high IRP-2 content. Degradation of ferritin occurs in lysosomes, which could lead to increased release of free iron and oxidative damage. These animals exhibit movement disorders which include ataxia, tremor and bradykinesia. Mice that are homozygous for a targeted deletion of IRP-2 and heterozygous for a targeted deletion of IRP-1 IRP + /-IRP2-/- develop severe neurodegeneration, characterized by widespread axonopathy and vacuolisation in several brain regions, including SN. Ultimately neuronal cell bodies degenerate in the SN activating microglia. Mice show gait and motor impairment when axonopathy is pronounced [165]. There is considerable evidence for abnormal metal homeostasis in AD. The presence of the 5 -UTR in the APP transcript may indicate that APP is a metalloprotein. Iron chelation has been proposed as a possible therapeutic approach for the treatment of AD. Indeed, as already stated earlier, APP was selectively down-regulated in response to iron chelation [98]. Chelation may also prevent iron-induced inflammation as well as A aggregation. The therapeutic advantages Met. Ions Life Sci. 1, 227–279 (2006)

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of various chelators, such as clioquinol (a copper chelator), desferrioxamine (an iron chelator) as well as polyphenols, e.g., epigallactocatechin-3-gallate (EGCG) and curcumin (a constituent of turmeric) [166], should be assessed in suitable animal models, for as yet, there are no details of changes in iron metabolism in any of these transgenic AD animal models.

8 CONCLUSIONS A logical therapeutic target to retard iron accumulation in specific brain regions would be chelation therapy. The problem is to get metal chelators to reach the appropriate target regions in the brain, where they will complex the iron and diffuse back out as the metal-chelate, without either interfering with the irondependent enzymes or by redistributing brain iron such that previously nontoxic iron becomes toxic by being moved to a different brain compartment. We showed some years ago, in iron-loaded rats, that while chelators were able to cross the blood–brain barrier, and reduce the levels of iron in specific brain regions, some of them also interfered with the metabolism of the neurotransmitter dopamine [97]. Increasing efforts are being made to target chelators, or to use the more sophisticated term, metal-protein-attenuating compounds (MPACs), to cross the blood–brain barrier. Recent examples include incorporating chelators in nanoparticles [167] and the use of lipophilic brain-permeable molecules and polyphenols (reviewed in [2]). Some success has been reported with the copper chelator clioquinol (5-chloro-7-iodo-8-hydroxyquinoline) in a phase 2 clinical trial in Alzheimer’s disease [168].

ACKNOWLEDGMENTS We gratefully acknowledge the support of the COST Chemistry Action D13 ‘New Molecules for Human Health’.

ABBREVIATIONS -CTF AD ADP AICD APO-TF APP AR-JP A BBB

-carboxy terminal fragment Alzheimer’s disease adenosine 5 -diphosphate amyloid precusor protein intracellular domain apotransferrin amyloid precursor protein autosomal recessive juvenile parkinsonism amyloid peptide blood–brain barrier

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CAPPD CASK CHIP CNS complex 1 Cp CSF Dcytb DFO DMT 1 E3 EGCG EPR Fp FRDA GH GPI GSH GSSG HNE HOLO-Tf Hsp40 Hsp70 IBR IRE IREG 1 IRP LB LC L-DOPA LGP LIP MAO MDA MPAC MPP+ MPTP MRI MTP1 MW NADH NFT NMDA

271

central APP domain class II PDZ protein, mammalian homologue of C. elegans Lin-2 carboxyl terminus of the Hsc70-interacting protein central nervous system NADH-coenzyme Q reductase ceruloplasmin cerebrospinal fluid duodenal cytochrome b desferrioxamine divalent metal transporter 1 ubiquitin protein ligase epigallactocatechin-3-gallate electron paramagnetic resonance ferroportin (=IREG1) Friedreich’s ataxia genetic hemochromatosis glycophosphatidyl inositol reduced glutathione oxidized glutathione 4-hydroxy-2-nonenal diferric transferrin heat shock protein of molecular weight 40 kD (a chaperone) heat shock protein of molecular weight 70 kD (a chaperone) in-between RING iron-regulatory element iron-regulated gene protein 1 (also known as ferroportin) iron-regulatory protein Lewy body locus coeruleus dihydroxyphenylalanine lateral globus pallidus labile iron pool monoamine oxidase malondialdehyde metal-protein-attenuating compound 1-methyl-4-phenylpyridinium 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine magnetic resonance imaging metal transporter protein 1 (=Fp, ferroportin, IREG1) molecular weight nicotinamide adenine dinucleotide (reduced) neurofibrillary tangle N -methyl-d-aspartate Met. Ions Life Sci. 1, 227–279 (2006)

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6-OHDA Pael-R PANK2 PD Pi PPi PS1 PTP 1 RING RNS ROS SIAH-1 SIAH-2 SN SNPC SOD Tf TFR THAL TSQ Ubl UCHL 1 UTR

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6-hydroxydopamine putative G-protein-coupled receptor protein pantothenate kinase 2 Parkinson’s disease inorganic phosphate inorganic diphosphate (=pyrophosphate) presenilin-1 protein metal transporter protein 1 (same as IREG1, ferroportin) really interesting new gene domain reactive nitrogen species (including nitric oxide and peroxynitrite) reactive oxygen species E3 ubiquitin-ligase (seven in absentia homologue) E3 ubiquitin-ligase (seven in absentia homologue) substantia nigra substantia nigra pars compacta superoxide dismutase transferrin transferrin receptor thalassemia quinoline congener which gives a fluorescent complex with Zn ubiquitin-like domain ubiquitin carboxy-terminal hydrolase untranslated region of an mRNA

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11 The Chemical Interplay between Catecholamines and Metal Ions in Neurological Diseases Wolfgang Linert,1 Guy N. L. Jameson,1 Reginald F. Jameson,2 and Kurt A. Jellinger3 1

Institute of Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9/163-AC, A-1060 Vienna, Austria <[email protected]> 2

3

Emeritus Professor, University of Dundee, Scotland, UK

Institute of Clinical Neurobiology, Kenyongasse 18, A-1070 Vienna, Austria

1 GENERAL INTRODUCTION 2 NEURODEGENERATIVE DISEASES 2.1 General Description 2.2 Specific Examples 2.2.1 Parkinson’s Disease 2.2.2 Alzheimer’s Disease 2.2.3 Huntington’s Disease 2.2.4 Amyotrophic Lateral Sclerosis and Other Diseases 2.2.5 Tauopathies 3 RELEVANT IN VITRO CHEMISTRY 3.1 The Catecholamines 3.2 Iron(III) and Catecholamines 3.2.1 Iron(III) and Noradrenaline 3.2.2 Iron(III) and 6-Hydroxydopamine 3.3 Iron(III), Catecholamines, and Oxygen 3.4 The Sulfur Content of Melanin Metal Ions in Life Sciences, Volume 1 © 2006 John Wiley & Sons, Ltd

Edited by Astrid Sigel, Helmut Sigel and Roland K. O. Sigel

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3.5 6-Hydroxydopamine and Ferritin 4 IRON AND PARKINSON’S DISEASE 4.1 General Features 4.2 Regional and Selective Increase of Brain Iron 4.3 Sources of Iron 4.4 Increased Iron and Parkinson’s Disease 4.5 Oxidative Stress 4.6 Treatment of Parkinson’s Disease with L-Dopa 4.7 Neuromelanin and Iron 4.8 Neuromelanin and Proteins 5 RELEVANT MANGANESE CHEMISTRY 6 MANGANESE AND MANGANOSIS 6.1 Exposure to Manganese 6.1.1 Relationship with Parkinson’s Disease 7 OTHER METAL IONS AND CATECHOLAMINES 7.1 Copper(II) 7.2 Nickel(II) 7.3 Vanadium 7.3.1 General Considerations 7.3.2 VO2+ , Adrenaline, and Oxygen 8 SUMMARY OF THE EFFECT OF METAL IONS ON AUTOXIDATION OF DOPAMINE 9 CONCLUSIONS ACKNOWLEDGMENTS ABBREVIATIONS AND DEFINITIONS REFERENCES

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1 GENERAL INTRODUCTION Many metal ions are as essential to life as other constitutive elements within living organs, such as fats, carbohydrates, and proteins. Today, research into the role of metal ions in neurological disorders is of particular interest because while many metal ions, such as Na, K, Ca, Mg, Fe, Co, Mn, Cu, and Zn are vital to the smooth functioning of cells, various organs and tissues, many are also harmful when not contained. Toxicologists and chemists have performed extensive research into the harmful properties of metal ions largely within the laboratory, while environmental Met. Ions Life Sci. 1, 281–320 (2006)

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disasters, such as Minamata and Camelot, have provided large amounts of data within specific populations. Currently, much attention is paid to the role of metal ions as pathogenic agents for several neurological disorders, in particular neurodegenerative diseases such as Parkinson’s, Huntington’s, and Alzheimer’s diseases, and amyotrophic lateral sclerosis to mention only a few. Theories that suggest metal ions are essential etiological agents in many neurodegenerative diseases have induced much progress in the research of their hitherto unknown molecular biological backgrounds, as for instance, recently documented [1].

2 NEURODEGENERATIVE DISEASES 2.1 General Description Neurodegenerative diseases are chronically progressive nervous disorders, morphologically described by progressive, often long-lasting, loss of specific vulnerable neuronal populations in the central and peripheral nervous system associated with gliosis and frequently with typical cytoskeletal protein aggregates forming intracytoplasmic and/or intraneuronal inclusions in neurons and/or glial cells. The reasons for such specific damage are largely unknown as are the general disease mechanisms and hence neurodegenerative diseases have traditionally been defined in terms of clinicopathological entities. A common feature of these conditions is a long run-in period until sufficient protein accumulates, followed by a cascade of symptoms over many years, with increasing disability finally leading to death. Recent molecular biological and genetic investigations have revealed that there are both overlap and intra-individual diversities between different entities in pathogenetic mechanisms. Genes, proteins, and metal ions involved in these conditions are being increasingly elucidated and hence the naming of the conditions after the essential toxic or deposited protein has become an option [2]. Most neurodegenerative disorders are now classified according to the major composition of their filamentous protein inclusions. Table 1 summarizes the most frequent neurodegenerative diseases according to current molecular biological criteria as well as their major morphology and essential clinical features.

2.2 Specific Examples The only disease that has been unambiguously related directly to metal ions (iron) and catecholamines is Parkinson’s disease and so this chapter deals almost exclusively with this condition. However, for the sake of completeness we will now describe the important clinical features of this and several related neurodegenerative diseases. Met. Ions Life Sci. 1, 281–320 (2006)

Mutated gene

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17q (Exon 10)

MAPT

MAPT

MAPT

MAPT

Pick’s disease

Progressive supranuclear palsy

Corticobasal degeneration

Polymorphism, H1/H1 genotype

Mutation Exon 10

Mutation Exon 10

21, 14, 1

APP, PS1, PS2

Alzheimer’s disease/ familial Frontotemporal dementia + parkinsonism (FTDP 17)

19

Chromosome

Tau (4R)

Tau (4R)

Tau (3R) Tau 3 + 4R

Tau 3 + 4R

 amyloid Tau 3 + 4R

 amyloid Tau 3 + 4R

Toxic protein, protein deposit

H1/H1 genotype

Advanced age

MAPT mutation

MAPT haplotype

ApoE  4

Risk factor

Multiple system degeneration, ballooned neurons

Multiple system degeneration, subcortical tau deposits

Lobarfrontotemporal atrophy; Pick bodies, Pick cells

Frontotemporal brain atrophy, nigral degeneration, tau deposits in neurons and glia

-Amyloid deposits (plaques, CAA); tau deposits (neurofibrillary tangles, threads); synapse and neuron loss, brain atrophy

Morphology

Classification of neurodegenerative diseases according to molecular biological criteria.

Tauopathies ± amyloid Alzheimer’s ApoE  4 disease/ sporadic

Disease

Table 1.

Parkinsonism, dementia, supranuclear gaze palsy Parkinsonism, apraxia, dementia

Frontal dementia

Dementia, behavioral disorders, aphasias, parkinsonism

Loss of higher cortical functions; memory loss; progressive dementia

Clinical features

284

Multiple system atrophy (MSA)

Dementia with Lewy bodies (Parkinson with dementia)

Parkinson’s disease sporadic

Parkinson’s disease juvenal/familial

Synucleinopathies

UCHL 1

Unknown

-Synuclein

Not identified

Striatonigral/ olivopontocerebellar atrophy, glial cytoplasmic inclusions

Cortical and subcortical Lewy bodies, nigral degeneration, Alzheimer pathology

ApoE  4

-Synuclein

-Synuclein

Subcortical (and cortical) Lewy bodies

Parkin

Not identified

Degeneration striatonigral and other systems

-Synuclein

SNCA polymorphism

1, 2, 4, 6, 12

SNCA (-synuclein), parkin, UCHL 1 PINK 1

.

Parkinsonism (MSA-P), cerebellar ataxia (MSA-C), autonomic disorders

Progressive dementia, parkinsonism, visual hallicinations, neurologic sensitivity

Instability

Rigidity, akinesias, tremor

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(Continued)

IT 15 HD huntingtin

AR (androgen receptor), CAG repeats

Ataxin; SCA 1-19, CAG repeats

Frataxin (PFRDA 1)

SCA 3HSD 1

Kennedy disease

Spinocerebellar ataxias

Friedreich’s ataxia

Machado-Joseph atrophy

Mutated gene

Huntington’s disease

Polyglutamine repeat extension

Disease

Table 1.

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2, 9, 15, 18, 21, 6, 8, 11, 14

4

Ataxin

Frataxin

Ataxin TB protein

Dysarthria, ataxia, cerebellar symptoms

Not identified Degeneration spinocerebellar tracts, intra-nuclear aggregates

Ataxia spasticity dysarthria nystaginus

Dysarthria, Not identified Degeneration cerebellar and spinal paraparesis, myocardiopathy posterior tract, demyelination spinal roots

Not identified Degeneration cerebellar, cortex and spinal posterior columns

Sensory Not identified Degeneration mot. neuropathy, neurons brainstem (VII, XII) and spinal reduced fertility cord, AR inclusions in neurons

AR

Clinical features

Not identified Striatal degeneration, Choriform hyperkinesias, HD nuclear and dementia cytoplasmic inclusions

Morphology

Huntingtin

Chromosome Toxic protein, Risk factor protein deposit

286

Creutzfeldt–Jakob disease (CJD) sporadic

Prion diseases

Amyotrophic lateral sclerosis sporadic

Amyotrophic lateral sclerosis familial

Motoneuron diseases

Dentatorubropallidoluysian atrophy (DRPLA)

PRNP 129 MM

ALS1-X

SOD-1

B-39

Multiple mutations

2, 9, 15, 18, 21

12

Proteaseresistent PrPres

SOD-1 (Cu/Zn SOD)

Atrophin-1

.

ppp polymorphism

Not identified

Not identified

Not identified

Progressive dementia, cerebellar symptoms, myoclonus, blindness

Spastic paraparesis bulbar syndrome

Ubiquitin + hyalin cell and nucleus inclusions

Spongiform encephalopathy, prion plaques

Spastic paraparesis bulbar syndrome

Ataxia, choriform movements, myoclonus, epilepsy

Degeneration central and periferal motoneurons

Atrophy of putamen, gl. pallidus, subthalamic nucleus, rubro, dentate nucleus; atrophin-1 inclusions in neurons and astroglia

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BRI

SER4 PPRO

Familial British/Danish dementia (familial)

Familial encephalopathy with neuroreserpin inclusions (FENIB)

Others

Codon 129

Creutzfeldt–Jakob disease (CJD) familial Variant CJD (nvCJD)

PRNPCodon 129

Mutated gene

(Continued)

Disease

Table 1.

13

20

Chromosome

Serpin 1 (neuroreserpin)

ABri/ADan

PrP protease resistent Protease-resistent PrPsc

Toxic protein, protein deposit

Not identified

Not identified

Not identified

Risk factor

Collins bodies in nucleus and cytoplasm in cortex, subcortex and white matter

Argyrophilic amyloid plaques and neurofibrillary tangles (hippocampus), cerebral amyloid angiopathy

Spongiform encephalopathy, diffuse PrP plaques, PrP in lymph nodes

Morphology

Dementia, restlessness, seizures, myoclonus

Dementia, sleep disturbances, ocular disorders

Depression, anxiety, dysarthria, dementia

Clinical features

288

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2.2.1 Parkinson’s Disease In Parkinson’s disease (PD), the most frequent extrapyramidal movement disorder in adults, neuronal loss in the substantia nigra (SN) and other subcortical nuclei is associated with widespread occurrence of cytoplasmic inclusions in subcortical and, less frequently, in cortical neurons, Lewy bodies, and neurites, formed from fibrillary -synuclein, parkin, and many other filamentous proteins [3,4]. Several epidemiological studies have implicated environmental factors with an increased risk of PD, in particular the association with long-term exposure to transition metals, such as iron, copper, and manganese, suggesting that exposure to external sources of metal may be involved in the etiology of PD [5]. Frequent Lewy bodies in both subcortical and cortical neurons occur in dementia with Lewy bodies (DLB), the second most frequent type of adult age dementia and parkinsonism, but DLBs are also seen in many other neurodegenerative diseases. In multiple-system atrophy (MSA), a sporadic movement disorder with parkinsonism and cerebellar dysfunctions, subcortical multi-system neuronal degeneration is associated with -synuclein containing cytoplasmic filamentous glial inclusions, called Papp–Lantos bodies, in oligodendroglia, and intraneuronal inclusions.

2.2.2 Alzheimer’s Disease In Alzheimer’s disease (AD), the most common type of dementia in advanced age, with genetically determined familial forms in around 5–7%, loss of cortical synapses and neurons is accompanied by extracellular deposition of A amyloid peptide in senile plaques and cerebral vessels (cerebral amyloid angiopathy). This loss is also accompanied by cytoskeletal lesions with paired helical filaments containing hyperphosphorylated microtubule-associated tau protein in neurons (neurofibrillary tangles), axons and dendrites (neuropil threads), and around amyloid forming dendritic plaques.

2.2.3 Huntington’s Disease In Huntington’s disease (HD), an autosomal dominant hyperkinetic disorder due to mutations of the IT-15 gene on chromosome 4p16.3 with expanded polyglutamine CAG repeats, neurodegeneration in man as well as in transgenic mice and mainly involving the striatum is associated with intranuclear and cytoplasmic neuronal aggregates of truncated ubiquitinated huntingtin, chaperons, and proteasomes [6]. Two main mechanisms of aggregation have been proposed: hydrogen bonding by polar-zipper formation or covalent bonding by transglutaminasecatalyzed cross-linking. In cell culture models of HD, aggregates are mostly stabilized by hydrogen bonds, but covalent bonds are also likely to occur. Met. Ions Life Sci. 1, 281–320 (2006)

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Nothing is known about the nature of the bonds that stabilize the aggregates in the brains of patients with HD and other neurodegenerative disorders. It seems that the nature of the bonds stabilizing the aggregates is one of the most important pathogenic questions [7]. Similar inclusions are seen in other rare autosomal dominant atypic polyglutamine disorders, e.g., dentatorubropallidoluysian atrophy (DRLPA) and various types of spinocerebellar ataxia (SCA), suggesting that these aggregations are a common pathogentic feature of the glutamine repeat neurodegeneration [8].

2.2.4 Amyotrophic Lateral Sclerosis and Other Diseases In amyotrophic lateral sclerosis (ALS), an adult neurodegenerative motorneuron disorder related to interactions of superoxide dismutase 1 (SOD 1) and neurofilaments, the neurons contain skein-like intranuclear ubiquitinated inclusions. In familial ALS, a group of dominantly inherited disorders, SOD 1 precipitates have been found [9,10]. The nature, time course, and molecular causes of cell degeneration and demise, the basic processes resulting in neurodegeneration, their relations with metal ions and to abnormal cytoskeletal protein aggregations are currently a matter of considerable scientific debate. Since similar processes seem to affect neurons of adults and older individuals, each producing a form of neurodegeneration, it is possible that certain common features are present that affect specific groups of proteins, and recent studies have provided insight into the basic processes common to neurodegeneration and into cell death programs in neurodegenerative disorders [11,12].

2.2.5 Tauopathies Another group of neurodegenerative disorders, referred to as tauopathies, shows cytoplasmic or intranuclear inclusions mainly composed of different biochemical forms of tau protein. Progressive supranuclear palsy (PSP) and corticobasal degeneration (CBC), two mainly sporadic rigid-akinetic movement disorders with dementia, show multisystem neuronal degeneration associated with neuronal and glial inclusions mainly composed of 4-R (repeat) tau protein, while AD and related disorders show neuronal inclusions containing 3 and 4 tau repeats. In Pick’s Disease, a rare presenile type of frontotemporal dementia (FTD), progressive cortical degeneration is associated with Pick bodies, cytoplasmic accumulation of 3-R tau protein, differing from that in AD and other tauopathies [13]. Other FTDs, such as frontotemporal dementia parkinsonism related to mutations of chromosome 17 (FTDP-17) also show different neuronal and glial tau inclusions due to mutations at exons 9–13 [14]. Met. Ions Life Sci. 1, 281–320 (2006)

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3 RELEVANT IN VITRO CHEMISTRY 3.1 The Catecholamines The catecholamines are an important class of organic molecules derived from catechol and tyrosine (Figure 1) that have many important roles to play in biological systems, for example as neurotransmitters. This is an outline of aspects of their interaction with metal ions that are important in the biochemical environment. Before dealing with specific catecholamines and metal ions we begin by a general survey of the topic: The catecholamines (Figure 1) contain two functional groups capable of coordinating metal ions, but only independently (so-called ambidentate ligands). The catechol function is particularly interesting as (a) it is capable of chelating a large number of metal ions and (b) it is relatively readily oxidized via two one-electron steps to a quinone that often reacts further by involving the side-chain amino group. These two reactions are often linked when the complexed metal ion is a transition metal in a higher oxidation state; for example, iron(III) forms complexes with the catechol function followed by one-electron transfer to yield the semiquinone and iron(II). Further oxidation by the iron(III) to yield the quinone follows (Figure 2). These redox reactions are of special importance in biological systems and so details of them form a large part of this chapter. If the metal ion is in a lower oxidation state, e.g., iron(II), then it can often bind dioxygen and thus act as a catalyst for the oxidation of the catecholamines (Figure 3). This reaction is a very important feature of the chemistry of the brain, leading as it does to considerable damage unless its features can be mitigated.

HO

HO +

NH3

HO

HO

dopamine

6-OHDA

CO2–

HO

NH3+

HO dopa

NH3+ OH

OH HO HO

OH HO

H3C

adrenaline (epinephrine)

NH2+

HO

NH3+

noradrenaline (norepinephrine)

Figure 1. The catecholamines under discussion: dopamine, 6-hydroxydopamine (6-OHDA), 3,4-dihydroxyphenylalanine (dopa), adrenaline (epinephrine), and noradrenaline (norepinephrine). Met. Ions Life Sci. 1, 281–320 (2006)

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HO HO

NH3+

+ M(n+1)+

O

+2H+

M(n+1)+ O

+M(n+1)+

O O

–2H+

NH3+

+ Mn+ fast

NH3+

O –

NH3+

O

+ Mn+

Figure 2. The general mechanism at low pH for the oxidation of catecholamines by transition metal ions, where overall charges have been omitted because they are pH dependent. Initial formation of a mono-complex followed by internal electron transfer. The semiquinone formed reacts rapidly with another metal ion to form the quinone.

O Mn+ O

NH3+

+

O O2 Mn+ O

O2

NH3+

O O

NH3+

+

Mn+

+

H2O2

Figure 3. Oxidation of catecholamines to quinones by dioxygen using a metal ion as catalyst, where overall charges have been omitted because they are pH dependent. The reactive species is a ligand–metal-ion–dioxygen complex.

Again this feature of the chemistry of the catecholamines is dealt with in some detail below. After oxidation to the quinone, however, the role of the side-chain is very important. The amino group can undergo internal Michael addition to form an indole ring (leucodopaminochrome, Figure 4) and, what is highly significant, regenerates the catechol function. This enables further oxidation (e.g., by initially binding a metal ion to the new catechol site, see Figure 2). The final molecular product is another, brightly colored quinone, e.g., dopaminochrome is formed from dopamine (Figure 4). Further oxidation leads to the formation of a crosslinked polymer known as melanin. The amino-containing side-chain also has important roles to play with respect to the binding of metal ions: the side-chains of catecholamines such as dopa and adrenaline contain additional binding sites (e.g., –OH) as well as the −NH2 making them chelating sites. Some metal ions, e.g., Cu2+ , bind equally well with Met. Ions Life Sci. 1, 281–320 (2006)

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Michael Addition

O

Oxidation O

HO

O +

NH3

dopaminoquinone

HO

293

N H

leucodopaminochrome

HO

N

dopaminochrome

Figure 4. Ring closure of the side-chain in dopaminoquinone codopaminochrome, which is further oxidized to dopaminochrome.

yields

leu-

both sites, often leading to polymer formation [15–18]. However, the binding of metal ions to the side chain seems to play no direct role in biochemical reactions, although it is important to note that these studies help to throw light on the binding sites in proteins.

3.2 Iron(III) and Catecholamines The simple aqua-cations of iron(III) are highly insoluble at physiological pHs and iron(II) is unstable with respect to oxidation to iron(III) and therefore, any iron present in biological systems must be complexed before it can be transported and utilized. Solubilization is maintained by the presence of a pool of low molecular weight complexes lying between where the iron is stored and where it is to be employed. Little is known about the chelators involved, but in mammals citrate seems a likely candidate, with nucleotides and amino acids also being suggested [19]. The reaction of the catecholamines with aqueous iron(III) has been the subject of many detailed kinetic studies [20–27] and, in general, they follow the reaction schemes presented in Figures 2 and 3 above. As well as the aqua ion, Fe3+ , the hydroxyanion, FeOH2+ , also reacts with the catecholamine to form the complex. What is particularly interesting is that these reactions are reversible and hence, oxidation can be prevented by the addition of a large excess of iron(II). The bis-complexes are of interest on two grounds. Firstly, they are unexpectedly stable towards internal electron exchange (indeed, catechol based bis-complexes were often used in the past for the colorimetric determination of iron(III)). Secondly, some bis-complexes of iron(III) bind dioxygen and, in some cases, even lead to the catalytic oxidation of the ligand [28]. Both of these effects are explicable by the fact that the catechol function is non-innocent (a term due to C. K. Jørgensen) and this means that the oxidation state of the central metal ion is made indeterminate because of internal electron transfer, and indeed, is acting in many ways as iron(II). At considerably higher pHs > 8, the also highly colored and relatively stable tris-complexes are formed, but the high pH required for their formation precludes their having any biological function. Met. Ions Life Sci. 1, 281–320 (2006)

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To date, two catecholamines, namely noradrenaline and 6-OHDA, have been shown not to follow the route of oxidation by iron(III) outlined above, i.e., via complex formation. The behavior of these two catecholamines will now be detailed separately.

3.2.1 Iron(III) and Noradrenaline A detailed kinetic study of the reaction between iron(III) and noradrenaline reported in 1998 by El-Ayaan et al. [25] was the first example of deviation from the basic kinetic scheme of initial formation of a complex prior to electron exchange. The kinetics were explained on the basis of the redox reaction taking place by parallel inner- and outer-sphere reactions, as illustrated in Figure 5. These authors also presented a detailed kinetic scheme for the subsequent formation of the indole ring. HO HO

–2H+

OH NH3+

+ Fe(III)

O Fe(III) O

+2H+

OH NH3+

Internal electron transfer

Outer sphere OH

O

NH3+

O +Fe(III)

fast

O O

+ Fe(II)

OH NH3+

+ Fe(II)

Figure 5. The oxidation of noradrenaline by iron(III) involves two parallel pathways at low pH, one involving complex formation, the other by outer-sphere electron transfer.

3.2.2 Iron(III) and 6-Hydroxydopamine The reaction via complex reaction is, in the case of 6-OHDA, virtually nonexistent – the complex is only detected as a transient species when the concentrations are very high. The reaction between iron(III) and 6-OHDA has been studied [21–23] over a wide pH range by means of NMR, spectroscopy, and stopped-flow methods. It was established that at low pH values three ketones are produced, namely the o-, p- and tri-ketones, and that these are all deprotonated Met. Ions Life Sci. 1, 281–320 (2006)

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HO NH3+ OH

HO

+

2Fe(III)

Outer-sphere redox reactions

HO

O

O

O O

NH3+

O

NH3+

OH

O

O

NH3+

Deprotonations O –O

O

NH3+

Figure 6. The reaction of 6-OHDA with iron(III) at low pH occurs almost exclusively by outer-sphere reaction and yields three quinones. All three deprotonate at physiological pH to yield the same quinone.

at physiological pHs (i.e., at pH ca. 7) to yield a single deprotonated quinone. These reactions are summarized in Figure 6. As will be detailed later (see Section 4.5, Figure 9), the reaction at pH 7 is extremely important in cell chemistry because 6-OHDA can be produced by a Fenton reaction [29–31] (involving iron(II) and H2 O2 ) and is able to reduce the iron in ferritin. The iron(II) can then be removed from the ferritin thus completing a redox cycle (see below).

3.3 Iron(III), Catecholamines, and Oxygen The catalytic oxidation by dioxygen of L-dopa has been studied by Martell and Jameson [28] and Jameson [32], but a complete quantitative interpretation of the kinetic data proved too difficult. The instrumental and computational methods available in the 1960s were just not up to requirements. However, three distinct pH ranges could be identified. At low pH values the reaction proceeds, as expected, by prior internal electron transfer to yield iron(II) which then acts as the catalyst whereas at higher pH values (ca. 5.5) the bis-complex of iron(III) also forms a complex with dioxygen, because the two bound noninnocent ligands transfer sufficient charge to the central iron to make it behave as iron(II) bound to two semiquinone radicals. Finally, at even higher pH values (ca. 7), the consumption of O2 ceases well above zero concentration and resaturation with oxygen results in a stable mixture. This is explicable on the basis that the accompanying precipitate of dark-brown melanin has the iron bound within the matrix, making it completely inaccessible. Met. Ions Life Sci. 1, 281–320 (2006)

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The fact that the iron(III) is trapped within this complex, cross-linked polymer might in fact provide a role for melanin in successfully isolating iron from the cell. However, the surface of this polymer is made up of many reactive fragments such as free radicals and catechol functions. The interaction of these surface fragments with metal ions has been widely investigated, e.g., with copper and zinc by Farmer et al. [33] and with iron by Double et al. [34]. The redox properties of neuromelanin are especially important because reducible ions can promote the dissolution of the melanin. Youdim et al. [35] draw attention to the fact that if iron(III) is the oxidant then the resulting iron(II) species could be responsible for the progression of PD via Fenton-like reactions producing hydroxyl radicals. As suggested below, one of the species that would result could well be 6-OHDA and this could possibly be the main species responsible for iron release from ferritin. In any case, it is perhaps ironic that ‘iron overload’ within the SN of parkinsonian patients might be the cause of the progression of the disease and the concomitant release of the iron from its protective sheath of melanin (the bleaching of the cells of the SN is one of the observable features of PD).

3.4 The Sulfur Content of Melanin 5-Cysteinyl-dopamine (5-cys-DA) is found in certain dopaminergic regions of the mammalian brain [36–38], including the caudate nucleus, putamen, globus pallidus, and SN. Indeed, chemical degradation of neuromelanin in the SN has suggested that 5-cys-DA is in fact a precursor of melanin [39–41], via its copolymerization during the oxidation of dopamine. During the progression of PD, however, increased ratios [42] of 5-cys-DA over dopamine are observed in the SN. It has therefore been postulated that, rather than being involved in melanin production, the formation of 5-cys-DA diverts production. In fact, with increasing molar excess of cysteine, the formation of melanin in vitro is decreased and ultimately stopped [39]. Moreover, due to its lower redox potential, 5-cys-DA can further oxidize and cyclize intramolecularly to form toxic dihydrobenzothiazines as shown by Zhang and Dryhurst [43]. Although 5-cys-DA can be formed by nucleophilic attack of cysteine on dopaminoquinone (Figure 7) and the mechanism has been studied in detail [44–47], concentrations of cysteine in the brain are generally low. Alternatively, glutathione, present in much higher concentrations, could attack to form 5-glutathionyl-dopamine which could then undergo enzymatic cleavage by -glutamyl transpeptidase and peptidase to form 5-cys-DA. Evidence for the presence of 5-glu-DA has not been found, but elevated activity of -glutamyl transpeptidase has been reported [48] in the SN of those patients that suffered from PD. Met. Ions Life Sci. 1, 281–320 (2006)

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HO HO

cysteine

O NH3+

HO dopamine

+

NH3+

HO

NH3

S

dopaminoquinone

+H N 3

O

CO2–

5-cysteinyldopamine glutathione peptidase 5-glutathionyldopamine γ-glutamyl transpeptidase

Figure 7. Dopaminoquinone can react with cysteine to form 5-cys-DA. This product can also arise from the reaction of dopaminoquinone with reduced glutathione followed by enzymatic cleavage of the bound tripeptide.

3.5 6-Hydroxydopamine and Ferritin By making use of the intense color of the deprotonated quinone of 6-OHDA, Jameson et al. [49] were able to demonstrate that this catecholamine can enter the ferritin and reduce the iron present, reaching redox equilibrium. Importantly, they also demonstrated that this alone did not explain the liberation of iron from this storage protein – an external complexing agent is required to remove the iron(II) produced, disturb the equilibrium within the protein, and thus continuously remove iron (Figure 8). It has been shown that dopamine itself is able to fulfil this role [50], but as has been pointed out above there are many other potential complexing agents present within the cell (e.g., citrate, amino acids, and nucleotides) that could also fulfil this role. These observations would further appear to add credence to the idea [35] that the neurotoxin 6-OHDA has an essential role to play in the progression of PD.

Figure 8. Schematic diagram illustrating the entrance of 6-OHDA into ferritin and reaching redox equilibrium with the iron core. This equilibrium is disturbed if a complexing agent is present. Met. Ions Life Sci. 1, 281–320 (2006)

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4 IRON AND PARKINSON’S DISEASE 4.1 General Features Parkinson’s disease, a brainstem type of Lewy body disease, is the most common neurodegenerative movement disorder in the elderly, with progressive degeneration of the dopaminergic nigrostriatal system and other neuronal networks caused by loss of pigmented neurons in the SN, zona compacta, and many subcortical nuclei associated with widespread occurrence of Lewy bodies [3]. The initiation of neurodegeneration may occur many years prior to the development of symptoms. Recent data point to a cascade of multiple noxious factors, including oxidative stress, formation of free radicals, proteomic stress, neuromelanin–iron interaction, impaired bioenergetics, mitochondrial dysfunction and nuclear DNA deficits and interaction between these factors, the two major primary and interrelated mechanisms implicated in the etiology of PD being oxidative pathology and mitochondrial dysfunction. Increments in indices of oxidative damage to proteins, lipids, and DNA, and correlated changes in the endogenous antioxidative systems are associated with neuronal damage in the PD brain. Age-related changes include alterations that favor increase in oxidative damage, such as pro-oxidative changes in mitochondrial pathways, decrease in antioxidant protective systems such as the glutathione system and changes in the cytoplasm, calcium concentration and membrane potential. Changes in mitochondrial function, in particular inhibition of complex I activity, have been proposed to be causally related to cell death in PD [51]. Complex I activity is readily restricted by oxidative damage, and the resulting change in respiratory function further stimulates oxidative mechanisms [52]. A common factor linking oxidative damage that is also believed to underly the Lewy body formation, and mitochondrial dysfunction, is the presence of a significant and pathological increase in the amount of iron in the degenerating SN [53].

4.2 Regional and Selective Increase of Brain Iron Iron is unevenly distributed within the brain, with the highest levels in the basal ganglia (substantia nigra, putamen, caudate nucleus, globus pallidus, red nucleus, and dentate nucleus [54]). Entry of iron into the mature brain is tightly regulated and, unlike other tissues, its turnover is extremely low and serum iron has little access to this organ [55]. Once inside the brain, iron is highly sequestered within organic storage forms, such as ferritin, with relatively little in a free and reactive form. Increase in brain iron is associated with a variety of neurodegenerative diseases and neurotoxin-induced neurodegeneration, suggesting pathogenic associations [56]. Increased regional brain iron has been identified in parkinsonian syndromes, such as PD, progressive supranuclear palsy, multiple system degeneration, in Met. Ions Life Sci. 1, 281–320 (2006)

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trinucleotide repeat disorders, e.g., Huntington’s disease, dentatorubropallidoluysian atrophy, and in dementia disorders, e.g., Alzheimer’s disease, and dementia with Lewy bodies. In these disorders, a striking feature of changes is the close association between increased tissue iron and degenerating brain region. In movement disorders, iron levels are increased in the basal ganglia, in AD it is associated with the pathological hallmarks (plaques and tangles) in the cortical regions [57,58]. In PD, significantly increased iron levels are only found in the degenerating SN [59], while in other movement disorders increased iron is also seen in other parts of the basal ganglia [60]. Total iron contents in most brain areas do not differ between PD and control brains [54], but an increase in total iron levels of about 77% was measured in PD substantia nigra compared to controls [61]. This increase was confined to the SN zona compacta, the region containing vulnerable dopaminergic neurons, but does not occur in the pars reticulata [62]. These data were confirmed by many research groups using different methods such as metal ion detection by inductively coupled plasma mass spectroscopy, X-ray microanalysis, histochemical techniques, etc. [63,64], and also by MRI studies, showing accumulation of iron in the nigrostriatal system in patients with PD [65,66].

4.3 Sources of Iron The sources of localized increased iron in PD brain are unknown, but several possibilities have been discussed. Firstly, the increase might result from an increased entry of peripheral iron into the SN via an altered blood–brain barrier, as observed in local inflammatory processes. Alternatively, rather than being the result of some pathological process, increased iron might enter the SN via normal iron regulatory systems. The major iron transport site, transferrin, is not up-regulated in SN in the brain, although specific polymorphisms in the transferrin gene have recently been associated with PD [67]. Another ironbinding protein, lactoferrin, is reported to be upregulated in surviving PD SN neurons [68] and increased numbers of receptors for this protein are present on PD SN neurons [69]. The translation of many proteins involved in iron regulation is controlled by two cytoplasmic proteins, called iron-regulatory proteins 1 and 2 (IRP 1 and 2), that bind to stem loop structures called iron-responsive elements of the protein’s mRNA. If iron is depleted, IRP binding results in decreased synthesis of proteins involved in iron storage and mitochondrial metabolism, such as ferritin and mitochondrial aconitase, and increased protein synthesis, such as the transferrin receptor, involved in iron uptake. However, whereas these proteins remain unchanged in the SN of PD [70], disturbances in iron metabolism are suggested to occur at multiple levels in PD [59], and may reflect genetic differences in iron-regulatory mechanisms [67]. Nevertheless, to Met. Ions Life Sci. 1, 281–320 (2006)

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date, no convincing explanation for the selective increase in iron in PD SN is available. Another possibility is that increased levels of iron might be derived from within the brain itself. SN is directly connected with the globus pallidus, one of the most iron-rich regions, via afferent -aminobutyric acid (GABA) pathways. However, no currently known mechanisms exist to explain movement of iron from one brain region to another. A further possibility is that the measured increases in iron reflect not an elevation in the total iron content, but rather redistribution from other cellular sources, e.g., increased nigral glial cells. Glia, predominantly oligodendroglia, but also microglia and astrocytes, are the major source of the iron storage protein ferritin. An increase in the number of iron-positive glial cells is associated with nigral degeneration in PD [64]. Despite increased numbers of ferritin-positive glial cells, total nigral ferritin concentration appears to be unchanged in PD [71,72]. The expression of ferritin mRNA has been shown to be increased in glial cells adjacent to degenerating SN neurons in PD, but no changes in binding activities of associated IRPs were detected within the PD dopaminergic neurons [70]. Histochemically, iron could not be demonstrated within the neuromelanin-containing neurons in SN, intracytoplasmic melanin, or extracellular melanin granules in the neuropil [63], although Lewy bodies contain iron [73,74]. Ferritin-bound iron results in oxidative, mediated membrane damage [75], and a dysregulation of ferritin homeostatic systems appears to occur in PD [71].

4.4 Increased Iron and Parkinson’s Disease The question of the temporal association between increased iron and neurodegeneration in PD is unclear. Earlier authors suggested that the increase in iron might be a late or secondary event in the disease process; Dexter et al. [76] reported unchanged nigral iron levels in cases of so-called incidental Lewy body disease, representing an early preclinical state of PD. Their conclusion that increased iron only occurs in advanced stages of neurodegeneration was supported by unchanged SN iron concentrations in ‘mild’ PD cases, assessed semi-quantitatively as a mild nigral neuronal loss [54]. However, more recent work using transcranial ultrasound indicates that increased iron can be demonstrated in the SN in PD patients even prior to the onset of clinical symptoms [77], suggesting that iron increase might occur earlier than previously thought. While both the sources and timing of increased iron in PD SN are still controversial, its presence appears to be involved in the two primary pathogenic pathways associated with neurodegenerative changes in PD, oxidative stress and Lewy body formation. Met. Ions Life Sci. 1, 281–320 (2006)

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4.5 Oxidative Stress The ‘oxidative stress’ hypothesis proposes an imbalance between the formation of cellular oxidants and the antioxidative processes. Decreased concentrations of antioxidant molecules such as reduced glutathione (GSH) through reduced activity of various enzymes including glutathione peroxidase and catalase occur in SN in PD. The early loss of systems designed to protect the brain, together with a site-specific increase of superoxide dismutase and monoamine oxidase (MAO) activity, may play an important role in priming the SN for oxidative damage in PD [78,79]. Oxidative stress, resulting from increased formation of hydrogen peroxide and oxygen-derived free radicals, can damage biological molecules and initiate a cascade of events, including dysfunction of mitochondrial respiration, excitotoxicity, and a fatal rise in cytosolic calcium [52]. Hydrogen peroxide is produced in human tissues by several enzymes, e.g., superoxide dismutase, l-amino acid-oxidase, glycollate oxidase, xanthine oxidase, and MAO. In dopaminergic nerve cells, it is mainly generated by MAO via deammination of dopamine, and non-enzymatically by auto-oxidation of dopamine. The interaction of hydrogen peroxide with the reduced forms of transitional metal ions, such as Fe(II) or copper(I) decomposes hydrogen peroxide to the highly reactive hydroxyl free radical via the Fenton reaction. In addition, hydroxyl radicals are produced in the mitochondria of nerve cells during oxidative phosphorylation. Hydroxyl radicals have a strong reactivity with almost every molecule found in living cells. These reactions include breakage of DNA, chemical alterations of the deoxyribose purine and pyrimidine bases, membrane lipids, and carbohydrates, leading to a cascade of events with subsequent damage to the mitochondrial electron transport system, disturbance of intracellular calcium homeostasis, induction of proteolysis by proteases, increased membrane lipid peroxidation, release of excitotoxic amino acids (glutamate, aspartate), and finally, cell death (reviewed in [3,79]). Several experimental models and human post-mortem studies in PD support the hypothesis that oxidative stress contributes to the loss of dopaminergic systems in PD [79]. Not only is the total nigral iron content increased in PD, but the ratio of Fe(II) to Fe(III) shifts from two to one in controls to one to two in PD brains [61]. This indicates an increased rate of synthesis of hydroxyl radicals. Postmortem studies also reported increased basal levels of thiobarbituric acid-reactive substances in SN of PD, a secondary product of lipid peroxidation, coupled with a decrease in polyunsaturated fatty acids, the substrates for lipid peroxidation [79]. In addition, there may be DNA damage as indicated by increased 8-hydroxydeoxyguanosine [80], a product of free-radical attack on guanidine in DNA. Further support for oxidative stress in PD comes from experimental models, e.g., the iron chelator desferrioxamine (desferal) and vitamin E protect rats against the 6-OHDA induced reduction of striatal dopamine content and decrease of dopamine-related spontaneous locomotor activity. Met. Ions Life Sci. 1, 281–320 (2006)

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The above findings indicate that the prevention of 6-OHDA-induced degeneration that is believed to induce nigrostriatal dopaminergic lesions via generation of hydrogen peroxide and hydroxyl radicals derived from it, presumably initiated by a transition metal such as iron. MRI and neurochemical and histochemical studies have shown that iron is increased in the striatum of 6-OHDAlesioned rats [81], and in vitro studies have shown that 6-OHDA reduces iron within ferritin enabling it to be removed by external complexing agents [49,82], while intranigral injection of Fe(III) produces neurotoxic effects similar to those observed with 6-OHDA [83]. In the MPTP model of parkinsonism, ADP depletion and ROS (reactive oxygen species) overproduction appear to occur soon after MPTP (1-methyl-4-phenyl1,2,3,6-tetrahydropyridine) application, subjecting the intoxicated cells to an early energy crisis and oxidative stress. Among the essential molecular pathways that are pivotal in triggering cell-death-related cascades, alterations in ATP synthesis and ROS production lead to the demise of the affected neurons [84]. Based on the reactions discussed above, it seems to be possible to produce a diagram that describes the reactions that can induce PD. This is depicted in Figure 9. Starting from the oxidation of dopamine by oxygen via dopaminoquinone and finally to melanin we know that peroxide is produced. Fluorescence spectroscopic investigations into unfixed postmortem brain materials show that

Figure 9. A plausible chemical explanation for the continuous production of cytotoxins during neurodegeneration. Met. Ions Life Sci. 1, 281–320 (2006)

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peroxides are present in the brain [85]. If any free iron(II) ions are present, the peroxide will react with dopamine via Fentons reaction to form 6-OHDA, which may, in the presence of a suitable chelator remove iron from ferritin (see Section 3.5). The quinones thus produced are able to then react with nucleophiles such as cysteine or glutathione to form toxic dihydrobenzothiazines (see Section 3.4). This instigates a series of cycles capable of continuously propagating the various cytotoxins.

4.6 Treatment of Parkinson’s Disease with L-Dopa Since its introduction in the late 1960s l-dopa or ‘levodopa’ (see Figure 1) has been generally accepted as the most effective treatment for the alleviation of the symptoms associated with PD. The effective chemical species required is dopamine, but being charged it cannot cross the membrane barrier: dopa is uncharged and can cross into the cells where it is decarboxylated to yield the required dopamine. However, not only is it unpleasant to take the large doses required, but it also gives rise to serious side effects (although these can be minimized by adding a blocker for decarboxylase and using the l-form of dopa exclusively). With reference to the cycle of events that may lead to the progression of PD (Figure 9), it can be shown that a continual increase in the dopamine content of the cells will result in ever increasing yield of the neurotoxin 6-OHDA. This has been confirmed by an examination of the urine of patients [86]. Thus, it is not surprising that levodopa treatment usually becomes inefficient after a few years and indeed assists in the advancement of the disease and accelerates the death of the patient. But it must not be forgotten that this therapy gives an extremely important improvement to the quality of life.

4.7 Neuromelanin and Iron Histochemical studies ascribe the localization of iron in the SN primarily to the glial compartment and changes in glia-associated ferritin have been described in PD. While some iron sequestration appears to occur in -synuclein in Lewy bodies, the importance of this iron for neurodegeneration is unknown. A more important source of intraneuronal iron involved in the oxidative pathway is neuromelanin, which characterizes the dopaminergic neurons in the human SN. Neuromelanin occurs as granules, possibly inactivated lysosomes, within the neuronal perikaryon. Neuromelanin is a dark polymer pigment produced in specific populations of catecholaminergic neurons in the brain. Interest in this pigment has increased in recent years because of a hypothesized link between neuromelanin and the special vulnerability of neuromelanin-containing neurons in cell death in PD. Unlike peripheral melanins, which are produced in specialized cells called melanocytes and may be transferred to other cell types, neuromelanin granules are stored in the cells in which they are produced. Met. Ions Life Sci. 1, 281–320 (2006)

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Neuromelanin granules display a unique, more heterogeneous appearance compared with peripheral melanin and are traditionally thought to result from a nonenzymatic synthetic pathway with no known pathway for neuromelanin catabolism. Recent data [34,56] indicate some regulations of neuromelanin synthesis and turnover. In the apparent absence of a role of tyrosinase in neuromelanin genesis, the search for an enzyme associated with neuromelanin production has yielded no likely candidate to date. An important role for neuromelanin is in binding (or encapsulating) metals, particularly potentially toxic cations such as iron, zinc, copper, manganese, chromium, cobalt, mercury, lead, and cadmium [87]. In particular, the interaction between iron and neuromelanin has been a focus of research because a marked accumulation of iron related to disease severity is reported in PD SN [54]. The cellular location of this apparent increase in iron is unclear, but a variety of changes in iron regulatory systems, such as the major iron-binding protein ferritin, occur in PD. As ferritin is primarily located in glia, rather than neurons [88], it is unlikely that this protein could regulate neuronal iron levels. By binding metals, neuromelanin may potentiate free radical formation or assist with protective scavenging of hydroxyl metals [89]. Reduction of neuromelanin in PD is likely to render the cell more susceptible to oxidative damage, and the level of redox activity detected in neuromelanin aggregates was significantly increased in PD and was highest in patients with most severe neuronal loss [90]. This change was not observed in tissue in the immediate vicinity of melanized neurons. Studies using synthetic dopamine–melanin have demonstrated that the binding of iron to neuromelanin is pH and concentration dependent and appears to be specific. Fe(III) could only be displaced from dopamine–melanin by compounds with iron-chelating capacity, such as desferrioxamine, but not by dopamine, spiperone, MPTP, and apomorphine [83]. These and other data obtained from a melanin model, concur with data from purified human neuromelanin isolated from the SN of controls, which exhibit Mössbauer spectra corresponding to highspin Fe(III) but not Fe(II) [91]. The capacity of neuromelanin to bind iron appears to be considerable, and is estimated to represent up to 20% of the total iron content in SN [92]. In SN tissue, neuromelanin is only about 50% saturated with Fe(III) suggesting that it maintains an important residual chelating capacity which can protect against iron toxicity [93]. Mössbauer spectrometry demonstrated the chelation of ferric iron by the neuromelanin polymer and suggested that the iron sites are arranged in a ferritin-like iron oxyhydroxide cluster form [91,92] and infrared spectroscopy confirmed that iron in neuromelanin is bound to oxygenderived phenolic groups in an octahedral configuration [94]. It is suggested that iron bound to neuromelanin represents a significant pool of intraneuronal iron inside the vulnerable dopaminergic neurons [92], and neuromelanin is thought to participate in scavenging metal-induced free radicals within the healthy brain. Neuromelanin can be considered as an endogenous iron-binding molecule in pigmented neurons [64,92,95]. It may therefore play a physiological role in Met. Ions Life Sci. 1, 281–320 (2006)

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intraneuronal iron homeostasis. Support for this theory comes from changes in neuromelanin in PD brain, where significantly less iron is bound to neuromelanin than that seen in normal brains [96]. This suggests that changes in iron binding to neuromelanin result in increased levels of intraneuronal free iron and in the subsequent cell damage observed in PD. Under pathological conditions, such as increased availability of Fe(III) in PD, neuromelanin is suggested to potentiate the formation of oxygen-derived radicals [97], and significantly enhanced cerebral membrane damage can be demonstrated in the presence of a neuromelanin Fe(III) complex [98]. Neuromelanin appears to be able to potentate or inhibit formation of oxygen-derived free radicals, playing a dichotomous role, depending on the cellular environment. The increased formation of a neuromelanin–Fe(III) complex in SN [64] is consistent with the hypothesis that neuromelanin acts as an intraneuronal pool of iron, which subsequently contributes to free radical production or other deleterious pathways. The absence of significant quantities of alternative iron-binding molecules, such as ferritin, in pigmented neurons, together with increased antioxidant capacity in the PD SN, suggest that an increase in the amount of iron available to interact with neuromelanin may result in interactions exerting a substantial influence upon oxidative-mediated pathways and, thus, cell survival. Whilst the cause of the increased iron concentration in the PD SN is unclear, the interaction of the metal with cellular constituents, such as -synuclein and neuromelanin, appears to be important for the development of the disease and oxidative-mediated neurodegeneration. These pathways may not represent the initial trigger of the disease process, but may reflect alterations in iron homeostasis and represent secondary, but important mechanisms involved in the progressive course of the disease. Recent data suggest that the development of iron-chelating substances, suitable for use in the CNS (central nervous system) may provide novel possibilities for therapeutic intervention in PD and other neurodegenerative disorders associated with increase in central iron levels.

4.8 Neuromelanin and Proteins In addition to binding metals, neuromelanin also contains and interacts with proteins. Recent studies of neuromelanin using 13 C-NMR demonstrated that the spectral pattern of neuromelanin isolated from PD brain exhibits a comparatively decreased melanin signal than that from control neuromelanin. This appeared to be due to the presence of a novel protease-resistant, lipoproteinic material not seen in the healthy brain [99]. Recent work has further shown that -synuclein, one of the major components of Lewy bodies in PD, is incorporated into the neuromelanin granule [100], suggesting an important interaction between neuromelanin and this lipoprotein. -Synuclein is a lipoprotein thought to be important in the transporting of diverse proteins and lipids [101]. Neuromelanin Met. Ions Life Sci. 1, 281–320 (2006)

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consists of 5–15% protein [89,102], and it will be important to determine all neuromelanin–protein interactions [103].

5 RELEVANT MANGANESE CHEMISTRY Although considerable work has been reported on the Mn(II)-catalyzed oxidation of dopamine, the results have proved difficult to unambiguously interpret. Herlinger [104] followed the reaction by use of an oxygen electrode and it is interesting to note that unlike iron [28] and vanadium [105] all the oxygen was consumed without precipitation of melanin. Thus, it appears that Mn(II) is a true catalyst over a large pH range and more importantly is not trapped within melanin. Herlinger [104] was able to establish that the reaction proceeds via initial formation of an LMnIIO2 complex, which is a strong oxidising agent, reacting with an LMn(II) complex to yield 2 Mn(III) complexes and H2 O2 . LMnIIO2 also reacts with free ligand to give LMn(III) and quinone and H2 O2 . Using EPR to follow semiquinone formation, Lloyd [106] also suggests the involvement of an Mn(III) species, but this takes the form of LMnIIIO2 as the reactive intermediate. However, Tyson and Martell in 1971 [107], came to the conclusion that in reactions with 3,5-di-tert-butylpyrocatechol no involvement of Mn(III) was indicated. Garner and Nachtman [108] have also shown that Mn(II) can initiate a Fentontype reaction with the peroxide produced and hence (as is the case with iron(II)), give rise to cytotoxic 6-OHDA (see Section 4.5).

6 MANGANESE AND MANGANOSIS 6.1 Exposure to Manganese Exposure to manganese can result to CNS damage that can be assessed by postmortem (neuropathologic) examination, neuro-imaging methods, use of biological markers, and neurological examination. Pathological studies indicate that brain damage from manganese involves multiple brain regions. In experimental animals chronically treated with manganese the brain lesions were diffuse, not only involving the basal ganglia but also the cerebral cortex as well as the cerebellum [109].

6.1.1 Relationship with Parkinson’s Disease Autopsy studies of patients who had suffered from manganism showed consistent atrophy of the basal ganglia with loss of neurons and gliosis. The lesions tend to Met. Ions Life Sci. 1, 281–320 (2006)

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focus in the pallidoluysian system, although the striatum and thalamus may be equally affected [110,111]. A review of previous autopsy cases by Yamada et al. [112] reported the main neuropathological change as a degeneration of the basal ganglia with pallidal lesions found in all examined cases. However, the caudate nucleus and putamen were often affected, whilst the SN, which is predominantly (exclusively) affected in PD, was only slightly damaged. Similar changes were reported in experimental manganese intoxication with the pallidum and subthalamic nucleus found with major lesions [113]. These observations claimed that there are some differences in the neuropathology between chronic manganese intoxication (CMI) and PD, a fact recently emphasised by Olanow [114]. Distribution of Mn in the brain in autopsy cases of CMI using flameless atomic absorption spectrometry showed no significant differences in average concentration between CMI, a PD case, and controls although there were some changes in the distribution, suggesting that the continuation of neurological disorders in CMI is not linked to elevated Mn concentrations in the brain [112]. Involvement of the SN and red nucleus has been reported in some cases of CMI, while Lewy bodies, a hallmark of PD, were identified in SN in one case. However, it is unclear whether or not the patient had PD [115]. More recent neuroimaging studies of symptomatic patients in CMI showed prominent involvement of the globus pallidus, sparing the dopaminergic system [116], while a recent study of a woman with severe liver failure and symmetric hyperintensity of the globus pallidus in MRI images showed increased serum manganese levels, and neuropathological examination of the globus pallidus was consistent with manganese neurotoxicity [117]. Recent positron emission tomography (PET) studies in chronic Mn intoxication, using 99m Tc-TRODAT-1, a tracer of dopamine transporter binding, showed a mild decrease of its uptake in the putamen and the ratio of putamen and caudate when compared with normal controls. Although the 99m Tc-TRODAT-1 shows a slight decrease in the putamen of manganese patients, the data indicate that the presynapic dopaminergic terminals are not the main target of CMI [118]. In workers from a ferromanganese factory in Taiwan who showed a progressive parkinsonian syndrome after exposure to elevated Mn concentrations, PET studies indicated early effects on presynaptic structures in the nigrostriatal system, including striatum and pallidum [119,120]. Follow-up of five patients with CMI and prominent rigidity, gait disturbances, foot tapping, and writing disorders, revealed that manganese levels had returned to normal and MRI did not show increased T-1 intensities, suggesting that clinical progression continues even 10 years after cessation of Mn exposure [121]. Subsequent PET studies showed preservation of pre- and postsynaptic nigrostriatal dopaminergic functions in patients exposed to manganese [122]. A population-based study on occupational exposure to several heavy metals and the risk of PD conducted in the Henry Ford Health Service revealed a lack of statistical significance for those with low prevalence of exposure, while these Met. Ions Life Sci. 1, 281–320 (2006)

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findings suggested that chronic occupational exposure to manganese or copper is associated with PD [123]. The pathophysiology of Mn has been reviewed by several authors, e.g., Normandin and Hazell [124], emphasizing that manganese induces damage to mitochondria and is a stimulation cofactor of DNA and RNA polymerases, inducing errors of DNA synthesis. Manganese exposure can play an important role in causing parkinsonian disturbances, possibly enhancing physiological aging of the brain in conjunction with genetic predisposition. An increased environmental burden of manganese may have deleterious effects on more sensitive subgroups of the population, with subthreshold neurodegeneration in the basal ganglia, generating a preparkinsonian condition.

7 OTHER METAL IONS AND CATECHOLAMINES 7.1 Copper(II) Most examinations of copper complexes with the catecholamines are confined to studies of the complexes formed. For example, Jameson and Neillie [15] and Gorton and Jameson [16–18] demonstrate that these complexes involve coordination to the side-chain as well as the catechol function leading to polymer formation. Like iron, copper is present in the gray matter of the brain and subserves many physiological functions. It is an essential element for the activity of a number of physiologically important enzymes. Enzyme-related malfunctions may contribute to severe neurological diseases: copper is an essential transition metal ion for the function of key metabolic enzymes, but its uncontrolled redox activity is a source of reactive oxygen species [125]. Copper is a component of cytochrome c oxidase, which catalyzes the reduction of oxygen to water, an essential step in the cellular respiration. Coppermediated oxidative stress is linked to mitochondrial dysfunction, which is a common feature of neurodegenerative disorders. Copper is a cofactor of Cu, Znsuperoxide dismutase which plays a key role in the cellular response to oxidative stress by scavenging reactive oxygen species, and is a constituent of dopamine -hydroxylase, a critical enzyme in the catecholamine pathway. Copper-binding enzymes play important roles in the establishment and maintenance of metal-ion homeostasis, in deficiency disorders with neurological symptoms (Menkes disease, Wilson’s disease) and neurodegenerative disorders (Alzheimer’s disease). Menkes and Wilson’s diseases (see Chapter 9) are inherited disorders of copper metabolism and the dramatic neurodegenerative phenotypes of both disorders underscore the essential nature of copper in nervous system development and the toxicity of the metal when neuronal copper Met. Ions Life Sci. 1, 281–320 (2006)

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homeostasis is perturbed. Ceruloplasmin contains 95% of the copper found in human plasma and inherited loss of the essential ferroxidase is associated with progressive neurodegeneration of the retina and basal ganglia [126]. Menkes and Wilson proteins have been characterized as copper transporters, and the amyloid precursor protein (APP) of AD has been proposed to work as a copper(II) transporter [127]. Copper-mediated oxidative stress is involved in neurodegenerative processes [126,128] with special impact on familial forms of amyotrophic lateral sclerosis. Furthermore, a possible role of copper reactivity in inducing critical steps in the apoptotic pathway leading to neurodegeneration is suggested [129,130] to occur in human brain during adult life [131]. It exists as free ions or bound to proteins; free ions and some bound ions can participate in oxidation–reduction (redox) reactions. Accumulation of redox active copper is central to many neurotoxicological hypotheses because it acts as catalyst for Fenton chemistry and catechol autoxidation, and can participate in redox chemistry with -amyloid peptides [132]. Copper(II) dependent A -mediated inhibition of human cytochrome c oxidase has been suggested to be an important contributor in the neurodegenerative process in Alzheimer’s disease [133]. Iron inhibits neurotoxicity induced by trace copper in a high reducing environment which may have implication for trace biometal interactions and free radical-mediated damage in neurodegenerative disorders, such as Alzheimer’s disease [134].

7.2 Nickel(II) As is the case with copper(II), nickel also often involves both coordination sites of catecholamines [135], although the amino side chain is here the most important.

7.3 Vanadium 7.3.1 General Considerations Vanadium is of biological importance because of its possible involvement in the manic depression cycle [136]. It forms complexes in oxidation states 2, 3 VOV4+  4 VO2+ , and 5 VO+ 2 , but the most detailed study has involved its role as a catalyst in the oxidation of the catecholamines by molecular oxygen (q.v.) – this work [105] also enabled some stability constants to be determined. Vanadium is an element classified in the group of heavy metals, very common in the natural environment, mainly in the heavy oils, carbon and bituminous materials, where it is associated with the heavy fraction. Vanadium emissions to the atmosphere are produced in areas around siderurgical industries, oil refineries, and cities that use fossil fuels for heating. Some clinical–epidemiological Met. Ions Life Sci. 1, 281–320 (2006)

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researches report a high incidence of congenital malformations of the CNS that could be associated with vanadium compounds [137]. In higher concentrations, vanadium may induce acute or chronic intoxication that damage biological structures and disorder biochemical systems. The toxic mechanism is suggested to be related, among others, to vanadium properties able to hinder a number of enzymatic systems. Vanadium salts may also be geotoxic and harmful at different phases of reproduction and development [138]. For example, vanadium in high concentrations may disturb phosphorus metabolism of nervous tissue with successive concentration-dependent decrease of alkaline and phosphatase activites in tissues, and inhibition of phosphoryl transfer reactions [139]. Recent studies revealed significant correlations between the vanadium levels in urine and serum of vanadium exposed subjects and cognitive deficits and neurobehavioral abilities, particularly visuospatial abilities and attention, measured by neuropsychological test batteries [140]. Cytological analysis in mice after inhalation of vanadium (sacrifice 1–8 weeks after inhalation) revealed drastic decrease of dendritic spines and of tyrosin hydroxylase-immunoreactive cells in the SN pars compacta, which decreased in a time-dependent mode. These findings may have functional importance after increased vanadium inhalation [141].

7.3.2 VO2+ , Adrenaline, and Oxygen Jameson and Kiss [105] decided that the vanadyl(IV) ion, VO2+ , would yield simpler kinetics than iron(III) in that the former is capable of two-electron transfer reactions. Adrenaline was used as the catecholamine since its protonation is considerably simpler than that of dopa. The complicated experimental rate equation was interpreted in terms of a series of reactions similar to those for the iron–dopa system for which rate and equilibrium data were calculated. The essential steps are: firstly, reduction of VO2+ via formation of the mono-complex yielding V(II) and the quinone by a two-electron step (there is no indication of the formation of free radicals during these reactions). This is followed by formation of the V(II) complex which then binds O2 , this leading to electron transfer from the catecholamine through the metal ion (see Figure 3) apparently yielding the quinone and peroxide in one step. However, the bis-complex (Figure 10) also binds dioxygen and two electrons are transferred through the complex (i.e., L2 VOO2 → L + Q + O2− 2 ). This almost certainly implies that the binding of the ligands not only leads to an

O O O V

HO +

CH3H2N

O

O

OH +

NH2CH3

Figure 10. Structure of the bis(adrenaline)vanadyl(IV) complex. Met. Ions Life Sci. 1, 281–320 (2006)

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120 Na2S2O4 added Percentage saturation of O2

100

VO 2+ added

80

60 more VO 2+ added 40

20

re-oxygenated

0 0

100

200

300

400

Time/mins

Figure 11. The consumption of oxygen upon addition of catalytic amounts of VO2+ to a solution of adrenaline shows that consumption ceases before all the dioxygen is used up. Addition of more VO2+ again leads to a premature cessation of consumption. Addition of dioxygen now leads to a stable solution. This shows the effect of the trapping of VO2+ within the precipitated melanin which is bright blue. (pH = 7 1 VO2+ = 1 77 × 10−4 and 0 98 × 10−4 M adrenaline = 10 mM).

effective reduction in the oxidation state, but also leads to a weakening of the V=O bond. It should be noted, however, that a study of the dopa-vanadyl system by Thompson [142] led him to postulate that the bis-complex involved V(III) and not VO2+ (this study, however, was not completed). It is important to note that, as is the case with iron(III), the reaction at about pH 7 ceases well above zero concentration of O2 and re-saturation with oxygen results in a stable mixture. This is illustrated in Figure 11 and it is interesting that the melanin precipitate is bright blue which suggests that it is VO2+ that is trapped within the melanin matrix.

8 SUMMARY OF THE EFFECT OF METAL IONS ON AUTOXIDATION OF DOPAMINE Table 2 shows the relative effects of metal ion catalysis on the autoxidation of dopamine. It is interesting that copper reacts via a half-order metal dependence [143] and this arises because, although the reaction is triggered by the Met. Ions Life Sci. 1, 281–320 (2006)

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Table 2. The effect of metal ions 100 M on the autoxidation of dopamine dopamine T = 10 mM pH = 7 60. Metal Ion Fe(II) Fe(III) Cu(II) VO2+ Ni(II) Mn(II) Co(II) a b

104 kobs s−1  5.6 6.7 8 6a 10.6 17.2 29 13 7a 46 2b

Half-order rate constant units 10−5 M1/2 s−1 . Possible half-order contribution.

initial electron transfer within a Cu(II) complex, the resulting Cu(I) does not bind to the catechol function but rather to dioxygen to form CuO+ 2 which is the active species. Note especially that at the relatively high pH of 7.6 all the metal ion species react considerably slower than at pH < 5. This is because it is mainly the biscomplexes that exist at higher pH.

9 CONCLUSIONS Metal ions are intrinsically linked to neurological disorders. As outlined above (Sections 2, 4, and 6) there is much evidence for a link between long term exposure to transition metals and several disorders. Furthermore, there are observable changes in metal ion distribution during and after the onset of several diseases. So although just one of many factors to be investigated, an understanding of metal ion interactions is very important and indeed, in this chapter we have tried to show that the chemistry of metal-ion catecholamine interactions has helped enormously with the understanding of the molecular basis of neurodegenerative diseases. However, there are still few clear-cut answers. This is in part due to the catecholamines themselves having a complicated pH dependent redox chemistry with many possible reaction pathways. Even initial complex formation depends upon the metal ion concerned, with iron and manganese binding exclusively to the catechol function while the softer copper and nickel bind also to the amino group. If bound to the catechol group to form the mono-complex (at low pH), internal electron transfer can occur (depending on the metal ion concerned) and the complex then breaks up with formation of the more easily oxidizable semiquinone. Met. Ions Life Sci. 1, 281–320 (2006)

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However, 6-OHDA and noradrenaline are also oxidized by iron(III) before formation of a colored complex due to the low redox potentials of these catecholamines. With the exception of 6-OHDA, at higher pHs stable bis and tris complexes hinder electron transfer through ‘non-innocent’ ligands. This ability of 6-OHDA to be oxidized by iron(III) at all pHs seems to explain the neurotoxicity of this compound in particular. The inherent instability of 6-OHDA means that if it is produced in vivo (as it can be through the in vitro oxidation of dopamine), then it will be present in extremely low concentrations. The specificity of the damage observed in PD, concentrated in neurons with high dopamine concentrations, and the long time over which the damage is caused means that such a mechanism seems feasible. The relative redox stability of the bis and tris complexes means that metal ion-catalyzed oxidation of the catecholamines by dioxygen becomes important. These reactions have been shown to be very complicated, with true catalysis often not occurring, and this is an area open to far more research. Especially since the conditions are oxidizing enough to lead to complete oxidation of the catecholamine to the insoluble polymer melanin. There, metal ion interactions are again important and still poorly understood. In vivo, these reactions become yet more complicated due to the complexation of the metal ion, affecting its redox potential, and the presence of nucleophiles such as cysteine can attack the intermediate quinone leading to complicated polymers, i.e., neuromelanin. Despite their limitations, it does seem that the study of the in vitro chemistry of the interactions of metal ions with catecholamines has been able to shed considerable light on the probable reactions taking place in vivo. Now is the time for these studies to be refined in such a way as to approach more closely the conditions appertaining to real biological systems. In concluding this chapter we would like to draw attention to a more general problem directly related to such investigations. Based on our own experience, both scientists studying in vitro and in vivo systems need to be aware that the results presented from each side can often be apparently contradictory although at the same time internally consistent. One of the problems in this respect is that of communication. It is very important that both sides find a common language to bring their results under one roof before they can come a little closer to describing the actual chemistry of biological systems.

ACKNOWLEDGMENTS The financial support of the ‘Jubiläumsfond’ of the Austrian National Bank (Project 10668) and stimulating discussions with many colleagues within the COST D8 and D13 programmes over many years are gratefully acknowledged. Met. Ions Life Sci. 1, 281–320 (2006)

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ABBREVIATIONS AND DEFINITIONS 5-cys-DA 5-glu-DA 6-OHDA ABri/ADan AD ADP ALS APP AR ATP BRI CAA CAG CBC CJD CMI CNS DLB dopa DRLPA FTDP-17 FTD GABA GSH HD IRP MAO MAPT MPTP MRI MSA PD PET PRNP PrPsc PrPres PS 1 + 2 PSP ROS

5-cysteinyl-dopamine 5-glutathionyl-dopamine 6-hydroxydopamine amyloid peptides associated with familial British/Danish dementia Alzheimer’s disease adenosine 5’-diphosphate amyotrophic lateral sclerosis amyloid precursor protein androgen receptor adenosine 5’-triphosphate familial British dementia gene cerebral amyloid angiopathy cytosine–adenine–guanine repeats corticobasal degeneration Creutzfeldt–Jakob disease chronic manganese intoxication central nervous system dementia with Lewy bodies 3,4-dihydroxyphenylalanine dentatorubropallidoluysian atrophy frontotemporal dementia and parkinsonism related to mutations of chromosome 17 frontotemporal dementia -aminobutyric acid reduced glutathione Hungtington’s disease iron regulatory protein monoamine oxidase microtubule-associated protein tau gene 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine magnetic resonance imaging multiple system atrophy Parkinson’s disease positron emission tomography human prion protein gene prion protein resistent prion protein presenilin 1 + 2 progressive supranuclear palsy reactive oxygen species

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SCA SN SNCA SOD UCHL

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spinocerebellar ataxia substantia nigra -synuclein gene superoxide dismutase ubiquitin carbonyl hydrolase

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12 Zinc Metalloneurochemistry: Physiology, Pathology, and Probes Christopher J. Chang and Stephen J. Lippard∗ Department of Chemistry, Massachusetts Institute of Technology, 77 Masachusetts Avenue, Cambridge, MA 02139, USA

1 INTRODUCTION 2 ZINC IN THE BRAIN 2.1 Tissue Concentrations, Distribution, and Speciation 2.2 Transport, Trafficking, and Homeostasis 2.3 Labile Zinc as a Signal or Toxin 3 ZINC SENSING FOR NEUROSCIENCE APPLICATIONS 3.1 Overview of Zinc Detection Methods 3.2 Requirements for Effective Fluorescence Imaging of Neuronal Zinc 4 BIOMOLECULE FLUORESCENT PROBES FOR ZINC 4.1 Peptide Sensors 4.2 Protein Sensors 4.3 Nucleic Acid Sensors 5 SMALL-MOLECULE FLUORESCENT PROBES FOR ZINC 5.1 Intensity-based Fluorescent Sensors with Ultraviolet Excitation 5.1.1 Quinoline Sensors 5.1.2 Dansyl Ionophores 5.1.3 Coumarin Fluorophores 5.1.4 Lanthanide-containing Probes 5.2 Intensity-based Fluorescent Sensors with Visible Excitation, Including Xanthenones 5.2.1 Zinpyr Sensors 5.2.2 ZnAF Dyes Metal Ions in Life Sciences, Volume 1 © 2006 John Wiley & Sons, Ltd

Edited by Astrid Sigel, Helmut Sigel and Roland K. O. Sigel

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5.2.3 Visible Excitation Calcium Probes Adapted for Intensity-based Zinc Sensing 5.2.4 Moderate-affinity Zinc Fluorophores 5.2.5 Materials-based Probes 5.3 Fluorescent Sensors for Ratiometric Detection of Zinc 5.3.1 Ultraviolet Calcium and Magnesium Probes Adapted for Ratiometric Zinc Sensing 5.3.2 Benzazole Probes 5.3.3 Ratiometric Zinc Sensing with Visible Excitation 6 CONCLUDING REMARKS AND FUTURE DIRECTIONS ACKNOWLEDGMENTS ABBREVIATIONS REFERENCES

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1 INTRODUCTION Metal ions are essential for the growth and development of the human mind, but disruption of their homeostasis can have devastating effects on cognition and behavior [1]. Elucidating metal ion functions in the brain and central nervous system (CNS) poses a grand challenge for understanding the neurological signals and cascades that induce thought, learning, and memory, as well as the pathological pathways that lead to neurodegeneration. Before this ultimate goal can be realized, however, large expanses of fundamental inorganic chemistry in the brain and CNS remain to be discovered. Metalloneurochemistry is the study of metal ions in brain and CNS at the molecular level [2,3]. Included in this unique discipline intersecting the fields of bioinorganic chemistry and neuroscience are studies of metal ions as neurotransmitters and second messengers, as cofactors for the biosynthesis of neuronal signaling agents and for management of cellular redox chemistry, and as neurotoxins when their homeostasis is compromised. Significant advances have been made recently in several forefront areas of research at the bioinorganic chemistry/neuroscience interface. Nowhere is this progress better illustrated than by work on the roles of potassium and sodium ions in neurotransmission. Neurochemical potentials induced by the transport of these inorganic ions through membrane-spanning channels transmit information and regulate cellular function [4–6]. The 2003 Nobel Prize in Chemistry shared by MacKinnon recognizes the landmark advances made on the structure and mechanism of K+ channels [7–17]. These biological machines provide a four-fold symmetric pocket of Met. Ions Life Sci. 1, 321–370 (2006)

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carbonyl donors from Thr-Val-Gly-Tyr-Gly domains that selectively coordinate and transport K+ ions over their smaller Na+ counterparts at a rate of 107 to 108 s−1 [7,10,11]. The role of calcium ions in neuronal signal transduction is another area of metalloneurochemistry that remains a vibrant subject of research. Calmodulin [18–21], calcineurin [22–29], and the synaptotagmins [30–39] are some of the proteins that manage the spatial and temporal distribution of Ca2+ in intra- and intercellular communication in the brain and CNS. Although progress in metalloneurochemistry has been greatest for s-block metal ions, the emerging relationship between cellular mismanagement of zinc, copper, and iron and neurodegenerative disorders, including Alzheimer’s disease (AD), Parkinson’s disease (PD), prion diseases, and familial amyotrophic lateral sclerosis (FALS), has stimulated interest in understanding the roles of d-block metal ions in neurological health and disease [1,2,40]. Zinc in particular is receiving much attention in the neuroscience community [41–45]. Specific parts of the brain like the hippocampus accumulate millimolar concentrations of labile Zn2+ under normal conditions [46,47], and the movement of these ionic pools is connected to cognitive functions like learning and memory [41,43,45] and has been implicated in neurotoxicity [43,48]. Zn2+ is a ubiquitous and mobile metal ion in neuronal systems, and mounting evidence suggests that the scope and diversity of Zn2+ signals in the brain and CNS may potentially rival those of Ca2+ [41,43]. Elucidating the complex roles of Zn2+ in neurobiology is predicated on the ability to track this analyte in living cells, tissue, and in vivo with high spatial and temporal resolution. For a spectroscopically silent metal ion like Zn2+ , these efforts require the development of new, noninvasive tools for imaging this otherwise undetectable species in neuronal systems. Fluorescent probes are ideally suited reagents for achieving this goal. The success of related tools for investigating Ca2+ in the brain and CNS [49–54] highlights the potential impact of Zn2+ -based optical imaging in living neuronal systems. In this review, we present a brief overview of neuronal Zn2+ physiology and pathology as well as a survey of Zn2+ -responsive fluorescent sensors developed for interrogating its metalloneurochemistry up to June 2004. We limit our discussion to probe designs that have been utilized successfully or have the potential to be applicable for biological studies, for several reviews on fluorescent Zn2+ sensors are already available [3,42,55–59].

2 ZINC IN THE BRAIN 2.1 Tissue Concentrations, Distribution, and Speciation Transition metals are not trace elements in the CNS, because the brain requires unusually large amounts of these nutrients for normal function [1,40]. Concentrations of zinc, copper, and iron in the gray matter (0.1–0.5 mM) [1,60] are Met. Ions Life Sci. 1, 321–370 (2006)

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on the same order of magnitude as magnesium [1], up to 102 times greater than neurotransmitters like acetylcholine and the monoamines [61], and up to 104 times higher than common neuropeptides [61]. Zinc is the most abundant transition metal in the brain after iron [46], and neurons accumulate the highest cellular concentrations of Zn2+ in the body [62]. The distribution of Zn2+ in the brain and CNS is variable from region to region. The hippocampus, amygdala, neocortex, and olfactory bulb are particularly rich in Zn2+ [46]. Increases in both the total amount of Zn2+ in the brain and in the concentration of Zn2+ per gram of wet brain tissue occur in early postnatal development, after which the brain maintains constant levels of Zn2+ throughout adult life [46]. Neuronal Zn2+ stores can be grouped into one of two types: a nonexchangeable, tightly bound pool and a chelatable, ionic pool. Zn2+ is an essential cofactor for the metabolism of all cells [62–64], and approximately 90% of the Zn2+ in neurons plays familiar structural and/or catalytic roles in transcription factors and enzymes [62–65]. In addition to these relatively static stores of Zn2+ , pools of labile Zn2+ are present in presynaptic nerve terminals throughout the brain and CNS. Millimolar concentrations of ionic Zn2+ are stored within the vesicles of certain glutamatergic neurons [46,47]. These zinc-enriched neurons are not uniformly distributed in the brain, but are concentrated in selected forebrain regions including the hippocampus, amygdala, and neocortex [46]. The olfactory bulb also contains two types of zinc-enriched terminals [66]. Concentrations of ionic Zn2+ in the hippocampus, the center of learning and memory in the brain, approach 0.3 mM in the mossy fiber terminals that project from the dentate granule cells to the hilar and CA3 pyramidal neurons [46]. The localization of such large amounts of labile Zn2+ in specific areas of the brain suggests a specialized role for Zn2+ in CNS function.

2.2 Transport, Trafficking, and Homeostasis The high concentrations and complex storage patterns of Zn2+ in neuronal tissue demand that the brain maintains strict control over its cellular Zn2+ homeostasis. The concentration of Zn2+ in the extracellular cerebrospinal fluid (CSF) 015 M [46] is two orders of magnitude less than in blood 11–23 M [60], and the uptake of Zn2+ across the blood–brain barrier (BBB) is homeostatically controlled. The entry of Zn2+ into neurons appears to overlap with routes of Ca2+ entry, and at least four specific pathways of Ca 2+ -like neuronal Zn2+ influx have been identified [67]. These include (i) voltage-dependent Ca2+ (VDC) channels; (ii) Ca2+ -permeable channels gated by certain types of kainate or 2-amino-5methyl-4-isoaxazolylpropionate (AMPA) receptors; (iii) Ca2+ -permeable channels gated by N -methyl-D-aspartate (NMDA) receptors; and (iv) transportmediated exchange with intracellular Na+ . In addition, zrt/irt-like (ZIP) proteins transport Zn2+ and other metal ions across the plasma membrane [68]. Met. Ions Life Sci. 1, 321–370 (2006)

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Several proteins have been characterized that mediate the complex distribution of the abundant and diverse intracellular Zn2+ pools in brain [2]. The zinc transporters (ZnTs) are members of the solute carrier 30 (SLC30) family of carrier proteins [68,69], originally known as the cation diffusion facilitator (CDF) family [70]. These integral membrane proteins, which have counterparts in all organisms, help maintain Zn2+ homeostasis by facilitating Zn2+ efflux from the cytoplasm to either various intracellular compartments or across the plasma membrane. ZnT-1 is a ubiquitously expressed member that is responsible for dietary uptake of Zn2+ [71], and ZnT-6 and ZnT-7 appear to transport Zn2+ from the cytoplasm to the trans-Golgi network (TGN) [72,73]. Also, expression of ZnT-1, ZnT-2, or ZnT-4 cDNAs in mammalian cells can confer resistance to Zn2+ toxicity [68,69,74]. Of particular interest to zinc metalloneurochemistry is ZnT-3, which has been suggested to be the primary protein responsible for loading Zn2+ into synaptic vesicles [68,69,75]. This protein is expressed exclusively in the brain and testis and is particularly abundant in the hippocampus, amygdala, neocortex, and olfactory bulb [75], where chelatable Zn2+ occurs in high concentrations. Electron microscopy experiments establish that the localization of ZnT-3 is coincident with synaptic vesicles that accumulate Zn2+ , and ZnT-3 knockout mice show no evidence for chelatable Zn2+ in these regions [76]. The ZnT-3 null mice also display markedly decreased amounts of deposited amyloid plaques when crossed with transgenic animals carrying the APP2576 gene compared to wild-type APP2576 mice [77]. These plaques, which are characteristic of AD and related neurodegenerative disorders [78], suggest an important contribution of ZnT-3 to their pathology. The metallothioneins (MTs) are another vital class of proteins involved in zinc metalloneurochemistry [2,79–81]. These cysteine-rich proteins have several functions in the brain and CNS, including detoxification of heavy-metal contaminants [62,81,82] and protection against oxidative stress [79,83–85]. MT-1 and MT-2 are expressed in all mammalian tissues [79,80], and MT-3 is a brainspecific member of this family, which is particularly abundant in zinc-enriched neurons [86]. MT-3 coordinates seven Zn2+ ions under normal conditions and maintains its cluster structure with fewer than seven ions bound [87]. Metal ion binding proceeds in a noncooperative manner, and MT-3 can house up to nine Zn2+ ions. MTs provide some of the most thermodynamically stable Zn2+ sites in eukaryotes with dissociation constants on the order of Kd = 01 pM, yet they can exchange Zn2+ readily with proteins having weaker Zn2+ affinities [79,88,89]. By comparison, carbonic anhydrase (CA), which also binds Zn2+ tightly with a Kd = 4 pM, exchanges Zn2+ extremely slowly with a half-life on the order of 1 year [90]. This unique combination of thermodynamic stability and kinetic lability with Zn2+ points to a buffer-like function for MT-3 and its isoforms in the brain and CNS [79,81,91]. In line with this suggestion, patients with AD show downregulation of MT-3 and increased levels of labilized Zn2+ [92,93]. Met. Ions Life Sci. 1, 321–370 (2006)

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Finally, a synergistic relationship is emerging between the neuronal trafficking of Zn2+ and nitric oxide (NO) [2]. NO is an important and versatile signaling molecule with far-ranging physiological and pathological functions [94], including a proposed function as a retrograde neurotransmitter [95–98]. Relevant cellular targets of NO and its metabolites include transition metal ions, tyrosine and other aromatic amino acid residues, including cysteine thiol groups [99]. Cysteine thiols at structural and/or catalytic sites of proteins react with NO directly or through transnitrosation to form S-nitrosothiols [79,100–103]. In the case of Zn2+ -dependent metalloproteins, formation of S-nitrosocysteine adducts labilizes Zn2+ from the polypeptide scaffold [79,100–102,104,105]. In neurons, NO is capable of inducing Zn2+ release from MT-3 with concomitant formation of a disulfide bond, a reactivity that suggests that MT-3 may be responsible for converting NO signals into Zn2+ signals [2,101,104]. On the other hand, uncontrolled reaction of reactive nitrogen oxide species (RNOS) with Zn–S bonds in MT and zinc fingers can lead to toxic levels of labilized Zn2+ [101,102,106,107]. These results underscore the complexity of small-molecule chemistry in the cellular environment, and further studies of Zn2+ /NO cross-talk will provide interesting insights into neuronal metabolism.

2.3 Labile Zinc as a Signal or Toxin The brain has a unique capacity for storing and mobilizing large amounts of ionic Zn2+ , and labile pools of this ion can exert diverse physiological and pathological effects in the CNS [43]. Vesicular Zn2+ has received particular attention. The abundant Zn2+ -containing terminals in the mossy fiber pathway are an attractive system for characterizing the functions of labile Zn2+ stores [41,46]. Many plausible functions for this ionic pool have been suggested, but none has been definitively established. The most widely accepted proposal is that Zn2+ acts as a modulator of synaptic transmission [41–43]. Work from several laboratories using living hippocampal slices indicate that chelatable Zn2+ is released into the synaptic cleft from presynaptic terminals along with glutamate upon synaptic activity or membrane depolarization [108–115], where it can transverse the cleft and permeate the postsynaptic neuron [43,114]. Although the release of vesicular Zn2+ into the synaptic cleft has been questioned recently [116], this study utilized juvenile animals that do not appear to have fully developed mossy-fiber circuits. The movement of ionic Zn2+ gives rise to three classes of proposed signals: Zn2+ -SYN, Zn2+ -TRANS, and Zn2+ -INT [43]. Zn2+ -SYN is a conventional, transmitter-like synaptic signal between the presynaptic bouton and the postsynaptic dendrite. Application of Zn2+ to cultured neurons can affect various agonist-gated and voltage-gated ion channels, and studies with living hippocampal slices support the notion that endogenous Zn2+ modulates the function of NMDA and Met. Ions Life Sci. 1, 321–370 (2006)

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GABA (-aminobutyric acid) receptors [114,115,117,118]. Zn2+ -TRANS is analogous to transmembrane Ca2+ signals and comprises a transmembrane flow of Zn2+ ions from the extracellular space through gated, ion-permeable channels like VDCCs and into the postsynaptic neuron [113,114]. Activation of AMPA receptors depolarizes the plasma membrane and allows entry of Zn2+ through VDCCs [67,119,120]. Recent findings propose that translocation of Zn2+ into CA3 pyramidal neurons is required for induction of long-term potentiation (LTP), a form of synaptic plasticity implicated as a cellular mechanism of learning [113]. Once inside the postsynaptic neuron, Zn2+ can interact directly with many cytosolic proteins to propagate the Zn2+ -INT signal. Included among these proteins are kinases such as protein kinase C (PKC) [121], calcium/calmodulindependent protein kinase II (CaMKII) [122], and the Src kinase family [123]. More speculatively, Zn2+ -INT signals can then be mobilized between cytosolic and organelle pools [2,107,124]. The combined results suggest that Zn2+ is a unique messenger substance that is released presynaptically and moves freely into postsynaptic neurons, but the precise functions of vesicular and synaptic Zn2+ are insufficiently understood. Not even their coordination environment in the presynaptic vesicles is known. In contrast, uncontrolled pools of labile Zn2+ are implicated in acute excitotoxic neuronal death after epileptic seizures, head trauma, and cerebral ischemia and reperfusion [48,125,126]. Delayed cell death in certain neurons after transient global ischemia and reperfusion is accompanied by an accumulation of chelatable Zn2+ , and the hippocampus is vulnerable in the CA1 and dentate gyrus regions [127–129]. Administration of synthetic Zn2+ chelators or metal-binding proteins before or immediately after traumatic injury can prevent neuronal death [130–132]. Sources of labilized, neurotoxic Zn2+ include vesicular [125,133] and nonvesicular [134] stores. The molecular details of Zn2+ -triggered neurotoxicity are not fully understood, but studies suggest that Zn2+ enters neurons through Ca2+ -permeable channels [126] and triggers cell death through several mechanisms. Included are cytotoxic upregulation of glutamate receptor activity [123], downstream generation of reactive oxygen species [135,136], and cross-talk with nitric oxide signaling pathways [79,101,102,107,137,138]. In particular, recent work has strengthened the link between elevated levels of Zn2+ and mitochondrial dysfunction [107,124]. Finally, the long-term disruption of labile Zn2+ homeostasis is connected to the pathology of AD and several other neurodegenerative disorders [1,43,139–141]. The aggregation of -amyloid plaques in the extracellular space of the brain is characteristic of AD [78], and several lines of evidence suggest that synaptic Zn2+ may contribute to the deposition of Alzheimer’s plaques in vivo. The early stages of AD attack the hippocampus [142]. Abnormally high levels of chelatable Zn2+ are found in amyloid plaques taken from the brains of AD patients and transgenic mice [143–145], and Zn2+ can induce aggregation and precipitation of the A (-amyloid) peptides that are a primary constituent of these Met. Ions Life Sci. 1, 321–370 (2006)

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senile plaques [1,146,147]. Moreover, ZnT-3 null/tg2576 mice that lack synaptic Zn2+ display reduced propensity for forming amyloid deposits [76]. Taken together, these results implicate a causative role for Zn2+ in neurodegeneration. Along these lines, the zinc–copper chelator clioquinol (CQ), 5-chloro-7-iodo-8hydroxyquinoline, inhibits brain deposition of A plaques in mice and shows promise as an oral therapeutic for AD [139,148,149].

3 ZINC SENSING FOR NEUROSCIENCE APPLICATIONS 3.1 Overview of Zinc Detection Methods The emerging importance of ionic Zn2+ in neurological signaling and disease, and ongoing debates about its proposed functions, has generated interest in tools and tactics to track Zn2+ in biological environments at the molecular level [2,42,58,59]. Zn2+ is a challenging analyte to monitor because of its closed-shell, spectroscopically silent d10 configuration. Conventional spectroscopic techniques such as electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) are ineffective owing to the absence of unpaired electrons and NMR-active nuclei, respectively. Electronic absorption spectroscopy is also difficult to apply because of the lack of d–d electronic transitions. As a result, methods for detecting biological Zn2+ have largely relied on invasive histochemical procedures. The most prominent of these traditional approaches is Timm’s staining, which identifies labile Zn2+ stores in fixed biological samples by treatment with sodium sulfide, silver, and hydroquinone to precipitate zinc sulfide [150]. In addition, colorimetric indicators for Zn2+ like diphenylthiocarbazone (dithizone) have existed for over 50 years [42,46]. When introduced by intravital or intraperitoneal injection, dithizone binds Zn2+ to form a bright red complex. This stain is capable of labeling loosely bound stores of Zn2+ in the brain, particularly in the hippocampus [42,46,151]. The Timms’ and dithizone methods have also helped identify chelatable Zn2+ stores throughout the body, including in the islets of Langerhans in the pancreas [152,153], secretory cells of the salivary gland [154], and prostatic fluid ducts [155]. The above-mentioned detection methods are invaluable for studying general zinc biochemistry, but are invasive techniques that are restricted to use in postmortem samples. The development of alternative approaches to monitor Zn2+ in cultured cells, tissue, or in vivo is thus crucial for understanding its complex roles in neurophysiology and neuropathology. The task calls for noninvasive tactics that provide both temporal and spatial data and minimize perturbation by the probe itself. Fluorescent sensors are ideally suited to meet this challenge. They are valued for their utility in living systems, their ability to report dynamic changes in analyte concentrations, the speed and sensitivity of their response, and spatial imaging with high resolution [156,157]. Met. Ions Life Sci. 1, 321–370 (2006)

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3.2 Requirements for Effective Fluorescence Imaging of Neuronal Zinc The criteria required for sensing Zn2+ in biological environments create significant design challenges. First and foremost is a selective fluorescence response to Zn2+ over competing biological analytes, especially abundant cellular metal ions like Ca2+ and Mg2+ . Sensors must bind rapidly and reversibly to Zn2+ and have dissociation constants Kd  close to the median concentration of the analyte. Detection systems that allow modular tuning of Kd values are particularly useful because labile pools of biological Zn2+ can vary over a wide range of concentrations. Biological compatibility is also essential. Zn2+ probes should be air-stable, water-soluble, and relatively insensitive to changes in ionic strength and pH. Both hydrophobic and hydrophilic sensors are useful for intracellular and extracellular applications, respectively. In addition, lipophilic, cell-permeable dyes are useful for probing vesicular and organelle stores of Zn2+ . Ideal optical properties include visible excitation and emission profiles to minimize cell and tissue damage and avoid autofluorescence from native cellular species, and high extinction coefficients  and quantum yields   to afford large brightness  ×  values. For spatial and temporal resolution, probes that report Zn2+ by an increase in emission intensity or a shift in excitation and/or emission profiles are superior to those that quench their emission upon Zn2+ recognition. In particular, sensors that undergo a wavelength shift in excitation or emission maxima can, in principle, provide accurate and quantitative determinations of Zn2+  by using ratiometric fluorescence imaging [51]. We now present a selection of tools for the fluorescence detection of Zn2+ in biological environments and their application for interrogating zinc metalloneurochemistry. Pertinent optical and biological properties of the zinc probes are displayed in Table 1.

4 BIOMOLECULE FLUORESCENT PROBES FOR ZINC 4.1 Peptide Sensors Owing to its unique electronic and structural preferences, Zn2+ is the only metal ion essential for all six classes of enzymes as well as several families of regulatory proteins [62,64,65]. Accordingly, nature has evolved numerous types of active sites to coordinate Zn2+ with high affinity and selectivity. In particular, zinc-finger proteins with a Zn2+ -binding Cysx Hisy x = 2 to 4 y = 4 − x chelate are the most widely occurring domains in the human genome and coordinate Zn2+ with picomolar affinity [62–64]. Several peptide systems have exploited this motif for the fluorescence detection of aqueous Zn2+ . An early study employed a zinc-finger peptide modified with a Trp residue and a 5-(dimethylamino)-1-naphthalenesulfonyl (dansyl) or coumarin fluorophore. Met. Ions Life Sci. 1, 321–370 (2006)

1 2 3 4 5 7 8 9 10 11 12 13 14 15 16 17 18 19 20 25 26 28 29

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0.2 nM

50 M

02 M 035 M

0.14 nM 65 nM 3 nM 1 pM 17 M 10 nM-1 M 08 M 2 nM 03 M 09 M 013 M

Kd for Zn2+ 333 333 333 495, 578 350 360 330 495 390(380) 435(420) 365 466 415 365, 540 470 460, 530 432 440 379 334 370 360 353

exc /nm Free Zn2+  560(525) 560(525) 560(525) 521, 596 500 500 560(450) 505 528(492) 602(558) 615(535) 504 510, 560 600, 617 538 519, 573 480, 535 480, 535 447 495 490 498 533

em /nm Free Zn2+ 

0004

01

008055 10096 03310 008610 00110 08810

Free Zn2+ 

Optical, thermodynamic, and biological properties of fluorescent zinc sensors.

(ZNS1) (FS04DNS) (FS02DNS) (Fl/Rh Zn finger) (Oxine peptide) (Sox peptide) (CA dansylamide) (CA Fl sulfonamide) (CA ABD-M) (CA ABD-N) (CA dapoxyl) (CA BTCS) (CA FRET) (CA FRET) (MBP) (Alexa 488/546 MT) (ZS1) (CFP/YFP MT) (GFP barrel) (TSQ) (Zinquin A) (TFLZn) (Oxine)

Probe

Table 1.

Genetically encodable Genetically encodable Genetically encodable Permeable and vesicle-permeable Impermeable, permeable ester Permeable and vesicle-permeable

Impermeable Impermeable Impermeable Impermeable Impermeable Impermeable Impermeable Impermeable

Cellular/membrane partition

1 1 1 2 3 3 3 4 4 4 4 4 5 5 6 6 6 6 6 8 8 8 8

Structure in figure

330

30 32 33 35 36 37 38 40 41 42 43 44 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62

(NO2 -Oxine) (SO3 H-Oxine) (di-SO3 H-Oxine) (SO2 NMe2 -Oxine) (di-SO2 NMe2 -Oxine) (di-SO2 NBu2 -Oxine) (Dansylethylcyclen) (Coumarin cyclen) (Coumarin 4-DPA) (Cou 343 3-DPA) (Eu APTRA) (Tb APTRA) (Tb DTPA bis-DPA) (ZP1) (ZP2) (ZP3) (ZPF1) (ZPC11) (ZPBr1) (ZPF3) (ZP4) (ZP5) (ZP6) (ZP7) (ZP8) (ZnAF-1) (ZnAF-2) (ZnAF-1F) (ZnAF-2F)

06 M 2.6 nM 0.7 nM 0.5 nM 0.7 nM 0.9 nM 1.1 nM 0.9 nM 0.8 nM 0.65 nM 0.5 nM 0.5 nM 0.5 nM 0.6 nM 0.78 nM 2.7 nM 2.2 nM 5.5 nM

05 M

0.55 pM

425 355 356 354 353 369 330 345 343 400(431) 262 262 260 515(507) 498(490) 502(492) 533(525) 534(527) 534(528) 520(510) 506(495) 504(495) 506(495) 505(495) 500(489) 489(492) 490(492) 489(492) 490(492)

460 515 493 499 464 465 553(528) 448 450 484(505) 700 545 490, 546, 587 531(527) 522(517) 521(516) 547(544) 550(549) 549(547) 537(533) 521(515) 520(517) 519(517) 521(517) 516(510) 514 514 514 514

.

0.38(0.87) 0.25(0.92) 0.15(0.92) 0.11(0.55) 0.22(0.50) 0.25(0.36) 0.14(0.60) 0.06(0.34) 0.29(0.48) 0.10(0.34) 0.04(0.05) 0.03(0.35) 0.022(0.23) 0.023(0.36) 0.004(0.17) 0.006(0.24)

<0001 (0.058) (0.33) (0.24) (0.70) (0.44) 0.03(0.11) (0.26) 0.038(0.88) 0.64

vesicle-permeable vesicle-permeable vesicle-permeable vesicle-permeable vesicle-permeable vesicle-permeable vesicle-permeable

Impermeable, permeable ester

Impermeable, permeable ester

Permeable and Permeable and Permeable and Permeable and Permeable and Permeable and Permeable and Impermeable Impermeable Impermeable Impermeable Impermeable

Permeable Permeable Permeable

8 8 8 8 8 8 9 9 9 9 10 10 10 11 11 11 11 11 11 11 11 11 11 11 11 14 14 14 14

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63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 83 85 87 89 90 91 92

(Continued)

(FluoZin-3) (RhodZin-3) (ACF-1) (ACF-2) (RF2) (NG DCF) (NG PDX) (FluoZin-1) (FluoZin-2) (RhodZin-1) (X-RhodZin-1) (NaphImide PIDA) (QZ1) (PEBBLE) (QD L-cysteine) (FuraZin-1) (IndoZin-1) (ZnAF-R1) (ZnAF-R2) (Benzazole) (Benzazole amine) (Benzazole pyridine) (Benzazole DPA) (Zinbo-5) (CZ1) (ZNP1)

Probe

Table 1.

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34 M 30 M 0.79 nM 2.8 nM 31 M 0.8 mM 0.6 nM 0.8 pM 2.2 nM 0.25 nM 0.55 nM

135 M 1 M 30–40 M 78 M 21 M 23 M 11 M 008 M 33 M

15 nM 65 nM

Kd for Zn2+ 488 545 495 505 514(510) 505 492 491 492 548 575 442 505(498) 508 400 378(330) 350 359(329) 365(335) 299(327) 306(318) 309(297) 308(334) 337(376) 445, 505 503, 539

exc /nm Free Zn2+  515 575 515 525 539 535 521 520 521 589 604 550 524 530, 600 460 510 480(395) 532(528) 495 460(406) 469(420) 471(415) 460(406) 407(443) 488, 534 528, 624

em /nm Free Zn2+ 

00880031 017010 023022 032023 028017 009021 002010 021064 002005

0004021 0024078

036056

0005043

Free Zn2+ 

permeable permeable permeable permeable permeable permeable

ester ester ester ester ester ester

Permeable Permeable Impermeable, permeable ester

Impermeable, permeable ester

Impermeable, permeable ester Impermeable, permeable ester

Impermeable

Impermeable, Impermeable, Impermeable, Impermeable, Impermeable, Impermeable,

Impermeable, permeable ester Impermeable, permeable ester

Cellular/membrane partition 15 15 16 16 16 17 17 18 18 18 18 18 19 20 20 21 21 21 21 22 22 22 22 22 23 24

Structure in figure

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333

Me2N

SO2 NH

AcNH-YQCQYCEKR AcNH-YQCQYDEKR Ac

N H

O

ADSSNLKTHIKTKHS-CONH2

1

ADSSNLKTHIKTKHS-CONH2

2

ACDIHGKNFSQSDELTTHIRTHT-CONH2

3

Figure 1. Dansylated zinc finger scaffolds for fluorescence sensing of zinc.

Time-resolved fluorescence measurements demonstrate Zn2+ -dependent changes in photoinduced energy transfer efficiency from the Trp donor to the dansyl or coumarin acceptor [158]. ZNS1 (FS03DNS, 1, Figure 1) utilizes a ‘ZFY-swap’ zinc-finger template with replacement of an internal phenylalanine residue by a dansylated -amino alanine [159]. Coordination of Zn2+ to ZNS1 induces peptide folding, which shields the dansyl fluorophore from polar solvent. Burying the dansyl probe in a hydrophobic environment results in an increase in emission intensity upon excitation at 333 nm and a blue shift of the emission maximum from 560 to 525 nm. ZNS1 is a sensitive fluorescent probe and binds Zn2+ tightly Kd = 014 nM. However, this Cys2 His2 peptide is susceptible to oxidation under physiological conditions and forms an intramolecular disulfide bond. Secondgeneration derivatives FS04DNS (2) and FS02DNS (3), bearing Cys2 AspHis and CysHis3 coordination spheres, respectively, show enhanced oxidative stability at the expense of Zn2+ affinity and sensitivity [160]. The Kd values for 2 and 3 are 65 and 3 nM, respectively. The zinc-finger consensus peptide modified with fluorescein donor and lissamine acceptor dyes provides a scaffold for ratiometric Zn2+ sensing by fluorescence resonance energy transfer (FRET) [161]. In the absence of Zn2+ , Cys2 His2 peptide 4 (Figure 2) is unfolded and FRET from fluorescein to lissamine is minimized. Excitation of the fluorescein donor at 430 nm produces fluorescein and lissamine emission bands of comparable intensity centered at 521 and 596 nm, respectively. Upon addition of Zn2+ , the intensity of the fluorescein donor emission band remains unchanged, whereas a notable enhancement of lissamine acceptor emission is observed. Zn2+ binding induces peptide folding and brings the fluorescein donor and lissamine acceptor moieties closer together to increase FRET. Probe 4 features visible excitation and emission profiles and a picomolar sensitivity to Zn2+ , but like ZNS1, it contains a Cys2 His2 motif that is oxygen-sensitive. Met. Ions Life Sci. 1, 321–370 (2006)

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O

Et2N

+ NEt2

O

HO

O

CO2H SO3H

4 O

N

O

SO2 NH

ATKCPECGKSFSQ-C-SDLVKHQRTHTG

CO2–

Figure 2. Doubly-labeled zinc finger probes for ratiometric zinc detection by fluorescent resonance energy transfer.

Synthetic amino acids have also been employed in peptide-based designs for the fluorescence detection of Zn2+ (Figure 3). This approach markedly reduces the size of peptide chemosensor scaffolds, but currently available artificial amino acids require tissue-damaging ultraviolet excitation to induce fluorescence. Fluorescent -amino acids 5 and 6 containing 8-hydroxyquinoline (oxine) have been incorporated into heptapeptide motifs [162]. Excitation of the oxine chromophore at 350 nm produces emission centered at 550 nm. The hybrid peptide containing oxine 5 suffers from rapid oxidation, but the peptide with oxine 6 is capable of detecting sub-micromolar levels of Zn2+ at physiological pH and ionic strength; the latter provides an attractive starting point for further probe development. A versatile class of peptide-based probes has been prepared recently using the chelation-sensitive amino acid 8-hydroxy-5-(N,N-dimethylsulfonamido)-2methylquinoline (Sox, 7) [163]. Combining Sox with a -turn sequence to preorganize the Zn2+ binding site produces a family of modular scaffolds that require only six to eight amino acids and offer tunable Zn2+ affinities ranging from 10 nM to 1 M. A limitation for biological applications is that the Sox chromophore requires ultraviolet excitation at 360 nm.

HO

OH

N OH

N H

O 5

HO

N

OH

N H

O 6

N

SO2NMe2 OH

N H

O 7

Figure 3. Artificial amino acids for fluorogenic zinc sensing. Met. Ions Life Sci. 1, 321–370 (2006)

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4.2 Protein Sensors Protein-based biosensors provide sophisticated structural scaffolds that can, in principle, offer more selective recognition sites for Zn2+ compared to truncated peptide analogues. These macromolecular Zn2+ -responsive probes can be grouped into one of two classes: sensor systems that combine synthetic fluorophores with natural Zn2+ -binding proteins, and sensor systems that couple fluorescent proteins to an engineered Zn2+ -recognition site. The most well-developed protein-based probes for Zn2+ rely on carbonic anhydrase as a Zn2+ -binding scaffold and inhibition of its metal-bound (holo) form by fluorescent aryl sulfonamides as a signaling mechanism [164]. In the absence of Zn2+ , the aryl sulfonamide has negligible affinity for metal-free (apo) CA. The free fluorophore is surrounded by polar aqueous solvent and exhibits a low fluorescence intensity, short excited-state lifetime, and small anisotropy. Upon addition of Zn2+ , the aryl sulfonamide binds to the Zn2+ center of holo CA. Encasement of the aryl sulfonamide fluorophore in the hydrophobic environment of the protein triggers an increase in fluorescence intensity, lifetime, and anisotropy. Several sulfonamide reporters have been employed for CA-based sensing of Zn2+ , with excitation and emission profiles spanning the ultraviolet to visible range [57,165–172]. These probes, which include 8 through 12, can detect down to picomolar amounts of free Zn2+ in aqueous solution (Figure 4). This strategy has been extended to detect Zn2+ by FRET using CA mutants

HO

Dye HN Zn His

O

O

Me2N

SO2

CO2H

His His

SO2NH2

H N

HN

9 SO2NH2

H N 4 O

8 S hydrophobic CA pocket N OH

S N

O OH

HN

SO2NH2

N O N SO2NH2

10

11

O N

Me2N

SO2NH2

12 N S

Me2N

O

O

SO2NH2 13

Figure 4. Zinc reporters based on carbonic anhydrase and fluorescent sulfonamides. Met. Ions Life Sci. 1, 321–370 (2006)

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His

AcHN HO

His Zn NH O2S

His

SO3H

N N HO3S

14

O HN

OH

6N H

O

HO2C

O

O His

His Zn NH

NMe2

O

O2S

His

N

15

O O

HO3SCH2

N

NH

N

HO2C O

O

HO3SCH2

N

Figure 5. Carbonic anhydrase probes for zinc detection by fluorescence resonance energy transfer.

labeled with a fluorescent donor or acceptor in combination with an appropriate free aryl sulfonamide partner. Systems 14 and 15 (Figure 5) are illustrative of this approach [57,172]. Some of the CA biosensors have been applied to image release of labile Zn2+ from live hippocampal slices [111]. Maltose binding protein (MBP) has been exploited as a macromolecular template for Zn2+ sensing [173]. This periplasmic bacterial protein consists of a single polypeptide chain with two domains connected by a hinge region. Ligand binding to MBP triggers a conformational change of the protein from an open form to a closed form through a combined bending–twisting motion around the hinge. MBP labeled with N -([2-(iodoacetoxy)ethyl]-N -methyl)amino7-nitrobenz-2-oxa-1,3-diazole (IANBD) has been converted to Zn2+ sensor 16 with the aid of the iterative computational design program DEZYMER (Figure 6). Met. Ions Life Sci. 1, 321–370 (2006)

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O

HN

1329F MBP

S

N O O N

16

NO2 SO3– O

H 2N

SO3– NH2+

SO3– O

H N

CO2H O

O

N (CH2)5

NH

Cl

H N

SO3–H N+

CO2H

S

Cl Cl

O

O 17

O ECFP

Receptor

EYFP

R

N N

18: Receptor = zif268 motif 19: Receptor = hMT-2

Zn O O

R R

N

N 20 O

GFP

Figure 6. Protein-based zinc fluorophores.

The I329F mutant of MBP changes the specificity of ligand binding to this protein from maltose to Zn2+ , providing a His2 Glu2 site for Zn2+ with moderate affinity Kd = 035 M. The natural Zn2+ -buffering protein metallothionein has been modified for fluorescence detection of Zn2+ . Recombinant human metallothionein 2 (hMT2) has a free N-terminal amino group, and introduction of a S32C mutation into this protein provides a second site with orthogonal chemical reactivity for fluorophore labeling. Notably, this additional thiol perturbs neither the overall structure of the protein nor the formation of metal-thiolate clusters. Alexa 488 and Alexa 546 were attached specifically to the mutated hMT-2 at the N terminus and Cys32 positions, respectively, to afford protein probe 17 (Figure 6) [174]. FRET between the Alexa 488 donor and Alexa 546 acceptor allows monitoring of metal binding and conformational changes in the N-terminal -domain of the protein. Intrinsic fluorescent proteins like green fluorescent protein (GFP) and its enhanced yellow (EYFP) and cyan (ECFP) derivatives have been genetically modified with metal-binding sites for analytical detection of Zn2+ . These types Met. Ions Life Sci. 1, 321–370 (2006)

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of probes are promising for nonhuman in vivo optical imaging applications because they can be introduced noninvasively to cells and tissue by transfection and/or can be targeted to specific tissues, organelles, or other cellular compartments by genetic methods [157]. The design of zinc sensor protein 1 (ZS-1, 18, Figure 6) is based on the popular chameleon-type FRET probes for Ca2+ . ZS-1 contains ECFP and EYFP units connected through a 39-amino-acid linker corresponding to a zinc-finger motif from a mouse transcription factor, zif268 [175]. Upon excitation at 432 nm, the ratio of emission intensities at 535 and 480 nm increases from 3.5 to 6.7 in the presence of Zn2+ . A similar fusion construct using human type 2a metallothionein (hMT-2a, 19, Figure 6) as a hinged Zn2+ -recognition site sandwiched between ECFP and EYFP has been employed to study the interaction between MT and NO in live mammalian cells [176]. Zn2+ chelation to the MT hinge in 19 brings the fluorescent protein pair closer together to increase FRET. An improved ECFP/EYFP FRET system has been obtained by engineering a bidentate or tridentate Zn2+ site at the interface between heterodimers of ECFP and EYFP [177]. Upon excitation at 433 nm, the ratio of emission intensities at 530 and 475 nm increases by 8to 10-fold in the presence of millimolar concentrations of Zn2+ . Finally, a GFP modified with a tridentate tripyrrole ligand (20, Figure 6) exhibits fluorescence increases upon Zn2+ binding, whereas Cu2+ chelation results in fluorescence quenching [178].

4.3 Nucleic Acid Sensors Nucleic acid constructs are an emerging class of metal ion sensors that are potentially useful for reporting Zn2+ [179]. A promising approach for metal ion detection using these building blocks is in vitro selection. This combinatorial method allows screening of large polynucleotide libraries (up to 1015 members) to amplify sequences that bind a target analyte with high affinity and selectivity. The resulting sequences fall into one of two classes: aptamers, which recognize and bind analyte, and DNA/RNA/PNAzymes, which bind analyte and catalyze a subsequent chemical reaction. When appropriately labeled, these types of nucleotide-based platforms have been employed as fluorescent [180,181] and colorimetric [182] stains for Pb2+ and can be adapted readily for detection of Zn2+ [183]. Peptide nucleic acid (PNA) probes provide another potential approach for Zn2+ sensing. Recent work has demonstrated the Zn2+ -dependent binding of PNA probes to target DNA [184]. Conjugates 21 and 22 consist of a single-stranded PNA and a Zn2+ receptor linked by a naphthalene diimide fluorophore (Figure 7). In the absence of Zn2+ , the PNA conjugates connected to the complementary DNA strand have relatively low thermal stability. Zn2+ binding increases the stability of the PNA:DNA duplex with intercalation of the diimide dye. Met. Ions Life Sci. 1, 321–370 (2006)

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NH2 N N N

O O HN

O

O

N

N

O

O

O NH N

HN O O

NH O O

L

N NH O O O

L=

N

N

N

N

H

N N

N

H

21

H

22

Figure 7. Peptide nucleic acid conjugates bearing zinc receptors.

5 SMALL-MOLECULE FLUORESCENT PROBES FOR ZINC Although several biomolecule templates have been employed successfully for detecting Zn2+ in water and a few have been applied to cellular environments, systematic modifications to tune binding affinities, selectivities, and photophysical properties can be challenging with these probes. Other complications may arise from the use of relatively large bioconjugates, including structural perturbations at the cellular site of interest and decreased temporal resolution with macromolecules that require a large conformational change or complex formation between two large aggregates as part of the signaling mechanism. These issues can be addressed with the use of fluorescent sensors based on small molecules, which offer a versatile way of tuning binding and photophysical properties while producing the smallest possible conformational perturbation within the biological milieu.

5.1 Intensity-based Fluorescent Sensors with Ultraviolet Excitation 5.1.1 Quinoline Sensors Quinolines represent the earliest and most enduring examples of small-molecule fluorogenic agents for Zn2+ in biological environments (Figure 8) [42,46,185]. Met. Ions Life Sci. 1, 321–370 (2006)

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NH2

OH N

N

8-HQ (oxine, 24)

8-AQ (23)

CO2H

O2S

O2S

NH

MeO

N RO2C

TSQ (25)

OH N

Y

X

O2S

NH

N

N MeO

O

Zinquin A (R = H, 26) Zinquin E (R = Et, 27)

29: X = H, Y = H 30: X = NO2, Y = H 31: X = NO2, Y = NO2 32: X = SO3H, Y = H 33: X = SO3H, Y = SO3H

NH

TFLZn (28)

34: X = SO3H, Y = NO2 35: X = SO2NMe2, Y = H 36: X = SO2NMe2, Y = SO2NMe2 37: X = SO2NBu2, Y = SO2NBu2

Figure 8. Quinoline agents for fluorogenic zinc sensing.

These sensors are generally based on one of two common platforms, 8-aminoquinoline (8-AQ, 23) or 8-hydroxyquinoline (8-HQ, oxine, 24). 8-AQ, 8-HQ, and their derivatives are nonfluorescent owing to intramolecular hydrogen bonding between the 8-substituent and the heterocyclic nitrogen atom. This hydrogen bonding interaction couples photoinduced proton transfer with electron transfer quenching of the tautomerized fluorophore. Binding of Zn2+ alleviates this quenching pathway to restore the yellow–green fluorescence of these dyes. The first and most widely used fluorescent stain for detecting intracellular Zn2+ is the aryl sulfonamide derivative of 8-aminoquinoline, 6-methoxy-8-ptoluenesulfonamido-quinoline (TSQ, 25, Figure 8) [186]. TSQ is a non-toxic, membrane-permeable compound that exhibits a weak, pH-independent fluorescence in the absence of Zn2+ . The fluorescence intensity of the probe at 495 nm increases by over 100-fold in the presence of Zn2+ , and TSQ can detect 0.1 nM levels of this free Zn2+ in aqueous solution without interference from Ca2+ or Mg2+ . Because of these features, TSQ has been applied extensively to identify pools of labile Zn2+ in the CNS, pancreas, and other parts of the body [42,46]. Zinquin A (26), Zinquin E (27), and TFLZn (28) are secondgeneration TSQ derivatives developed to improve solubility and membranepermeability (Figure 8) [187–190]. Met. Ions Life Sci. 1, 321–370 (2006)

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Despite their ability to stain intracellular pools of labile Zn2+ , quinoline probes have a number of limitations for optical imaging applications. They require ultraviolet excitation that can damage biological samples and exhibit dim fluorescence × < 1×103 M−1 cm−1  for their Zn2+ -saturated forms [42]. Optical improvements have been made with oxine derivatives such as 29 to 37 (Figure 8), but none of these probes has both a visible excitation maximum and high quantum yield [191]. In addition, issues have been raised concerning the formation of Zn2+ -quinoline complexes under physiological conditions [192,193]. The probes can form mixed complexes with partially coordinated Zn2+ in cells and show a high degree of cooperative binding to generate a 2:1 probe:Zn2+ species. These coordination properties can generate uncertainty in measurements of ionic Zn2+ . Finally, with specific regard to neuronal systems, TSQ and its congeners will penetrate into vesicles, but have unstable partition coefficients over time, which limits their use in living samples [42].

5.1.2 Dansyl Ionophores Numerous probe designs for sensing Zn2+ have employed the 5-(dimethylamino)naphthalene-1-sulfonamide (dansyl) fluorophore because of the easy attachment in synthetic chemistry [55,56]. The optical properties of dansyl sensors generally limit their utility in cellular imaging applications, however. They require ultraviolet excitation  ∼ 350 nm and exhibit weak fluorescence in aqueous solution due to low quantum yields  ∼ 01 and extinction coefficients  ∼ 5×103 M−1 cm−1 . A notable exception is dansylamidoethylcyclen 38 (Figure 9), which has been used as a selective fluorescent stain for apoptotic cells by detecting increased levels of labilized intracellular Zn2+ [194,195]. Sensor 38 was designed to model the tight binding between CA and aryl sulfonamides. Coordination of Zn2+ to 38 results in a 5-fold increase in emission intensity at 528 nm with 330 nm excitation. Cyclen 38 binds Zn2+ tightly and its Kd value of 0.55 pM at pH 7.8 is comparable to those of Zn2+ -dependent enzymes and zinc-finger motifs. The free probe is cell-permeable and exhibits minimal fluorescence in live HeLa or HL60 cells owing to their low endogenous levels of ionic Zn2+ . Induction of apoptosis releases Zn2+ from intracellular stores, which is chelated by 38 and signaled by an increase in fluorescence intensity. The resulting Zn2+ complex is cell-impermeable and remains trapped for several hours to stain apoptotic cells selectively.

5.1.3 Coumarin Fluorophores Coumarins have been employed recently as platforms for cellular Zn2+ detection. These fluorescent reporters operate by photoinduced electron transfer (PET), Met. Ions Life Sci. 1, 321–370 (2006)

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Me2N R SO2 HN N

N

N

N

H

MeO

H

N

N

N

N

MeO

38 H

N

O

O

R

R 39: R = H 40: R = CH2CO2Et

N

N

N N

MeO N MeO

O

O

O

N

O

41

42

Figure 9. Zinc reporters with dansyl and coumarin fluorophores.

the most common mechanism employed for small-molecule metal ion sensing [196,197]. PET sensors consist of a fluorophore platform with an appended receptor. In the absence of analyte, electrons localized on a donor atom of the receptor, usually a nitrogen atom, participate in back electron transfer with the fluorophore excited state to quench emission. Metal ion coordination induces a change in the electronic structure of the fluorophore–receptor conjugate to alleviate PET quenching and restore fluorescence. The resulting ‘off-on’ fluorescent chemosensors offer a convenient method for detecting metal ions and other biological analytes with high spatial and temporal fidelity. For example, coumarin–cyclen conjugates 39 and 40 (Figure 9) are prepared readily by alkylation of 4-bromomethyl-6,7-dimethoxycoumarin [198]. Triester 40 forms a 1:1 complex with Zn2+ that induces a 4.4-fold increase in emission intensity at 448 nm upon excitation at 345 nm. A quantitative Kd determination is hampered by the extremely slow binding of Zn2+ to the macrocyclic receptor of 40, which occurs with a half-life longer than 60 min. Nevertheless, this probe is membrane-permeable and capable of detecting micromolar changes in intracellular Zn2+ in cultured GH3 rat pituitary tumor cells on the hour timescale. To enhance the kinetics of binding, a pair of coumarin sensors bearing an open-chain di-(2-picolyl)amine (DPA) chelator was synthesized (Figure 9) [199]. In methanol, the 4-substituted sensor 41 undergoes a 23-fold increase in emission intensity with excitation at 343 nm. Probe 41 forms a 1:1 Zn2+ complex with a Kd of 05 M, and quantum yields for the free and Zn2+ -bound forms are = 0038 and 0.88, respectively. In contrast, addition of Zn2+ to 3-substituted Met. Ions Life Sci. 1, 321–370 (2006)

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42 induces a red shift of both the excitation and emission profiles for the probe because of coordination of the metal ion to the coumarin carbonyl oxygen. Excitation/emission maxima for free and Zn2+ -bound forms are 400/484 and 431/505 nm, respectively. Unfortunately, this shift is solvent-dependent and is minimized in solvent systems containing increasing amounts of water, which competes with the carbonyl for Zn2+ binding.

5.1.4 Lanthanide-containing Probes Supramolecules that trigger sensitized lanthanide emission upon Zn2+ binding have been investigated as chemosensors for detecting aqueous Zn2+ . A distinct advantage of such probes is that the long-lived, millisecond luminescence lifetimes of lanthanide complexes allow emitted light to be collected after a delay time, which eliminates background emission from short-lived fluorescence and scattered light decay. However, current lanthanide sensors for Zn2+ have prohibitively short ultraviolet excitation wavelengths  < 300 nm that limit their value with cell and tissue samples. A series of lanthanide cyclen complexes with an appended o-aminophenolN ,N ,O-triacetic acid (APTRA) chelator have been utilized for sensing Zn2+ by a PET mechanism [200,201]. APTRA complexes 43 and 44 (Figure 10) excited at 262 nm exhibit 42% and 26% increases in emission intensity, respectively, upon the addition of Zn2+ . The Tb3+ probe 44 displays a moderate affinity for Zn2+ Kd = 06 M in the presence of physiological concentrations of Ca2+ and Mg2+ . A similar strategy has been pursued by linking two DPA groups to a Eu3+ (45) or Tb3+ (46) diethylenetriaminepentaacetic acid (DTPA) core (Figure 10) [202]. Excitation of DTPA complex 46 at 260 nm triggers a rise in luminescence intensity centered at 545 nm after Zn2+ addition. This Zn2+ -induced emission increase is ascribed to enhanced intramolecular energy transfer from the flanking pyridyl groups to the lanthanide ion, which are brought closer together by Zn2+ chelation. Tb3+ complex 46 binds tightly to Zn2+ Kd = 26 nM and

O

CO2H O N

N O O

H N

M N

O N

O

N

CO2H

N

N

N

N O

CO2H

O 43: M = Eu 44: M = Tb

N N

3+ CO2– M –O2C – CO HN 2 NH N N N O O

45: M = Eu 46: M = Tb

Figure 10. Lanthanide-containing zinc probes. Met. Ions Life Sci. 1, 321–370 (2006)

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is selective for Zn2+ in the presence of 5 mM concentrations of Ca2+ or Mg2+ . A family of related Gd3+ DTPA bis-amide complexes has been characterized as potential Zn2+ -responsive contrast agents for magnetic resonance imaging (MRI) [203,204].

5.2 Intensity-based Fluorescent Sensors with Visible Excitation, Including Xanthenones Fluorescent sensors that operate with visible excitation and emission wavelengths hold distinct advantages over their counterparts with shorter ultraviolet profiles. Excitation in the ultraviolet region can damage biological samples and trigger native cellular autofluorescence, particularly from nicotinamide adenine dinucleotide (NADH). The rising use of laser excitation sources for fluorescence microscopy has also increased the demand for chromophores with optical profiles in the visible region. In particular, xanthenones represent an attractive class of fluorescent dyes for optical imaging in biological samples. The flagship members of the xanthenone family are fluorescein and rhodamine. These bright fluorophores and their derivatives offer visible excitation and emission profiles  > 500 nm, quantum yields approaching unity, and high extinction coefficients  ∼ 8 × 104 M−1 cm−1 . In addition, these reagents can be modified synthetically to afford either cell-permeable or cell-impermeable versions. Because of these favorable optical and biological characteristics, our laboratory and others have been developing intensity-based sensors based on xanthenone platforms.

5.2.1 Zinpyr Sensors Zinpyr 1 (ZP1, 47, Figure 11) and Zinpyr 2 (ZP2, 48, Figure 11) are firstgeneration members of a family of PET probes that can detect intracellular Zn2+ by inducing a positive fluorescence response upon metal ion complexation [3,205,206]. ZP1 was obtained from a one-pot Mannich reaction between 2 ,7 -dichlorofluorescein (DCF) and the iminium ion condensation product of formaldehyde and DPA [205]. ZP2 was prepared using an alternative synthetic route involving the stepwise conversion of 4 ,5 -dimethylfluorescein to 4 ,5 fluoresceindicarboxaldehyde followed by reductive amination to install the DPA ligands [206]. Both probes feature optical properties characteristic of fluorescein. In the absence of Zn2+ , ZP1 has a quantum yield of = 038 with excitation and emission maxima at 515 and 531 nm, respectively. Addition of up to 1 equivalent of Zn2+ to ZP1 triggers a 3-fold increase in fluorescence intensity with a slight blue shift in excitation and emission maxima to 507 and 527 nm, respectively, owing to coordination of Zn2+ to the fluorescein phenol. The quantum yield for Zn2+ -bound ZP1 is = 087. This high quantum yield combined with Met. Ions Life Sci. 1, 321–370 (2006)

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X N

N

N N

N N

HO

O

O

N N

HO

CO2H

Y

O

O Z CO2H

Y

Y

HN

X

X Y

N

Y ZP1 (X = Cl, Y = H, 47)

ZP4 (X = H, Y = H, Z = Cl, 54)

ZP2 (X = H, Y = H, 48)

ZP5 (X = F, Y = H, Z = Cl, 55)

ZP3 (X = F, Y = H, 49)

ZP6 (X = Cl, Y = H, Z = Cl, 56)

ZPF1 (X = Cl, Y = F, 50)

ZP7 (X = OMe, Y = H, Z = Cl, 57)

ZPCl1 (X = Cl, Y = Cl, 51)

ZP8 (X = H, Y = F, Z = F, 58)

ZPBr1 (X = Cl, Y = Br, 52) ZPF3 (X = F, Y = F, 53)

Figure 11. Zinpyr family of fluorescent zinc sensors.

its large extinction coefficient  = 84 × 104 M−1 cm−1  establishes that ZP1 is almost 50 times brighter than TSQ and other traditional quinoline fluorogenic agents [42,186]. Similar Zn2+ -induced fluorescence enhancements are observed for ZP2. Both probes form tight 1:1 Zn2+ :ZP complexes (Kd = 07 nM for ZP1, Kd = 05 nM for ZP2) with high selectivity for Zn2+ over cellular concentrations of Ca2+ and Mg2+ because of the DPA receptors. Binding of a second Zn2+ ion to either sensor occurs with much lower affinity (Kd = 85 M for ZP1, Kd = 9 M for ZP2). ZP1 is cell-permeable and has been applied for detecting changes in intracellular Zn2+  in COS-7 cells and neuronal tissue. This lipophilic probe, unlike TSQ, is stable in vesicles and capable of resolving individual synaptic boutons in acute brain slices [42]. Despite these desirable features, the first-generation ZP dyes underscore a general challenge for applying PET probes in biological samples. Metal-binding atoms in receptors are often susceptible to protonation at physiological pH. This property produces interfering fluorescence from the protonated apo dye, which can limit the utility of any PET sensor for cellular imaging. ZP1 and ZP2 display relatively high pKa values for the tertiary nitrogen atoms responsible for PET switching and, as a consequence, show background fluorescence in their unmetalated forms (pKa = 84 and = 038 for ZP1, pKa = 94 and = 025 for ZP2). Strategies to address the issue of proton-induced interfering fluorescence Met. Ions Life Sci. 1, 321–370 (2006)

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are essential not only for improving tools for neuronal Zn2+ detection, but also for the general design of effective PET probes for biological media. Our group has explored several different tactics toward this goal. In one approach, we sought to reduce pKa values and proton sensitivity of the tertiary DPA nitrogen atoms responsible for PET switching by introducing electronwithdrawing substituents onto the fluorescein scaffold. To achieve this objective, we exploited the synthetic versatility of the fluorescein platform and its Mannich reactivity to prepare a library of ZP1-like sensors (49–53, Figure 11) [207]. A systematic survey of halogen substituents appended to the top xanthenone and/or bottom aryl ring of the fluorescein backbone provides a basis for tuning pKa values and attendant proton-induced interfering fluorescence of apo probes at physiological pH. ZP3 (49) is a representative member of the Zinpyr family with improved optical characteristics. Because of its fluorine substituents at the xanthenone 2 and 7 positions, apo-ZP3 exhibits markedly reduced pKa (6.8) and background fluorescence  = 015 values compared to dichloro-ZP1 and unsubstituted ZP2. ZP3 has excitation and emission maxima at 498 and 522 nm, respectively. Upon addition of up to 1 equivalent of Zn2+ , the fluorescence intensity of ZP3 increases by over six-fold  = 015−092, with concomitant blue shifts for both excitation  = 490 nm and emission  = 516 nm maxima. This probe has the highest brightness value reported yet for a Zn2+ -specific

Figure 12. Confocal fluorescence image of a live hippocampal slice stained with ZP3. Acute hippocampal slices from 90-day-old adult rats were incubated with 10 M ZP3 for 20 min at 37 C and then imaged. Data were collected with a 4× objective lens. Met. Ions Life Sci. 1, 321–370 (2006)

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chemosensor  × = 78 × 104 M−1 cm−1  and shows a 3-fold improvement in dynamic range over the parent compound ZP1. In line with other members of the ZP family, ZP3 binds Zn2+ tightly Kd = 07 nM with high selectivity over millimolar concentrations of Ca2+ and Mg2+ because of the DPA ligands. ZP3 is cell-permeable and capable of imaging changes in endogenous intracellular

Zn2+  reversibly in live hippocampal neurons and slices. This probe affords bright staining of the Zn2+ -rich mossy fiber circuit connecting the dentate gyrus and CA3 regions in acute hippocampal slices (Figure 12), as well as providing the first fluorescence detection of ionic Zn2+ in isolated dentate gyrus cultures (Figure 13) [207]. Another strategy for reducing background fluorescence on the ZP platform is substitution of the aliphatic-derivatized nitrogen PET switch with an aromaticsubstituted one, because the latter typically has lower pKa values. ZP4 (54, Figure 11) is the first monoderivatized member of the ZP family and integrates an aniline nitrogen atom into a chelating ligand containing a DPA moiety [208]. In the absence of Zn2+ , ZP4 displays a low quantum yield  = 006 with a pKa = 72. Excitation and emission maxima for apo-ZP4 are 506 and 521 nm, respectively. ZP4 binds a single Zn2+ ion with nanomolar affinity Kd = 065 nM and signals this event by a 5-fold increase in fluorescence intensity ( = 006

Figure 13. Confocal fluorescence images of live dentate gyrus neurons labeled with ZP3. Neurons were incubated with 10 M ZP3 for 20 min at 37 C and then imaged. Data were collected with a 40× objective lens. Met. Ions Life Sci. 1, 321–370 (2006)

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to 0.34) with blue shifts in both the excitation  = 495 nm and emission  = 515 nm maxima. Like other DPA-derivatized fluoresceins, ZP4 exhibits a Zn2+ -selective response in the presence of cellular concentrations of Ca2+ and Mg2+ . Staining experiments indicate that ZP4 cannot readily penetrate cell membranes. The relative impermeability of this probe may arise from several factors, including its charge at neutral pH, a dipole moment, or an inability to adopt a permeable lactone isomer. Because of this feature, ZP4 has been applied to image damaged neurons in intact hippocampal slices, providing the best histochemical method to date for staining neurons damaged by Zn2+ following seizure or blunt head trauma [2,209]. In an effort to improve the optical properties of the ZP4 motif, the nature of the substituent para to the aniline was varied to produce ZP5 (55), ZP6 (56), and ZP7 (57) (Figure 11) [210]. Although a comparison of probes with X = H, F, Cl, and OMe reveals unpredictable photophysical behavior, their modular synthesis illustrates the potential of monosubstituted fluorescein building blocks for rapid screening of sensor candidates. A recent addition to the ZP family is ZP8 (58, Figure 11) [211]. This probe combines design strategies used for ZP3 and ZP4 by employing an aniline-DPA receptor appended to a monosubstituted difluorofluorescein platform. ZP8 has a low quantum yield  = 003 in the absence of Zn2+ and exhibits an 11-fold increase in fluorescence intensity ( = 003 to 0.35) upon chelating one equivalent of this ion. The sensor binds Zn2+ tightly Kd = 06 nM with selectivity over millimolar concentrations of Ca2+ and Mg2+ .

5.2.2 ZnAF Dyes Another approach for Zn2+ -responsive PET probes relies on the attachment of DPA ligands to 5- or 6-aminofluorescein (AF) fluorophores (Figure 14). The direct attachment of the nitrogen PET switch to the dye results in sensors that display larger dynamic ranges because of reduced pKa values. ZnAF-1 (59) and ZnAF-2 (60) have pKa values of 6.2 and exhibit 17- and 51-fold increases in

HO

O

X

O X CO2H

6 NH 5 N

ZnAF-1 (5-sub, X = H, 59) ZnAF-2 (6-sub, X = H, 60) ZnAF-1F (5-sub, X = F, 61) ZnAF-2F (6-sub, X = F, 62)

N N

Figure 14. ZnAF probes for fluorescence zinc detection. Met. Ions Life Sci. 1, 321–370 (2006)

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fluorescence intensity upon addition of Zn2+ , respectively [212]. Quantum yields for the Zn2+ -bound forms of ZnAF-1  = 023 and ZnAF-2  = 036 reveal that these probes are dimmer than their ZP counterparts by a factor of 3 to 5. Excitation  ∼ 490 nm and emission  ∼ 515 nm maxima for these dyes track those of fluorescein, and the DPA chelators confer nanomolar affinity for Zn2+ (Kd = 078 nM for 59, Kd = 27 nM for 60) with high selectivity over millimolar concentrations of Ca2+ and Mg2+ . Second-generation sensors ZnAF-1F (61) and ZnAF-2F (62) contain fluorine substituents and display a markedly improved dynamic range over their parent counterparts [213]. ZnAF-1F and ZnAF-2F have pKa values of 4.9 and exhibit 69- and 60-fold increases in fluorescence intensity upon addition of Zn2+ , respectively. Quantum yields for the Zn2+ bound forms of ZnAF-1F  = 017 and ZnAF-2F  = 024 are slightly lower than their unsubstituted analogues, further limiting their brightness. ZnAF-2F is cell-impermeable, but its diacetyl derivative, ZnAF-2F DA can pass through cell membranes to provide cytosolic ZnAF-2F after ester hydrolysis. ZnAF-2F DA can stain pools of labile Zn2+ in acute rat hippocampal slices. The cellimpermeable ZnAF-2 has also been applied to monitor release of Zn2+ from hippocampal slices into the extracellular space [115].

5.2.3 Visible Excitation Calcium Probes Adapted for Intensity-based Zinc Sensing Ca2+ -responsive fluorescein and rhodamine sensors have been modified for fluorescence detection of Zn2+ . FluoZin-3 (63, Figure 15) [214] is a tetraanionic fluorescein Zn2+ sensor based on the Ca2+ probes Fluo-3 and Fluo-4 [215]. The BAPTA (bis-(o-aminophenoxy)-ethane-N ,N ,N  ,N  -tetraacetic acid) chelators of Fluo-3 and Fluo-4 bind Ca 2+ tightly Kd ∼ 300 nM and display even higher affinity for Zn2+ Kd < 1 nM. Removing one of the N -acetyl moieties lowers the overall ion affinity of the chelator structure, with the resulting FluoZin-3 probe

HO

O

O

F

Me2N OMe

F

O

OMe

O

NH CO2H

+ NMe2

O

O N

HO2C

O

NH CO2H

CO2H

FluoZin-3 (63)

N HO2C

CO2H

RhodZin-3 (64)

Figure 15. FluoZin-3 and RhodZin-3 zinc fluorophores. Met. Ions Life Sci. 1, 321–370 (2006)

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retaining physiologically relevant Zn2+ affinity for a 1:1 Zn2+ : probe complex Kd = 15 nM. FluoZin-3 has the highest dynamic range of any Zn2+ -responsive sensor to date, displaying a greater than 200-fold increase in fluorescence intensity upon Zn2+ addition with a Zn2+ -bound quantum yield = 043. However, the carboxylate receptors are not as selective as their DPA counterparts for Zn2+ . Additions of 100 M Ca2+ or Fe2+ to FluoZin-3 trigger fluorescence increases that are 49% and 78%, respectively, of the enhancements observed upon Zn2+ binding. FluoZin-3 is membrane-impermeable and has been used to monitor Zn2+ secretion from pancreatic -cells with high sensitivity (10–40 nM) and temporal resolution (16 ms/image) [216]. The higher sensitivity of FluoZin-3 allows detection of Zn2+ release up to 15 m away from the cell; for comparison, Zinquin is effective at a range of only 1 to 2 m. RhodZin-3 (64, Figure 15) is a rhodamine analogue of FluoZin-3 [217]. This probe displays a 75-fold increase in emission intensity upon formation of a 1:1 Zn2+ :probe complex Kd = 65 nM. RhodZin-3 exhibits no Ca2+ sensitivity at ≤40 M concentrations and modest sensitivity to Fe2+ Kd = 5 M. The acetoxymethyl (AM) ester of this sensor is membranepermeable and co-localizes with the mitochondrial marker, MitoTracker Green, in cultured cortical neurons. RhodZin-3 has been utilized to monitor endogenous Zn2+ release from mitochondria by protein-bound stores [124].

5.2.4 Moderate-affinity Zinc Fluorophores Because local flows of labile Zn2+ in the brain can approach concentrations of 0.3 mM, several Zn2+ -responsive probes with moderate Zn2+ affinity have been developed. ACF-1 (65, Figure 16) and ACF-2 (66, Figure 16) are Zn2+ probes that consist of a 6-hydroxy-9-phenylfluorone reporter coupled to a trimethylcy-

HO

O

O N

X

X

Et

N Me N

N

N

N

N

Et O

O Cl CO2H

N Me N Me

ACF-1 (X = H, 65)

RF-2 (67)

ACF-2 (X = Cl, 66)

Figure 16. Cyclen-containing ACF and Rhodafluor fluorescent sensors for zinc. Met. Ions Life Sci. 1, 321–370 (2006)

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clen receptor [218]. The ACFs feature visible excitation and emission maxima ( exc / em = 495/515 nm for 65, exc / em = 505/525 nm for 66) and display 14and 26-fold increases in emission intensity, respectively, upon saturation with Zn2+ . The detection limit of the ACFs under simulated physiological conditions is 500 nM with micromolar affinity. The Rhodafluor (RF) sensors employ a hydrid fluorescein/rhodamine chromophore [219]. RF2 (67, Figure 16) has a pyridyl-appended cyclen integrated into the fluorophore framework. This probe undergoes a modest increase in fluorescence intensity upon Zn2+ coordination  = 036 − 056 and has a micromolar affinity for this ion Kd = 135 M. The slow binding of Zn2+ to macrocyclic receptors like cyclen limits their use for real-time cellular imaging, and no biological applications of either the ACFs or RFs have been reported. Open-chain nitrogen chelates are anticipated to have faster kinetics of Zn2+ complexation. Newport Green (NG) DCF (68, Figure 17) is a first-generation sensor in this direction [215]. This probe features a DCF fluorophore and a DPA receptor connected through a phenylacetamide spacer. Newport Green DCF has excitation  = 505 nm and emission  = 535 nm maxima in the visible region and displays a 3.3-fold increase in fluorescence intensity upon Zn2+ binding with moderate affinity Kd = 1 M. This sensor actually shows higher fluorescence enhancements with Co2+ and Ni2+ compared to Zn2+ , but is insensitive to Ca2+ and Mg2+ . The diacetate form of Newport Green DCF is membrane-permeable and has been used to track Zn2+ influx into the cytosol through voltage- or glutamate-gated ion channels [220,221]. The Newport Green PDX derivative (69, Figure 17) shows an improved fluorescence response with slightly lower affinity for Zn2+ Kd = 30 − 40 M [222]. Carboxylates have also been integrated into moderate-affinity sensors for Zn2+ . FluoZin-1 (70, Figure 18) is a Zn2+ -responsive analogue of the Ca 2+ dye O

HO Cl

O

HO

Cl

F

O

O F

CO2H

CO

N

N

N

N

NH

N

NG PDX (69)

N

NG DCF (68)

Figure 17. Newport Green dyes for zinc reporting. Met. Ions Life Sci. 1, 321–370 (2006)

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HO

O

F

HO2C

N

O

HO

F

Cl

O

O Cl CO2H

OMe CO2H

CO NH

FluoZin-1 (70)

HO2C

N

CO2H

FluoZin-2 (71) + NMe2

O

Me2N

HO2C

OMe CO2H

N

RhodZin-1 (72)

N

O

HO2C

N

+ N

OMe CO2H

X-RhodZin-1 (73)

O NH N

Et–N O

CO2H CO2H

74

Figure 18. Moderate-affinity fluorescent zinc sensors with carboxylate receptors.

Fluo-4 that contains a phenyliminodiacetic acid (PIDA) receptor [222]. This fluorophore has visible excitation  = 491 nm and emission  = 520 nm maxima and exhibits a 200-fold fluorescence enhancement upon Zn2+ saturation Kd = 78 M. FluoZin-2 (71, Figure 18) is a Zn2+ -responsive Calcium Green analogue [222]. Sensor 71 shows a 12-fold increase in emission intensity with Zn2+ coordination and a slightly tighter binding affinity than FluoZin-1 Kd = 21 M. Because of their carboxylate groups, both FluoZin-1 and FluoZin-2 have moderate affinities for Ca2+ in the physiological concentration range (Kd = 8 mM for 70, Kd = 5 mM for 71). Analogous RhodZin-1 (72, Figure 18) and X-RhodZin-1 (73, Figure 18) probes have been prepared by using rhodamine and X-rhodamine fluorophores, respectively [222]. The PIDA group has also been installed onto a naphthalimide chromophore. Sensor 74 (Figure 18) has a broad Met. Ions Life Sci. 1, 321–370 (2006)

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N HN HO

O

O Cl CO2H

QZ1 (75)

Figure 19. Quinozin-1 (QZ1), a bright fluorescent probe with micromolar affinity.

absorption band between 370 and 510 nm with a maximum at = 442 nm [223]. Upon excitation at 442 nm, a corresponding broad emission band is observed between 480 and 700 nm with a maximum at = 550 nm. The naphthalimide probe undergoes a 53-fold fluorescence enhancement upon Zn2+ binding. A new class of quinoline-fluorescein conjugates has been developed as moderate-affinity Zn2+ sensors. Quinozin-1 (QZ1, 75, Figure 19) is a firstgeneration member of this family that exhibits fluorescein-like optical properties, a large fluorescence enhancement upon Zn2+ coordination, and insensitivity toward cellular concentrations of Ca2+ and Mg2+ [224]. This probe has visible excitation and emission maxima  exc / em = 505/524 nm and a low quantum yield  = 0024 in the absence of Zn2+ . Zn2+ complexation triggers a 44-fold increase in integrated fluorescence intensity  = 0024−078 with a slight blue shift of the excitation maxima  exc = 498 nm. QZ1 binds Zn2+ rapidly with moderate affinity Kd = 33 M.

5.2.5 Materials-based Probes Two different types of materials have been employed for Zn2+ detection in biological environments. The PEBBLE (probe encapsulated by biologically localized embedding) strategy utilizes polymer spheres to encapsulate soluble fluorophores, resulting in increased photostability and protection from protein interference. These sensors can be introduced into cells by microinjection. A PEBBLE for Zn2+ (Figure 20) was constructed by entrapping a Zn2+ -sensitive dye (Newport Green) and a reference fluorophore (Texas Red Dextran) in an acrylamide polymer matrix [225]. The dynamic range of PEBBLE 76 for Zn2+ is 4–50 M. In another approach, water-soluble CdS quantum dots (QD) were capped with l-cysteine groups for Zn2+ binding (Figure 20) [226]. The conjugated QDs exhibit an emission increase in the presence of Zn2+ with a lower detection limit of 08 M. The luminescence enhancement of QD 77 by Zn2+ is attributed to activation of surface states. Met. Ions Life Sci. 1, 321–370 (2006)

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L-cysteine

PEBBLE Newport Green

CdS

Texas Red 76

77

Figure 20. Materials-based probes for zinc. PEBBLE (probe encapsulated by biologically localized embedding) and quantum dot examples are shown.

5.3 Fluorescent Sensors for Ratiometric Detection of Zinc Fluorescent probes that report ionic Zn2+ in aqueous media by an increase in emission intensity are of considerable practical utility for interrogating its metalloneurochemistry. These intensity-based sensors are not ideal for applications that require quantitative measurements of changes in intracellular or extracellular

Zn2+ , however, because of variations in sample thickness, excitation intensity, emission collection efficiency, and artifacts associated with probe concentration and environment. Ratiometric sensors that shift their excitation and/or emission profiles in response to Zn2+ can, in principle, eliminate the need for correction factors to negate these limitations, providing accurate determinations of Zn2+ ]. These probes afford simultaneous detection of free and bound states using dualexcitation and/or dual-emission measurements.

5.3.1 Ultraviolet Calcium and Magnesium Probes Adapted for Ratiometric Zinc Sensing Traditional fura and indo fluorophores employed for cellular Ca2+ and Mg2+ imaging have been modified for ratiometric sensing of Zn2+ . These probes operate by a photoinduced charge transfer (PCT) mechanism. In PCT sensors, a fluorophore that contains an electron-donating group (donor) is conjugated to an electron-withdrawing group (acceptor). Internal charge transfer (ICT) from the donor to the acceptor leads to a polar excited state where the Stokes’ shift is influenced by the microenvironment around the fluorophore. Metal ion binding can trigger an observable change in this fluorescent CT state. The design strategy for turning ratiometric Ca2+ and Mg2+ sensors into Zn2+ -responsive probes involves substitution of tetracarboxylate BAPTA or tricarboxylate APTRA chelators with a dicarboxylate PIDA receptor. The PIDA ligand confers a micromolar affinity for Zn2+ that is insensitive to millimolar concentrations of Ca2+ and Mg2+ [215]. Met. Ions Life Sci. 1, 321–370 (2006)

ZINC METALLONEUROCHEMISTRY

HO2C

355

N O

O

N OMe

CO2H CO2H

FuraZin-1 (78)

N N H

HO2C

MeO

CO2H CO2H

IndoZin-1 (79)

N N

OMe

N

O

R =

NH

O

N CO2H ZnAF-R1 (80)

CO2H

ZnAF-R2 (81)

R

Figure 21. Ratiometric fura and indo zinc fluorophores with ultraviolet excitation.

FuraZin-1 (78, Figure 21) is a Zn2+ -responsive Fura-2 analogue that exhibits an excitation wavelength shift from 378 to 330 nm in the presence of increasing Zn2+ concentrations [222]. This excitation-ratiometric probe has an emission maximum at 510 nm. IndoZin-1 (79, Figure 21) is a Zn2+ -responsive Indo-1 analogue that shows an emission wavelength shift from 480 to 395 nm with increasing amounts of Zn2+ [222]. This emission-ratiometric probe has an excitation maximum at 350 nm. Both sensors exhibit moderate affinity for Zn2+ (Kd = 34 M for 78, 30 M for 79) and good selectivity over cellular concentrations of Ca2+ and Mg2+ . FuraZin-1 has been used to monitor Zn2+ levels in yeast cells and has helped clarify the role of the ZRC1 protein in storage and detoxification of excess Zn2+ during Zn2+ shock [227]. In a similar approach, DPA-based ligands have been coupled to benzofuran and fura chromophores to produce ZnAF-R1 (80, Figure 21) and ZnAF-R2 (81, Figure 21), respectively [228]. ZnAF-R2 undergoes an excitation wavelength shift from 365 to 335 nm upon Zn2+ complexation with an emission maximum at 495 nm. ZnAF-R2 binds Zn2+ tightly Kd = 28 nM and selectively reports this ion in the presence of 5 mM concentrations of alkali and alkaline earth cations. The quantum yields for the free and Zn2+ -bound forms of the probe are = 017 and 0.10, respectively. The lipophilic ethyl ester derivative of ZnAF-R2 is cell-permeable and has been applied for imaging changes in intracellular Zn2+  by addition of exogenous Zn2+ in RAW 264.7 macrophages.

5.3.2 Benzazole Probes Benzazoles are another class of fluorophores that have been developed for ratiometric detection of Zn2+ . Representative members of this family include the Met. Ions Life Sci. 1, 321–370 (2006)

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HN N

HN SO2

O CO2Y

X MeO

OMe

O

OH N H N

82: X = H, Y = Et 83: X = H, Y = H 84: X = CH2NMe2, Y = Et 85: X = CH2NMe2, Y = H 86: X = CH2N(CH2py)(Me), Y = Et 87: X = CH2N(CH2py)(Me), Y = H 88: X = CH2DPA, Y = Et 89: X = CH2DPA, Y = H

Zinbo-5 (90)

N

Figure 22. Benzazole sensors for ratiometric zinc sensing with ultraviolet excitation.

2-hydroxyphenyl derivatives of benzimidazole, benzoxazole, and benzothiazole. The signaling mechanism for these benzazole probes is based on the Zn2+ induced inhibition of excited-state intramolecular proton transfer (ESIPT). In the absence of Zn2+ , ESIPT is mediated by hydrogen bonding between the 2-hydroxyphenyl group and the heterocyclic nitrogen atom. Zn2+ coordination displaces this hydrogen bond, resulting in a shift in excitation and/or emission profiles [229]. This strategy has been employed to obtain Zn2+ -selective ratiometric fluorescent sensors 82–89 (Figure 22) [230]. A similar design approach provides the Zinbo family of ratiometric probes. Zinbo-5 (90, Figure 22), which employs an aminomethylpyridine chelating moiety, displays both excitation (337–376 nm) and emission (407–443 nm) wavelength shifts upon formation of a 1:1 Zn2+ :dye complex [231]. Quantum yields for the free and bound fluorophore are = 002 and 0.10, respectively. Zinbo-5 binds Zn2+ tightly Kd = 22 nM and shows no fluorescence response to alkali and alkaline earth metal ions at concentrations up to 5 mM. Because it requires ultraviolet excitation that can be damaging to biological samples, two-photon excitation at 710 nm was employed for ratiometric imaging in fixed fibroblast cells with Zinbo-5.

5.3.3 Ratiometric Zinc Sensing with Visible Excitation A desirable research direction for ratiometric imaging is to extend the excitation profiles of probes into the visible region. In one approach to this goal we recently reported an esterase-mediated system for ratiometric imaging of intracellular Zn2+ . The design for this strategy involves the coupling of a Zn2+ sensitive dye with a Zn2+ -insensitive dye through an ester linker. Introduction of the two-fluorophore probe into cells and subsequent cleavage of the ester linkage by intracellular esterases will then liberate a Zn2+ -responsive sensor Met. Ions Life Sci. 1, 321–370 (2006)

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N

N

N

N

N

N

HO

O

O

CZ1 (91) Cl O

H N

O N

O

Cl CO2H O

O

cell membrane esterase processing

N

N

N N

O

N N

HO

O

O

OH N

O

Cl

O HO

Cl CO2H

H N O

Figure 23. Coumazin-1 (CZ1), a two-fluorophore system for ratiometric sensing that is processed by intracellular esterases.

to measure changes in intracellular Zn2+  and a free dye for optical calibration. Coumazin-1 (CZ1, 91, Figure 23) is a two-fluorophore prosensor containing a Zn2+ -insensitive coumarin 343 fragment and Zn2+ -sensitive ZP-type sensor [232]. CZ1 is membrane-permeable and exhibits negligible fluorescence  = 004. Treatment of CZ1 with an in vitro esterase hydrolyzes the ester linkage to afford coumarin 343 and a PET-based Zn2+ sensor analogue of ZP1. Excitation of the coumarin fragment at 445 nm yields emission at 488 nm, and excitation of the ZP fragment at 505 nm yields emission at 534 nm. The ratio of ZP to coumarin intensity collected at 534 nm and 488 nm, respectively, varies from 0.5 in the absence of Zn2+ to 4.0 in the presence of a two-fold excess of Zn2+ . The ZP fragment binds Zn2+ tightly Kd = 07 nM with high selectivity over cellular concentrations of Ca2+ and Mg2+ . Experiments with HeLa cells demonstrate that CZ1 can be loaded into live cells and report changes in intracellular Zn2+  by ratiometric imaging. The carboxylate functionality on the Met. Ions Life Sci. 1, 321–370 (2006)

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N N HN

N

HO

O O ZNP1 (92) CO2H

OH O

O

O HO

CO2H

naphthoxyquinone

O

CO2H

phenoxynaphthoquinone

Figure 24. Zin-naphthopyr-1 (ZNP1), a tautomeric probe for ratiometric detection of zinc, and limiting naphthoxyquinone and phenoxynaphthoquinone resonance forms for the semi-napthofluorescein backbone.

bottom ring of the fluorescein chromophore provides a versatile synthetic handle for the preparation of a variety of ZP-sensor conjugates [233]. Another tactic for developing Zn2+ -responsive ratiometric sensors is based on incorporation of a Zn2+ receptor onto a hybrid fluorescent dye. Zin-naphthopyrl (ZNP1, 92, Figure 24) is a hybrid sensor based on a seminaphthofluorescein platform that can attain fluorescein- and naphthofluorescein-like forms [234]. Upon excitation at 499 nm, apo-ZNP1 exhibits two emission bands of comparable intensities at 528 and 604 nm. Upon addition of Zn2+ to form a 1:1 Zn2+ :ZNP1 complex, excitation at 499 nm produces a fluorescence spectrum with one dominant emission band centered at 624 nm with a minor component at 545 nm. The ratio of naphthofluorescein- to fluorescein-like emission intensities  624 / 528  varies from 0.4 to 7.1 upon Zn2+ coordination, an 18-fold increase. ZNP1 binds Zn2+ tightly Kd = 055 nM, and the ratiometric response is highly selective for Zn2+ over cellular concentrations of Ca2+ and Mg2+ . The lipophilic derivative ZNP1-Ac is membrane-permeable, and the ZNP1 produced after intracellular esterase processing is capable of detecting NO-induced changes in intracellular

Zn2+  in live mammalian cells by ratiometric fluorescence imaging. ZNP1-Ac has also been applied to image NO-triggered labilization of cytosolic Zn2+ stores in live hippocampal neurons (Figure 25) [235]. Met. Ions Life Sci. 1, 321–370 (2006)

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Figure 25. Ratio confocal fluorescence imaging using the Zeiss LSM510 META system operating in the lambda mode. Fluorescence was collected in 10.7 nm optical windows centered at 612 and 526 nm. Grayscale figures depict the ratio of fluorescence intensities at these two emission wavelengths. Confocal fluorescence images display NO-triggered release of endogenous Zn2+ in live hippocampal neurons by a rise in the ratio of emission intensities collected at 612 and 526 nm. Cells were incubated with 20 M ZNP1-Ac for 20 min at 37 C (left), and ZNP1-stained cells were treated with 100 M SNOC (S-nitrosocysteine) and imaged after 15 min (middle) and 45 min (right).

6 CONCLUDING REMARKS AND FUTURE DIRECTIONS Significant advances have been made to provide the requisite methodology for understanding how zinc functions in the brain and CNS. Central to this goal is the development of new tools that can be utilized for studying Zn2+ in biological environments. We have highlighted recent progress in the design, synthesis, and characterization of fluorescent Zn2+ sensors and their application for real-time optical imaging in living systems. Because much of this work is still in its early stages, there are tremendous opportunities for scientists interested in problems at the bioinorganic chemistry/neuroscience interface. New tactics are required for expanding the repertoire of available imaging agents for Zn2+ and related analytes. Directions for future Zn2+ sensor design include improved ratiometric sensors for quantitation of endogenous Zn2+ pools, probes that span a wider range of Kd values, reagents that shift excitation/emission profiles towards the infrared region, and responsive fluorophores that can be targeted to specific intracellular and extracellular locations. Of particular interest for the characterization of vesicular Zn2+ and its synaptic translocation are lipophilic probes with moderate binding affinities and fast response times. In addition, new instrumental advances for Zn2+ detection in vivo are emerging to complement these improved reagents, including optical microscopes with multiphoton excitation and the fabrication of fiber-optic probes. Taken together, these results demonstrate the value of fluorescent probes for Zn2+ and show their potential for imaging this ion in real time and space in living systems. As the tool kit for Zn2+ metalloneurochemistry continues to grow, so will our insight into how Zn2+ contributes to the function of our minds (see also the Addendum in [236]). Met. Ions Life Sci. 1, 321–370 (2006)

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ACKNOWLEDGMENTS This work was supported by the National Institute of General Medical Sciences (GM 65519 to S.J.L.). C.J.C. thanks the Jane Coffin Childs Foundation for a postdoctoral fellowship.

ABBREVIATIONS A AD AF AM AMPA APTRA 8-AQ BAPTA BBB CA CaMKII CDF CNS CQ CSF CT CZ dansyl DCF dithizone DPA DTPA ECFP EPR ESIPT EYFP FALS FRET GABA GFP hMT 8-HQ IANBD

-amyloid Alzheimer’s disease aminofluoresecein acetoxymethyl 2-amino-3-(3-hydroxy-5-methyl-4-isoaxazolyl)propionate o-aminophenol-N,N,O-triacetic acid 8-aminoquinoline bis-(o-aminophenoxy)-ethane-N ,N ,N  ,N  -tetraacetic acid blood–brain barrier carbonic anhydrase calcium/calmodulin-dependent protein kinase II cation diffusion facilitator central nervous system clioquinol = 5-chloro-7-iodo-8-hydroxyquinoline cerebrospinal fluid charge transfer Coumazin 5-(dimethylamino)-1-naphthalenesulfonyl residue 2’,7’-dichlorofluorescein diphenylthiocarbazone di-(2-picolyl)amine diethylenetriaminepentaacetic acid enhanced cyan fluorescent protein electron paramagnetic resonance excited-state intramolecular proton transfer enhanced yellow fluorescent protein familial amyotrophic lateral sclerosis fluorescence resonance energy transfer -aminobutyric acid green fluorescent protein human metallothionein 8-hydroxyquinoline N -([2-(iodoacetoxy)ethyl]-N -methyl)amino-7-nitrobenz-2-oxa1,3-diazole

Met. Ions Life Sci. 1, 321–370 (2006)

ZINC METALLONEUROCHEMISTRY

ICT LTP MBP Me MRI MT NADH NG NMDA NMR NO oxine PCT PD PEBBLE PET PIDA PKC PNA QD QZ RF RNOS SLC Sox TGN TSQ VDCC ZIP ZNP ZnT ZP ZS

361

internal charge transfer long-term potentiation maltose binding protein methyl group magnetic resonance imaging metallothionein nicotinamide adenine dinucleotide Newport Green N -methyl-d-aspartate nuclear magnetic resonance nitric oxide 8-hydroxyquinoline photoinduced charge transfer Parkinson’s disease probe encapsulated by biologically localized embedding photoinduced electron transfer phenyliminodiacetic acid protein kinase C peptide nucleic acid quantum dot Quinozin Rhodafluor reactive nitrogen oxide species solute carrier 8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline trans-Golgi network 6-methoxy-8-p-toluenesulfonamido-quinoline voltage-dependent calcium channel zrt/irt-like proteins Zin-naphthopyr zinc transporter Zinpyr zinc sensor

REFERENCES 1. 2. 3. 4.

A. I. Bush, Curr. Opin. Chem. Biol., 4, 184–191 (2000). S. C. Burdette and S. J. Lippard, Proc. Natl. Acad. Sci. USA, 100, 3605–3610 (2003). S. C. Burdette and S. J. Lippard, Coord. Chem. Rev., 216–217, 333–361 (2001). C. Miller, Curr. Opin. Chem. Biol., 4, 148–151 (2000). Met. Ions Life Sci. 1, 321–370 (2006)

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13 The Role of Aluminum in Neurotoxic and Neurodegenerative Processes Tamás Kiss,1 Krisztina Gajda-Schrantz,1 and Paolo F. Zatta2 1

Department of Inorganic and Analytical Chemistry, Bioinorganic Chemistry Research Group of the Hungarian Academy of Sciences, University of Szeged, P.O. Box 440, H-6701 Szeged, Hungary 2

CNR Institute for Biomedical Technologies, Metalloproteins Unit, Department of Biology, University of Padova, Viale G. Colombo, 3, 35121 Padova, Italy

1 INTRODUCTION 2 CHEMICAL FORMS OF ALUMINUM IN BIOLOGICAL SYSTEMS 2.1 Free Ions and Mononuclear Ions 2.2 Low Molecular Mass Complexes 2.3 Reversible Macromolecular Complexes 2.3.1 Interactions with Non-enzyme Proteins 2.3.2 Interactions with Enzyme Proteins 2.4 Irreversible Macromolecular Complexes 3 ALUMINUM LOADING IN HUMANS 3.1 Aluminum Metabolism 3.2 Speciation of Al(III) in Blood Serum 4 TOXICOLOGY OF ALUMINUM IN ANIMALS AND HUMANS 4.1 Experimental Toxicology 4.2 Biochemical Toxicology 4.2.1 Acetylcholinesterase 4.2.2 Monoamine Oxidase 4.2.3 Carbonic Anhydrase Metal Ions in Life Sciences, Volume 1 © 2006 John Wiley & Sons, Ltd

Edited by Astrid Sigel, Helmut Sigel and Roland K. O. Sigel

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4.2.4 Na+ /K+ -ATPase 4.2.5 ATP-dependent Enzymatic Activities 4.3 Human Toxicology 5 ALUMINUM AND ALZHEIMER’S DISEASE 6 GENERAL CONCLUSIONS ACKNOWLEDGMENTS ABBREVIATIONS REFERENCES

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1 INTRODUCTION Aluminum derives its name from the Latin alumen (bitter salt), KAlSO4 2 · 12H2 O. This was used as an astringent in ancient medicine in Greece and later in Rome. Al is the most abundant metallic element in the Earth’s crust, its concentration in the oceans is below 1 g Al/L. Until recently, most natural waters contained insignificant amounts of Al. Any free Al(III) usually disappeared into the sediment as a hydroxide. Because of the low bioavailability of Al, despite its ubiquity, it was long considered innocuous for living systems. With the advent of acid rain, however, Al began to escape from its minerals and dissolve in fresh waters. At pH < 6 the Al(III) concentration increases sharply in lakes, where micromolar amounts may occur [1]. Although there are some vague indications about the beneficial biological role of Al [2,3], these are far too limited to permit any firm conclusion. In contrast, it has been known for more than a century that Al forms complexes that are neurotoxic to animals [4], and more recently, to man. The concentration of Al found in the tissues reflects the geochemical environment of the individual and of locally raised food products. The ingestion of foods, beverages, and pharmacological products is the major route by which most humans are exposed to Al. The average Al intake in Europe is approximately 2–10 mg/day, but only a very small proportion of this Al 5–10 g is absorbed. The contents of Al in various biological fluids and tissues are reported in Table 1. Under normal conditions, i.e., with a normal daily load and a healthy organism, this amount is well tolerated, in consequence of the efficient defense mechanisms, which involve the formation of poorly soluble phosphates and hydroxides in the gastrointestinal (GI) tract. These compounds are easily excreted and are therefore not harmful. Furthermore, most of the Al(III) absorbed is eliminated in the urine (probably in the form of a citrate complex). However, in the event Met. Ions Life Sci. 1, 371–393 (2006)

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Table 1. Normal human aluminum values of biological tissues and biological fluids. Biological tissues (mol/kg dry weight) Spleen 96 ± 7

Liver 152 ± 63

Muscle 44 ± 44

Heart 37 ± 30

Lung 1592 ± 1269

Brain 81 ± 48

Biological fluids (nmol/L) Plasma 88 ± 48

Cerebrospinal fluid 111 ± 37

Bile 252 ± 167

Urine/24h 263 ± 37

of an abnormally high Al load and/or an impaired renal function, an excess of Al(III) can be absorbed and transported to various target organs, where it may accumulate and exert harmful effects, e.g., osteomalacia in the bones, microcytic anaemia in the red blood cells, or neurodegenerative diseases in the brain. The toxicity of aluminum is greatly affected by its bioavailability, i.e., the multitude of forms in which the element exists in the environment. Additionally, partly independently of the chemical form in which it is absorbed, it will interact with many endogenous or exogenous potential Al binders in the various biofluids and tissues. These interactions influence the biological effects of Al. Accordingly, speciation studies may help towards an understanding of the effects of Al in biological systems. Hundreds of papers, books [5–11] and reviews [3,12,13] have been published on the toxic properties of Al. Conferences and conference series have been devoted to this hot topic, but our knowledge remains rather contradictory and the situation relating to the neurotoxicity of Al is still very confused. Al(III) is a typical hard metal ion, and the most likely binding sites of Al3+ in biosystems are therefore O donors, especially negatively-charged O donors. Carboxylate, phenolate, catecholate, and phosphate are the strongest Al(III) binders. Biomolecules containing such functions may be involved in the uptake and transport processes, and also in the biological and physiological actions of Al(III) in living systems. Small organic acids, e.g., citric acid, are important low molecular mass (l.m.m.) binders in biological fluids [9]. Transferrin plays an essential role in Al(III) transport in the plasma. Organophosphates are important Al(III) binders, e.g., ATP, which is widely distributed in living systems; numerous reactions which take place in living organisms involve ATP. Catecholamines occur in fairly high concentrations in the brain and may have an important role in binding Al(III). Phosphorylated peptides and proteins, which are frequently observed among neuronal degradation products in various neurological disorders, may play a role in the accumulation of Al(III) in the brain. In addition to the stability of metal ion complexes, an important and often overlooked feature is the rate of ligand exchange out of and into the metal ion coordination sphere. Kinetically, ligand-exchange reactions for the Al(III) ion and Met. Ions Life Sci. 1, 371–393 (2006)

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its complexes may be characterized as sluggish. Ligand-exchange reactions will definitely occur in vivo. However, the rate of ligand exchange may be comparable to the rates of transport and other biological processes; thus, biological fluids may not attain their true thermodynamic species distribution. This is particularly true for the formation of polynuclear complexes. The reactions of oligonuclear and/or mixed hydroxo complexes of Al(III) can be extremely slow, resulting in a nonequilibrium state of Al(III)–ligand systems in biology. Accordingly, the time dependence of the speciation of Al(III) complexes, which may provide a nonequilibrium species characterization of Al(III) in biological fluids and tissues, may be more relevant to biological systems [14,15]. This chapter reviews the role of Al in neurotoxic and neurodegenerative processes. Since the small species are the ‘dynamic or mobile units’ through which Al(III) can be translocated in vivo, it is of particular importance to know which of them predominate under specific circumstances. Accordingly, the complexes of relevant l.m.m. and high molecular mass (h.m.m.) biomolecules, that occur in biological systems, will first be briefly reviewed. This will be followed by a discussion of its interactions with h.m.m. constituents of biological fluids and tissues, focusing mostly on oligopeptides and proteins. A short review on the Al metabolism in humans precedes a description of Al speciation in the blood serum, and the importance of the role of time in biospeciation is demonstrated. After the toxicology of Al(III), the involvement of Al(III) in neurodegenerative disorders mostly in Alzheimer’s disease (AD) is discussed.

2 CHEMICAL FORMS OF ALUMINUM IN BIOLOGICAL SYSTEMS The behavior of Al(III) species in cells and biological fluids can be described in terms of four different forms: (i) free or mononuclear ions; (ii) l.m.m. complexes; (iii) reversible macromolecular complexes; and (iv) irreversible macromolecular complexes.

2.1 Free Ions and Mononuclear Ions In solutions more acidic than pH ∼ 5, the main mononuclear species is AlH2 O6 3+ . As the pH increases, mononuclear and (depending on the concentration) various polynuclear species are formed. Neutral solutions give a precipitate of AlOH3 , which redissolves in the form of AlOH4 − . Al(III)-containing samples readily form supersaturated metastable solutions [14]. For this reason simple inorganic Al(III) salts are not suitable for toxicological studies; instead, well-defined complexes formed with l.m.m. ligands are recommended [15], according to a fixed protocol as suggested by Zatta et al. [16]. Met. Ions Life Sci. 1, 371–393 (2006)

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The speciation and stability of the Al(III)-fluoro complexes have been critically reviewed [17,18]. It has been clarified that AlF4 − , proposed to be a tetrahedral phosphate analogue, is a hexacoordinated octahedral species, containing two molecules of water. Accordingly, its involvement in biological signalling processes as a phosphate analogue should be carefully considered [19]. With inorganic phosphate at neutral pH, Al(III) forms a poorly soluble compound which appears to be a mixed phosphate–hydroxo complex with variable proportions of phosphate and hydroxide [20,21]. Such species provide one of the major modes of elimination of ingested Al(III). Diphosphate, triphosphate and other oligophosphates can form stable six-membered mono and bis chelates with Al(III) via the coordination of adjacent phosphate groups [9,15]. They bind Al(III) about four orders of magnitude more strongly than monophosphate. It has been demonstrated that silicic acid significantly reduces the biological availability and toxicity of Al(III) [22,23], forming hydroxyaluminosilicates [24,25]. Exley [26] carried out detailed studies to explain the role of Al(III)silicates in the biogeochemical cycle of aluminum and its potential involvement in AD [27,28].

2.2 Low Molecular Mass Complexes Simple -amino acids have been found unambiguously to influence the speciation 27 of AlH2 O3+ Al 1 H and 13 C NMR studies in the weakly acidic pH 6 [29,30]. range indicated that they bind in a monodentate way through the carboxylate group, and the amino group is in the protonated form [29,31]. The tridentate Asp, containing two COO− and one central NH2 donor groups, is a significantly stronger Al(III) binder [29]. In small peptides, the terminal and side-chain COO− groups of Asp and Glu proved to be not sufficient to keep Al(III) in solution and to prevent the precipitation of AlOH3 at physiological pH [32]. At the same time, for a larger peptide of neurofibrillary tangle (NFT) origin, Ac-LysSerProValValGluGly, 1:1 complexes in various protonation states have been detected by pH potentiometry and NMR [33]. Phosphorylated Ser-, Thr- or Tyr-containing peptides and their derivatives occur in biological systems, mostly in the NFTs [34,35]. As the phosphate groups of these proteins are fairly basic pKa ∼ 6 − 7, they can bind Al(III) quite strongly [21,36,37]. The complexes of Al(III) with nucleotides and other organic biophosphates have been exhaustively reviewed by Kiss et al. [38] and Nelson [39]. The basic terminal phosphate of the nucleotides pK ∼ 6 is the primary binding site for Al(III) [9,38–41]. Nucleic acids bind Al(III) at least 105 times more weakly than do nucleotides; therefore, DNA can hardly compete with ATP or other nucleotides or biophosphates for Al(III) [36]. Catechol derivatives, including catecholamines, have higher affinity for Al(III) in the neutral and basic pH range, forming the octahedral tris complex AlL3 [14]. Met. Ions Life Sci. 1, 371–393 (2006)

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Aliphatic hydroxycarboxylic acids, such as lactic, malic, citric, tartaric, saccharic, etc. acids, are of special interest in Al(III) biospeciation, as they can exist in the biological fluids and tissues of the human body [15]. Citrate is the most extensively investigated ligand, but, despite the huge number of studies, rather contradictory data have appeared in the literature as concerns the complexes existing at equilibrium [42–44]. The main reason is that the speciation is strongly time-dependent. Mononuclear 1:1 and 1:2 complexes, formed in fairly fast reactions, react to form oligonuclear species [44].

2.3 Reversible Macromolecular Complexes As a consequence of the chemical similarities of Ca(II), Mg(II), and Fe(III) with Al(III), proteins which bind and/or transport these metal ions, are also expected to interact with Al(III) [45,46].

2.3.1 Interactions with Non-enzyme Proteins The chemical speciation of Al(III) is obviously a critical point in attempts to assess which specific compounds cross the blood–brain barrier (BBB) and contribute to the neurotoxicity of Al(III). The most notable papers on this subject deal mostly with the interactions of Al(III) with transferrin, albumin, calbindin, and calmodulin [47]. Some relevant interactions will be discussed later.

2.3.2 Interactions with Enzyme Proteins Table 2 in Ref [47] lists enzymes on which the effects of Al(III) have been investigated. Compilation of such studies are to be found, for example, in the paper by Ganrot [12] and the special issue of Life Chemistry Reports [8]. It may be seen from that table that Al(III) influences (inhibits or activates) not only acid–base enzymes, but also some redox enzymes. Most of them are either activated by Mg(II) or Ca(II) or use nucleotides (mostly ATP) as a cofactor. The effect of Al(III) in these enzymatic processes is generally the displacement of metal ions or its strong interaction with the nucleotides, through their phosphate functions. The effects of Al(III) on enzyme proteins are reviewed in some detail in [47], while the effects of Al(III) on some brain enzymes are discussed in Section 4.2.

2.4 Irreversible Macromolecular Complexes Al(III) is a rather sluggish metal ion, but its practically irreversible binding in biological systems is nevertheless rather rare. It may occur, however, in molecular aggregates, when the exchange reactions of Al(III) are slowed down because of Met. Ions Life Sci. 1, 371–393 (2006)

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the formation of hydrolyzed oxo- or hydroxo-bridged Al clusters wrapped around by organic compounds. Al(III) accumulated in the brain in NTFs and senile plaques (SPs) may represent such complexes. The quantitative information available on these interactions is rather limited. Al(III) coordination has also been detected for larger oligopeptides, such as soluble amyloids A 1–40 and A 6–25 [48,49]. Recent experiments point to the basic contribution of A aggregation to the etiology of AD [50,51]. Besides Al(III), several transition metal ions, such as Zn(II) and the redox-active Cu(II) and Fe(III), may also be involved in amyloid binding [52]. The Al(III) binding sites of phosphorylated and nonphosphorylated fragments of neurofibrillary proteins [53,54], yielding the -pleated sheet structure, are the Glu or phosphorylated Ser. The high stability of these Al(III) complexes [48,49] may be relevant to the mechanism through which the neurotoxin Al(III) could give rise to the formation of NFTs. Interestingly, the CD- and IR-detected interaction could not be measured by multinuclear NMR, suggesting some outersphere coordination between AlOH3 particles and the proteins in these aggregates [33].

3 ALUMINUM LOADING IN HUMANS 3.1 Aluminum Metabolism Human exposure to Al is unavoidable through drinking water, pharmacological products, dust, cosmetics, etc., as may be seen in Figure 1. The metabolism of aluminum is rather complex, though a more precise knowledge becomes possible following the relatively recent utilization of the radioactive isotope 26 Al. In spite of its natural abundance, Al accumulates very poorly in living organisms (0.1%) due to the efficient protection of natural physiological barriers (Figure 1), i.e., the cutaneous, respiratory, GI, BBB, bone marrow, etc. barriers [55]. Use of the radiotracer 26 Al has led to the basic feature of Al biokinetics/bioavailability being unraveled: Al is predominantly retained in the skeleton, with some deposited in the brain [56]. Following 26 Al citrate injection, about 83% of the dose was found to be excreted in the urine and less than 2% in the feces during 13 days; it was noteworthy that the t1/2 of the retained 26 Al was 7 years confirming the limited ability of systemic aluminum to be eliminated via the GI tract [57]. Yokel et al. [58] reported that 26 Al concentrations in the rat brain were decreased only slightly from 1 to 35 days after systemic 26 Al injection, either in the absence or in the presence of the aluminum chelator deferroxamine, suggesting prolonged brain Al retention. The GI tract is a formidable, but not impervious barrier to the entry of Al, and the metal speciation is of paramount importance in Al uptake and excretion. Physiologically, the pH plays a central role in the solubility of Al compounds, and it is likely that absorption occurs in the acidic medium of the stomach and Met. Ions Life Sci. 1, 371–393 (2006)

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Figure 1. Metabolic pathways of the absorption and transport of aluminum from the external environment to various compartments (organs and tissues) of the body. The various biological barriers strongly protect from metal ion accumulation that could be detrimental in terms of toxicological impact.

proximal duodenum. Moreover, the combination of aluminum and citrate (which form a neutral complex at pH ∼ 5 [15]) considerably enhances the GI absorption of Al [5]. Other factors that affect Al absorption are fluoride and silicon. The former forms stable complexes, apparently decreasing Al excretion; this aspect warrants further studies. Silicon has been proposed to form insoluble compounds with Al, thereby decreasing absorption [59–61], but the clinical evidence is not unambiguous. Feinroth et al. [62] proposed an energy-dependent process for Al absorption through use of an everted gut sac from the rat small intestine; however, whether the GI absorption of Al is related to a concentration gradient of ionized aluminum or to an active transport mechanism, is still rather unclear.

3.2 Speciation of Al(III) in Blood Serum Absorbed Al(III) is transported by the bloodstream. Different speciation models [15,20,63,64] have been reported in the literature, in connection with the Met. Ions Life Sci. 1, 371–393 (2006)

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actual chemical forms of Al(III) in the blood serum. As concerns the protein fraction, all models agree that most of the Al(III) is bound to transferrin. Albumin, another potential carrier protein, is a much weaker metal ion binder and is not assumed to be able to compete for a significant amount of Al(III) [9,65]. The picture is much more controversial as concerns the more mobile l.m.m. Al(III) binders [15,20,63,64]. The reason is the lack of reliable speciation data. In the Al(III)-phosphate system, under model conditions in the mM concentration range, precipitation occurs at pH > 3 5, but extrapolation from the experimental data to physiological conditions may be rather dangerous [21]. Further, there are rather slow oligomerization reactions in the Al(III)-citrate system, which are strongly concentration-dependent. At mM concentrations, a trinuclear species Al3 CitH−1 3 OH4− predominates in a wide pH range including the physiological pH, in a thermodynamic equilibrium state [42]. This trinuclear complex is formed, in a rather slow process, via the interactions of mononuclear 1:1 and 1:2 complexes, which exist at the beginning of complex formation and also predominate at high excesses of the ligand [63]. Electrospray ionization mass spectrometry (ESI-MS) measurements have proved that the halflife of formation of the trinuclear species at about 10 M Al(III) is ∼80 min [64]. It is reasonable to question whether the thermodynamic equilibrium approach provides a realistic description of the Al(III) speciation under physiological conditions: when the Al(III) concentration rarely exceeds a few M, the ligand excess for potential binders is at least 1000- to 10,000-fold, and, in consequence of the continuous metabolism, biological fluids are open systems, which never reach true thermodynamic equilibrium. The problem of time-dependent speciation is a real challenge in Al(III) bioinorganic chemistry [43]. Harris et al. [64] used difference UV spectroscopy as the basic method, supported by pH-metric and ESI-MS measurements. They reported a model for the biospeciation of Al(III) in serum at ∼10 M Al(III), which is much closer to the biologically relevant value, ∼01–0 3 M [66,67]. Their speciation model (Table 2) indicates that 93% of the total Al(III) is bound to transferrin. Of the pool of l.m.m. Al, 88% would be bound to citrate, 8% to hydroxide and only ∼2% to phosphate [64]. The speciation models reported so far ignored the formation of ternary complexes between citrate and phosphate. Recently, the time-dependent speciation in the Al(III)-citrate A3− -phosphate B3−  ternary system has been monitored by pH-potentiometry and multinuclear NMR spectroscopy [63]. Table 2 presents the species distribution of Al(III) under plasma conditions. At physiological pH, phosphate seems to be the more efficient Al(III) binder, although citratebound Al(III) also occurs in significant concentration, in accordance with the results of Bell et al. [68], who detected Al(III)-bound citrate in native serum by means of 1 H NMR. Bantan et al. [67] studied the speciation of l.m.m. Al(III) complexes in serum from healthy volunteers by means of fast liquid chromatography and electrospray techniques. In agreement with the results discussed Met. Ions Life Sci. 1, 371–393 (2006)

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Table 2. Speciation of Al(III) with blood serum components at pH ∼7 4 and 25  C. % of Al(III) bound cAlIII = 10 M 64 High molecular mass components Albumin Transferrin Low molecular mass components Phosphate Citrate Citrate–phosphate

93 0 1 6

cAlIII = 1 mM 63

77 14 5 4

above [63], they found that the main l.m.m. Al(III) species present in the serum are binary Al(III)-citrate, Al(III)-phosphate and ternary Al(III)-citrate-phosphate complexes. The distribution of these complexes varied from individual to individual.

4 TOXICOLOGY OF ALUMINUM IN ANIMALS AND HUMANS 4.1 Experimental Toxicology As the brain is the recognized target for Al(III) toxicity, a number of studies have been carried out to establish a correlation between the internal Al load and the functions of the central nervous system (CNS). The neurotoxicity of Al has been known for more than a century, on the basis of observations following the subcutaneous administration of Al salts (e.g., Al(III)-tartrate or -lactate) to rabbits, cats, and dogs. The resulting neurodegeneration was particularly severe in the lower cranial nerves [4]. Brain autoptic examination revealed nerve cell degeneration with inflammatory reactions. New interest was aroused in Al neurotoxicity when Klatzo et al. [69] produced convulsions with neurofibrillary degeneration (NFD) by injecting Holt’s adjuvant, (mainly AlPO4 ) intracranially into rabbits. This NFD was apparently similar to that observed in AD, but the ultrastructure revealed different features [70]. Numerous experiments have revealed that Al(III) induces NFD in aluminumsensitive animals, e.g., cats, ferrets, rabbits, and dogs. However, rodents such as hamster, guinea pig, rat, and mouse, similarly to monkeys and man, appear to be remarkably resistant [2]. Al(III) is known to alter the permeability of the BBB substantially. The observed enhancement of the permeability is correlated with the basal permeability of the substances and with their lipid solubility. This suggests that Al(III) is Met. Ions Life Sci. 1, 371–393 (2006)

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able to enhance lipid-mediated nonsaturable membrane diffusion (for reviews on Al(III) and BBB permeability see [55,58,71]). It has further been demonstrated that Al(III) is able to alter the BBB function either permanently or transiently, depending on the physicochemical properties of the metal coordination sphere of the compound applied [69]. Johnson et al. [72] reported that the injection of Al(III) into rabbits produced profound changes in the architecture of the cytoskeletal proteins, with the consequent production of neurofibrillary pathology in the neurofilament proteins [73]. They demonstrated that the tau proteins, a family of proteins associated with the cytoskeletal filaments, were the component relevant to Al-induced NFT formation. Tau and NFT proteins are deeply altered in a number of neurodegenerative diseases called tauopathies. In this connection, Shin et al. [74] demonstrated that Al(III) modulates the properties of injected PHFtau proteins in the rat brain by retarding the proteolysis of these proteins and favoring the accumulation of amyloidogenic material as well as the co-deposition of amyloid, ubiquitin, and apolipoprotein E, all pathological features observed in AD brains. These data are explained in terms of the ability of Al(III) to bind selectively to domains in PHTtau that contain phosphorylated Ser or other residues, and this in turn justifies the in vivo aggregation and delayed proteolysis of PHFtau . Recently, Praticò et al. [75] fed transgenic mice (Tg 2576) overexpressing human amyloid precursor protein, and the wild-type as a control, with a diet enriched in 2 mg/kg Al(III)-lactate. They observed that the Tg mice displayed elevated concentrations of soluble and insoluble amyloid A 1–40 and A 1–42 in the cortex and hippocampus as compared with mice fed with a normal diet. These data are indicative as to how Al(III) may effectively act in reproducing in vivo one of the most relevant features observed in AD, the overaccumulation of amyloids. Some authors have proposed that an abnormal accumulation of -amyloids observed in the AD brain is due to a reduced activity of proteolytic enzymes [76,77]. In this connection, it is worth mentioning that Al(III) seems to inhibit the proteolytic activity of chymotrypsin and trypsin [78,79]. However, the molecular mechanism that could explain such an inhibition is still a matter of discussion.

4.2 Biochemical Toxicology As discussed briefly above, and comprehensively in [47], the interactions of Al(III) with non-enzyme proteins will affect the mobility of this metal ion and more importantly the structures of the proteins with which it interacts (see Section 2.4). At the same time Al(III) is able to either inhibit or activate many enzymatic pathways (see, e.g., [12,47]). It has recently been demonstrated that Al(III) is able to modify the activities of those key enzymes that have been observed to be altered in AD and affected in other neurological processes. Met. Ions Life Sci. 1, 371–393 (2006)

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4.2.1 Acetylcholinesterase Acetylcholinesterase (AChE) is a key enzyme that possesses multiple molecular forms differing in the number of catalytic subunits. It is present in both the CNS and the peripheral nervous system. AChE is strongly activated in AD, and this is the reason why an anticholinesterasic therapeutic approach has been carried out for many years though with modest results. It has been postulated that the cholinotoxic effect produced by Al(III) is not simply due to a direct interaction between Al(III) and the cholinergic protein, but is a consequence of a neurodegenerative effect [80]. In vitro, Al(III) has a strong activating effect on AChE. This increase in enzymatic activity can be attributed to a structural modification produced by the interaction between Al(III) and AChE, as has been demonstrated by spectroscopic measurements [81].

4.2.2 Monoamine Oxidase Monoamine oxidase (MAO) is present in two isoforms, called A and B, and catalyzes the oxidative amination of various amines, an activity that is enhanced in the brain with ageing. In particular, the activity of MAO-B was found to be significantly elevated in the hippocampus and cortex gyrus of AD patients [82]. In neuroblastoma cells, Al(III) is able to strongly activate both MAO-A and B [83] and could be related in this respect with the elevation observed in AD.

4.2.3 Carbonic Anhydrase A carbonic anhydrase (CA) dysfunction impairs cognition and has been proposed to be associated with mental retardation both in AD and in ageing [84].

4.2.4 Na+ /K+ -ATPase The Na+ /K + -ATPase activity has recently been shown to be strongly altered in a rat AD model; this is most probably due to an alteration in the lipid environment of the membrane [85]. It has been shown that the activities of these two enzymes are also strongly altered by the Al(III) ion. In fact, while CA is markedly inhibited, Na+ /K + -ATPase is strongly activated [86].

4.2.5 ATP-dependent Enzymatic Activities It is worth bearing in mind that Al(III) is significantly complexed in biological systems, and this phenomenon is amplified when Mg2+ is depleted. There is little doubt that Mg2+ can be replaced by a variety of divalent cations such as Met. Ions Life Sci. 1, 371–393 (2006)

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Mn2+  Co2+  Ni2+ , and Zn2+ [87]; in biological systems, however, Mg2+ cannot be replaced by these divalent cations as their overall concentration is 104 times lower. Since Al(III) binds to phosphate significantly more strongly than does Mg2+ [15,20,21], there is the possibility that Al(III) occupies Mg2+ sites in various cell compartments; these sites could include phosphorylated tubulin [87]. Moreover, it is useful to remember that a Mg2+ depletion is a rather common physiological feature in the aged [88]. Al3+ binds 560 times more strongly than Ca 2+ to the surface of phosphatidylcholine vesicles [89]. Stability constants have been determined for ATP, ADP, AMP, 2,3-DPG and HOPO2− 3 and Al(III) [41,46,90]. Al(III) is able to strongly influence all enzymes that depend on the ATP metabolism [22,72,91]. It is worth mentioning that the glucose metabolism, which is strictly related to the ATP metabolism, has been observed to be altered in AD. Glucose is the major source of energy in the CNS, and any impairment in cerebral glucose oxidation can be expected to result in deficits in both acetylcholine synthesis and ATP formation, which might also contribute to alter the amyloid precursor protein (APP) processing and enhance the susceptibility to Al(III) neurotoxicity [92]. Thus, the interaction between Al(III) and ATP is relevant in influencing many cellular and enzymatic processes, e.g., the synthesis of acetylcholine, glutamate, aspartate and GABA [93], and, at least in in vitro and in vivo models Al is able to modify all these processes.

4.3 Human Toxicology Al toxicity symptoms include constipation, colic, decreased appetite, nausea, skin ailments, twitching leg muscles, increased perspiration, fatigue, motor paralysis, local numbness, and fatty degeneration of the kidneys and liver, resulting from decreased levels of calcium and phosphorus. Measurable concentrations of Al in the brain tissue of human embryo, which gradually increase during gestation, reaching the highest level in the newborn, have long been reported [94]. The blood of premature infants contains more Al than that of those delivered at term, and at delivery the newborn blood contains a higher level of Al than that of the mothers [95]. There is a general consensus that Al accumulates with aging [96,97]. After these and other paradigmatic cases, it is well established that Al can lead to neurological, skeletal, and hematological disorders [5]. Neuropsychological assessments suggest detrimental effects of Al on short-term memory, learning, and attention. Exposure to high levels of aluminum can affect the human CNS, as observed among welders exposed to Al fumes for several years [98,99]. A recent examination of 64 former aluminum dust-exposed workers suggested a possible role of the inhalation of aluminum dust in preclinical mild cognitive impairment (MCI) which might be a prelude to AD or an AD-like neurological Met. Ions Life Sci. 1, 371–393 (2006)

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deterioration [100]. Event-related potential (ERP-P300), mini-mental state examination (MMSE), MME-time, clock drawing test (CDT) CDT-time and CDTscore as preclinical tests suggested 10 g/L as a cut-off level of serum aluminum with a concomitant high blood level of iron to compete with Al accumulation. In this connection, it has been clearly demonstrated in rats that Al selectively accumulates in the brain via inhalation in a metal speciation-dependent way [101]. Al accumulation and compartmentation in the CNS in rats exposed to inhalation of Al(III)-acetylacetonate (highly lipophilic) for 2 weeks led to the presence of the metal ion in the brain cortex, the hippocampus, the entorhinal area, the olfactory bulb as well as in the Purkinje cells of the cerebellum, and the white matter. This was significant in that it demonstrated the mobility of the metal ion in the brain once the external barrier was crossed, together with the relevance of metal speciation in defining the accessibility and the vulnerability of the protective physiological barriers. The olfactory pathways as a route of entry of metal ions in the brain have recently been reviewed by Tjalve and Tallkvist [102].

5 ALUMINUM AND ALZHEIMER’S DISEASE Metal ions have been implicated as potential risk cofactors in several neurodegenerative disorders (for recent reviews, see, e.g., [103,104]). However, only dialysis encephalopathy, non-iron-dependent microcytic anemia and osteomalacia have been clearly attributed to aluminum in chronic dialysis treatment [105]. Several books and excellent reviews are available on this issue [10,106–110], and for this reason it will not be treated specifically in this chapter. There is now a general consensus, based on morphological, statistical, and epidemiological results, that in subjects undergoing chronic dialysis the pathognomy related to Al(III) accumulation is predominantly due to the utilization of aluminum-containing products, present either in the dialytic solutions or in the pharmacological treatment. On the other hand, it is extremely informative that the available results from a wide range of literature suggest that Al does not cause any increase in AD morphology, at least not in terms of bioavailable Al in the drugs utilized in long-term hemodialysis. Iatrogenic fatal encephalopathy confirms the extraordinary neurotoxicity of Al/Al compounds in direct contact with the CNS parenchyma [111,112]. In this context, it appears of paramount importance to distinguish the concept of neurotoxicity from neurodegeneration [113]. While the clinical neurotoxicity of metal ions results in neurodegeneration, the pathophysiology of AD, and other diseases such as Parkinson disease, amyothrophic lateral sclerosis, etc., is characterized by the accumulation of certain redox active metal ions (e.g., iron and copper) in the CNS, which eventually leads, among others, to free radical-mediated oxidative stress, the altered misfolding of certain proteins, the modification of enzymatic activities, and eventually neuronal death [113]. It remains to be established whether an abnormal accumulation is the primary or Met. Ions Life Sci. 1, 371–393 (2006)

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secondary event in the pathogenic processes. However, the aluminum hypothesis in AD has been and continues to be controversial [11,114–118]. Many questions have been raised, but no clear answer has been given as concerns the possible link between Al and AD. In spite of numerous uncertainties, this issue remains of great scientific interest, even if the AD scientific community largely tends to ignore it (see, for instance, the AD website: www.alzforum.org). Here we merely refer to some papers [69,119–124] illustrating the rather contradictory history of the involvement of Al in AD. The recent book edited by Exley [11] compiles the scientific evidence that Al has abundant possibilities to interfere with the normal neurochemistry/neurobiology of the brain so as to induce or enhance AD-type disorders. As mentioned above, Al(III) can enhance the formation of NFTs through interactions with the (hyper)phosphorylated proteins involved in the pathogenesis of tangle formation. Furthermore, as Al(III) can interact strongly with phosphates, polyphosphates and nucleotides, it can affect numerous enzymatic processes involving nucleotides. This basic chemical evidence cannot be ignored, especially since some of the results have been confirmed biologically by in vitro or even in vivo studies. After many years of studies and thousands of publications, no unequivocal evidence emerged on the role of Al(III) in the pathogenic changes, signs, and symptoms observed in AD and other neurodegenerative diseases. However, some epidemiological studies do seem to indicate a potential correlation between aluminum in drinking water and the occurrence of AD [125,126]. Al is currently and commonly utilized in nephrological treatment to reduce hyperphosphatemia, without any particular manifestation of AD in nephropathic patients on chronic dialysis. An interesting case of aluminum encephalopathy has recently been reported from our laboratory, involving a subject not in dialysis who developed the classical Al encephalopathy, after taking Maalox (aluminum hydroxide), a common antacid. Autoptic examination revealed an overaccumulation of Al in several cellular elements in the brain, and the clinical case ended fatally in a relatively short time [127]. Individual sensitivity to aluminum accumulation must therefore be seriously considered as an issue of paramount importance. Among the features that characterize AD, amyloid aggregation, NFT, and hyperphosphorylation of tau proteins are the most common. NFTs persists in the extracellular space after neuronal death, together with APP-containing dystrophic neurons, microglia, astrocytes, P-component, cholinesterase, extracellular matrix proteins, metal ions, etc. All these materials define senile plaques (SPs). There is now a vast literature on the role of metal ions in producing protein misfolding and concurring on protein (amyloid in AD) aggregation and precipitation in several neurodegenerative diseases (for an up to date recent review, see [128]). Several recent papers have reported the aggregating effect of Al(III) on amyloids [75,129,130] (Figure 2). The reduced availability of ATP due to its interaction with Al(III) has been also invoked as the cause of damage to the function of the endoplasmic reticulum Golgi apparatus/trans Golgi network, generating Met. Ions Life Sci. 1, 371–393 (2006)

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Figure 2. Electron microscopy: amyloid fibrils formed in the presence of 10 M Al(III) at neutral pH. (A) 1 M amyloid fragment 1–38; (B) 1 M amyloid fragment 1–42.

misfolded/malfolded proteins retained by the cell. In conclusion, Al is able to accelerate the formation of fibrils with misfolding. It has been hypothesized [116] (Figure 3) and demonstrated [74,75] that hyperphosphorylated tau could be the relevant site of Al accumulation. It is reasonable to think that in AD phosphorylated filaments, which may be aberrant, are an ideal site for the binding of Al in cells which have suffered an influx

0

0 0

AI 0 0 0

Microtubules

0H

P 0– 0

0 TAU

0– 0 0 0 AI 0 0 0

TAU

H0 0

P 0

0

Microtubules

Figure 3. Hypothetical interaction between Al(III) and hyperphosphorylated tau proteins in AD as a possible beginning of protein aggregation. Met. Ions Life Sci. 1, 371–393 (2006)

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of Al ions. Consequently, Al may stabilize the phosphorylated proteins and the filaments are likely to be cross-linked by Al (Figure 3). NFT may be a result of the cross-filaments and the plaque initiated intracellularly would finally appear as an extracellular body [131].

6 GENERAL CONCLUSIONS • The neurotoxicity of Al has been established in both in vivo and in vitro models, and also in humans undergoing clinical intoxication, e.g., patients with an impaired renal function, with the potential consequence of the development of mild cognitive impairment and dialysis encephalopathy. • Experimental work has demonstrated that many factors can influence the uptake and fate of Al in the human and the animal brain (e.g., age, renal function, pharmacological treatment, diabetes, parathyroidectomy treatment, calcium depletion, environmental exposure, phosphate metabolism, nutritional state, chemical speciation, etc.). • The presence of Al(III) has been clearly demonstrated in the NFTs in the AD brain through the use of different instrumental analyses and procedures. However, no conclusive proof exists so far for a direct connection between Al and AD. • The accumulation of Al in the brain of patients with dialysis encephalopathy is greater than that observed in the brains of patients with AD; moreover, the metal distribution in the various topological areas, its localization within the neurons and the mechanism of action all appear to be different in AD than in dialysis encephalopathy. • The molecular basis of Al(III) neurotoxicity is still largely unknown. In AD, environmental factors can contribute considerably to the accumulation of Al(III) and thus to an acceleration/aggravation of the pathological condition. • Primary lesions in AD and in dialysis dementia have been postulated to be due to an impairment of the BBB permeability, which allows Al to reach the brain in a metal speciation-dependent way. Consequently, Al(III) might alter the synthesis and metabolism of various chemical compounds in various biochemical pathways, as observed in demented brains. • Al has deleterious effects on many biochemical processes at far less than the mM level, e.g., in tubulin aggregation 10−10 M InsP3 -evoked Ca2+ signalling 10−6 M, amyloid aggregation <10−6 M. • As a phosphate binder, Al(III) is able to enhance the aggregation of neuron degradation products through the formation of NFTs and SPs. • Al(III) is able to alter the activities of proteolytic enzymes profoundly and this could alter the metabolism of the amyloids significantly. • It is important to note that during the International Conference on Metals and the Brain in 2000, the general consensus was reached that aluminum exposure Met. Ions Life Sci. 1, 371–393 (2006)

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and intoxication should be avoided as much as possible not only in subjects affected by neurological disorders or undergoing chronic dialysis treatment or parenteral nutrition, but also as a recommendation for the general population (www.bio.unipd.it/∼zatta/aluminum.html).

ACKNOWLEDGMENTS This work was supported financially by the Hungarian National Research Fund (OTKA No. T037385); K. G.-S. thanks the Ministry of Education, Hungary, for the Békésy György scholarship (BÖ 9/2003).

ABBREVIATIONS 2,3-DPG A AChE AD ADP AMP APP ATP BBB CA CDT CNS ERP ESI-MS GABA GI h.m.m. InsP3 l.m.m. MAO MCI MME MMSE NFD NFT PHF SP

2,3-diphosphoglycerate -amyloid acetylcholinesterase Alzheimer’s disease adenosine 5’-diphosphate adenosine 5’-monophosphate amyloid precursor protein adenosine 5’-triphosphate blood–brain barrier carbonic anhydrase clock drawing test central nervous system event-related potential electrospray ionization mass spectrometry -aminobutyric acid gastrointestinal tract high molecular mass inositol-1,4,5-triphosphate low molecular mass monoamine oxidase mild cognitive impairment mini-mental examination time mini-mental state examination neurofibrillary degeneration neurofibrillary tangle paired helical filament senile plaque

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14 Neurotoxicity of Cadmium, Lead, and Mercury Hana R. Pohl, Henry G. Abadin, and John F. Risher Agency for Toxic Substances and Disease Registry (ATSDR), US Department of Health and Human Services, Division of Toxicology, Mailstop F-32, 1600 Clifton Road, Atlanta, GA 30333, USA

1 INTRODUCTION 2 CADMIUM 2.1 Toxicokinetics and Mechanism of Action 2.1.1 Toxicokinetics 2.1.2 Mechanism of Action 2.2 Neurotoxicity Studies in Humans 2.3 Neurotoxicity Studies in Animals 3 LEAD 3.1 Toxicokinetics and Mechanism of Action 3.1.1 Toxicokinetics 3.1.2 Mechanism of Action 3.2 Neurotoxicity Studies in Humans 3.2.1 Neurological Signs and Symptoms in Adults 3.2.2 Behavioral Function in Adults 3.2.3 Peripheral Nerve Function in Adults 3.2.4 Neurological Signs and Symptoms in Children 3.2.5 Behavioral Function in Children 3.2.6 Electrophysiological Evidence of Neurotoxicity in Children 3.2.7 Peripheral Nerve Function in Children 3.3 Neurotoxicity Studies in Animals Metal Ions in Life Sciences, Volume 1 © 2006 John Wiley & Sons, Ltd

Edited by Astrid Sigel, Helmut Sigel and Roland K. O. Sigel

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4 MERCURY 4.1 Toxicokinetics and Mechanism of Action 4.1.1 Toxicokinetics 4.1.2 Mechanisms of Toxicity 4.2 Neurotoxic Effects in Humans 4.3 Neurotoxic Effects in Experimental Animals 5 CONCLUSIONS ABBREVIATIONS REFERENCES

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1 INTRODUCTION Cadmium, lead, and mercury are very important metals from the public health viewpoint. Cadmium, lead, and mercury (metallic and inorganic) are released to the environment from hazardous waste sites and from the burning of fossil fuels, mining, smelting, and industrial activities. In the United States, release of cadmium from human activities is estimated in the range of 4000 to 13 000 tons per year, with major contributions from mining and the burning of fossil fuels. Food and cigarette smoke are the primary sources of cadmium exposure for the general population. Mining and smelting operations and commercial uses have led to the widespread distribution of lead in the environment and consequential exposure to human populations. In the United States, pollution control measures and bans on the use of lead in many commercial products have resulted in significant reductions in exposure. Past uses, however, continue to pose human health threats from exposure to, among other things, lead-based paint in older housing, contaminated soils from the use of leaded gasoline, leaching of lead into drinking water from lead-soldered joints in plumbing systems, and soil contamination near mining and smelter sites. Worldwide, lead consumption is mostly due to the production of lead–acid batteries. This is also true for the United States where battery production comprises over 80% of the consumption. Lead also has the highest recycling rate of any metal, with over 75% of the lead requirements in the United States being met by recycling, mostly from lead–acid batteries. Although most of the world production of mercury is generated by mercury mines, most of the mercury produced in the United States comes from secondary production sources (i.e., recycling). Recycling includes the processing of scrapped mercury-containing products, and industrial waste, and scrap. Three general chemical forms of mercury exist: metallic mercury, inorganic mercury, Met. Ions Life Sci. 1, 395–425 (2006)

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and organic mercury compounds. For the general population, the most important pathway of exposure to mercury is ingestion in food, especially in fish and other seafood.

2 CADMIUM 2.1 Toxicokinetics and Mechanism of Action 2.1.1 Toxicokinetics Cadmium exists in air primarily as fine suspended particulate matter. Alveoli are the major site of absorption for an estimated 50–100% of deposited particles <01 m in size [1]. Only about 5% of the larger particles >10 m deposited in the upper respiratory tract are actually absorbed. Most ingested cadmium (about 95%) passes through the gastrointestinal tract without being absorbed. Absorption from the gut appears to take place in two phases, uptake from the lumen into mucosa, then transfer into the circulation. Factors affecting cadmium absorption include metal–metal (e.g., iron, calcium, chromium, magnesium, zinc) and metal–protein interactions (glutathione, sulfhydryl-containing enzymes) in the body and in the food or water. Levels of other metals and proteins can vary with age and physiological status, and affect cadmium kinetics. Cadmium absorption is known to increase with iron or calcium deficiency and increased fat in the diet (i.e., longer residency times for absorption to occur). Cadmium is not well absorbed by the skin (about 0.5%). Following absorption from any route of exposure, cadmium distributes widely throughout the body, with the major portion ending up in the liver and, especially in the kidney. Average cadmium concentrations in the kidney are near zero at birth, and rise with age to a peak (typically around 40–50 g/g wet weight) between ages 50 and 60, after which kidney concentrations plateau or decline. Liver cadmium concentrations also begin near zero at birth, increase to typical values of 1–2 g/g wet weight by age 20–25, then increase only slightly thereafter [2,3]. The choroid plexus represents the blood–cerebrospinal fluid barrier for heavy metals including cadmium. Cadmium tends to accumulate in the choroid plexus at concentrations much higher than those found in the brain [4,5]. Cadmium does not undergo any direct metabolic conversion, but the cadmium +2 ion readily binds to anionic groups in proteins and other molecules [1]. When absorbed, cadmium in plasma circulates bound primarily to albumin and subsequently to metallothionein. However, upon entering the liver, cadmium forms a complex with metallothionein that is released back into the bloodstream. Metallothionein, which is also found in the lungs and gastrointestinal tract, is a low-molecular-weight protein capable of binding as many as seven cadmium atoms per molecule. Metallothionein is inducible in most tissues by exposure to cadmium, zinc, and other metals, as well as organic compounds and a variety Met. Ions Life Sci. 1, 395–425 (2006)

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of other physiologic stresses [6]. Metallothionein-bound cadmium is readily filtered by the renal glomerulus and reabsorbed from the glomerular filtrate by the proximal tubule cells. The complex is catabolized by tubular lysosomes releasing free cadmium. That in turn stimulates the synthesis of metallothionein in the tubular cells; cadmium is recaptured, and remains in kidney cells bound to the metallothionein until the cellular capacity to produce metallothionein is exceeded. This process is important in protection against chronic cadmium nephrotoxicity [7]. Of the cadmium that is absorbed into the body, most is excreted very slowly, with urinary and fecal excretion being approximately equal. Half-times for the human kidney have been estimated at between 6 and 38 years, and for the human liver at between 4 and 19 years [8]. The placenta is only a partial barrier to fetal exposure to cadmium. Cadmium concentration was found about half as high in cord blood as in maternal blood in several studies, including both smoking and nonsmoking women [9,10]. Cadmium can be excreted in human milk at levels from 5–10% of the levels in maternal blood [11].

2.1.2 Mechanism of Action The toxic effects of cadmium are generally attributed to free cadmium ions (i.e., cadmium not bound to metallothionein) [12]. Metallothionein present in the choroid plexus is likely responsible for sequestering of cadmium that results in lower cadmium levels in the brain [13]. In the process, cadmium affects the choroid plexus itself; cadmium-induced toxicity is characterized by the loss of microvilli and a rupture of the apical surface. Cadmium can directly affect the function of the nervous system; it was demonstrated to have synaptic blocking effects at both adrenergic and cholinergic synapses [14]. However, cadmium does not interfere with the transmission of action potentials into presynaptic nerve terminals. Rather, cadmium blocks transmitter release by competing for calcium receptor sites located on the outer surface of the presynaptic nerve terminal [14]. The effect of cadmium can be reversed by increasing the extracellular calcium concentrations [15]. Cadmium-induced injury in the cerebral microvessels is likely associated with oxidative stress [16]. Free cadmium ions may induce various adverse effects including inactivation of metal-dependent enzymes, activation of calmodulin, and initiation of production of oxidation derivatives [6,17]. Reduced activity of enzymes, in particular of glutathione reductase and glucose-6-phosphate dehydrogenase, was reported in brain regions of growing rats exposed to cadmium [18]. Cadmium also inhibited the activity of antioxidant enzymes, including catalase, manganese-superoxide dismutase, and copper/zinc-superoxide dismutase [19,20]. The inhibition correlated with increased cadmium levels in corresponding brain regions. The biochemical changes were not observed in hippocampus, a finding Met. Ions Life Sci. 1, 395–425 (2006)

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agreeable with no increases of cadmium levels in that region [18,20]. Cadmiuminduced lipid peroxidation was observed in tissues from various organs, including the brain. Because cadmium does not undergo redox reaction, it was speculated to cause iron-induced lipid peroxidation by displacing iron from its biological binding sites [21]. Increases in lipid peroxidation in brain homogenates of rats exposed to cadmium were dose dependent [22]. Curcumin, a naturally occurring antioxidant, protected against the peroxidation. In addition, reaction of cadmium with certain sulfhydryl groups can alter cellular metabolism and function which in turn can cause cellular damage. For example, rats treated with cadmium (i.p.) for 30 days had a decrease in reduced glutathione (GSH) levels and an increase in oxidized glutathione (GSSG) levels in various brain regions [18]. Further, the production of free radicals may alter mitochondrial activity and genetic information [23]. For example, a chronic low-dose cadmium exposure of rats caused increases in single-strand breaks in the brain cells [24]. An in vitro experiment with brain cell cultures from Pleurodeles waltl, demonstrated that cadmium caused DNA strand breaks and inhibited the repair of oxidative DNA damage [25].

2.2 Neurotoxicity Studies in Humans Kidneys are the primary target of cadmium toxicity. Studies on neurotoxicity associated with exposure to cadmium in humans are limited. An occupational study reported a modest correlation between cadmium exposure and decreased performance on neurobehavioral tests for attention, psychomotor speed, and memory in a small cohort of 31 men exposed to cadmium in workplace air (average exposure = 145 years) [26]. Another occupational study found slowing of visual motor functioning on neurobehavioral testing, increase of complaints indicative of peripheral neuropathy and problems with equilibrium and concentration ability in a cohort of 42 cadmium-exposed workers as compared with their matching controls [27]. The findings correlated with cadmium in urine samples. In addition, workers chronically exposed to cadmium fumes that induced renal damage also had impairment in olfactory function [28]. Several epidemiological studies attempted to link environmental exposure to cadmium and neurological effects. These studies used hair cadmium as an index of exposure. End points that were affected included verbal IQ in a cohort of 149 rural Maryland children [29], acting-out and distractibility in a cohort of 80 rural Wyoming children [30], and disruptive behavior in a cohort of 40 Navy recruits [31]. The usefulness of the data from these studies is limited because of the potential confounding effects of lead exposure; in fact, some effects were seen only in conjunction with hair lead contents [30]. In addition, lack of control for other possible confounders, including home environment, caregiving, and parental IQ levels, and inadequate information on cadmium exposure levels can be noted in the studies. Met. Ions Life Sci. 1, 395–425 (2006)

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2.3 Neurotoxicity Studies in Animals Information regarding neurotoxicity following inhalation exposure is limited. Acute inhalation exposures to cadmium caused neurological effects in rats (tremors and reduced activity) only at high levels (132 mg Cd/m3 and 112 mg Cd/m3 , respectively) that also induced mortality [32]. Continuous exposure at lower levels 0105–1034 mg Cd/m3  was not neurotoxic [33]. In contrast, the evidence for neurotoxicity is quite strong in oral exposure studies. Motor activity decreased by 50% in rats after a single oral dose of 50 mg Cd/kg/day [34]. Similarly, decreased motor activity was reported in rats following low-dose exposures to cadmium in drinking water for up to 24 weeks [35] or in their feed for 60 days [36]. Other neurobehavioral changes observed in rats after 55–60 days exposure to 5 mg Cd/kg/day in the feed included increased passive avoidance [37] and increased voluntary ethanol consumption [38]; both effects being indicative of increased anxiety in the animals. Rats that received a higher dose of 40 mg Cd/kg/day by gavage for 14 weeks exhibited aggressive behavior [39]. Degenerative changes in the choroid plexus have been found in mice exposed to 1.4 mg Cd/kg/day in drinking water for 22 weeks [40]. Peripheral neuropathy has been reported in rats after a 31-month exposure to 3.6 mg Cd/kg/day in drinking water [41]. A shorter exposure caused weakness and muscle atrophy. Increased brain dopamine levels and decreased enzyme activities were observed in rats exposed to 24 mg Cd/kg/day for 90 days in drinking water [42]. Nerve cell or brain damage following parenteral exposure indicating neurodevelopmental effects in the offspring have also been reported [43,44]. Four-day-old rats were more susceptible to lesions in the corpus callosum, caudate putamen, and cerebellum than were 8-week-old adults following a single subcutaneous injection of cadmium chloride. Postexposure brain lesions and hyperactivity were also evident in newborns at dose levels that produced no such effects in adults [43].

3 LEAD 3.1 Toxicokinetics and Mechanism of Action 3.1.1 Toxicokinetics Once deposited in the lower respiratory tract, particulate lead is almost completely absorbed, and all chemical forms of lead seem to be absorbed [45,46]. The extent and rate of gastrointestinal absorption are influenced by physiological states of the exposed individual (e.g., age, fasting, nutritional calcium and iron status) and physicochemical characteristics of the medium ingested (e.g., particle size, mineralogy, solubility, lead species). Gastrointestinal absorption appears to be higher in children than in adults [47,48]. Met. Ions Life Sci. 1, 395–425 (2006)

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Following absorption, lead is distributed to the blood and soft tissues with final accumulation in the bone where it accounts for about 90% of the body burden in adults, and about 70% in children [49]. The reservoir of bone lead in adults can serve to maintain lead levels in the blood long after exposure ends [50–52] and serve as a source of exposure to the fetus during catabolization of the maternal skeleton [53–55]. Two physiological compartments appear to exist to varying degrees for lead in cortical and trabecular bone. In one compartment, bone lead is essentially inert, having a half-life of several decades. A labile compartment exists as well that allows for maintenance of the equilibrium of lead between bone and soft tissue or blood [56–58]. Of the lead in blood, 99% is bound within the red blood cells with 50% bound to hemoglobin [45]. The half-life of lead in adult human blood has been measured as 36 days by Rabinowitz et al. [57] and 28 days by Griffin et al. [59].

3.1.2 Mechanism of Action The mechanism of lead neurotoxicity is not well understood although several mechanisms have been proposed. Lead Pb2+  may substitute for Ca2+ in Ca2+ dependent processes involved in synaptic transmission that are essential for learning, memory, growth, and differentiation of nerve cells, and motor function. Lead blocks the voltage-regulated calcium channels, inhibiting the influx of Ca2+ and release of neurotransmitter that follows the depolarization of presynaptic nerve terminals, and has been shown to inhibit depolarization-evoked neurotransmitter release and to affect dopamine uptake and release [60–63]. Intracellularly, Pb2+ was shown to activate norepinephrine exocytosis at concentrations three orders of magnitude lower than Ca2+ [64]. In addition, Pb2+ has also been shown to activate binding of synaptotagmin I at similar concentrations [65,66]. Synaptotagmin is a Ca2+ binding protein involved in neurotransmitter release [67]. Disruption of neurotransmitter release is also possible via Pb2+ binding to protein kinase C, a modulator of neurotransmitter release, with a high affinity for Pb2+ . Pb2+ has also recently been shown to activate calmodulin, another Ca2+ -binding protein involved in the neurotransmitter exocytosis [68,69]. In glutamatergic systems, lead inhibited depolarization-evoked glutamate release. In addition, in the astroglia, lead inhibited high-affinity glutamate uptake and glutamine synthetase activity that catalyzes the formation of glutamine from glutamate [70,71]. The development of neural networks early in life involves pruning of excess synapses and is influenced by the pattern of neural activity occurring in response to experiences of the infant. This process may be modulated by lead-induced increases in basal neurotransmitter release or persistence in the synaptic cleft and decreases in depolarization-induced neurotransmitter release. Whether or not there is an effect on the development of neural networks, adaptation to Met. Ions Life Sci. 1, 395–425 (2006)

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these changes in neurotransmitter release may alter the efficiency of synaptic transmission and, thereby, contribute to some of the neurobehavioral deficits in children at low-level lead exposure [60,61,63]. Lead has been shown to decrease dopamine binding in the nucleus accumbens (mesolimbic dopamine system), which has been shown to be critical to the mediation of fixed-interval schedule-controlled behavior [72–74]. Lead inhibits the N -methyl-d-aspartate (NMDA) receptor complex activity, perhaps through a zinc allosteric site. Another mechanism by which lead may affect the nervous system is through its effect on neuronal cell adhesion molecules (NCAMs), membrane-bound cellrecognition molecules that regulate cell–cell interactions, including synapse formation. Chronic, low-level lead exposure impairs the desialylation of NCAMs during postnatal periods that coincide with synapse formation. This interference with the sialylation pattern may perturb synapse selection, thus contributing to learning deficits. The exact mechanism of interference with desialylation has not been determined, but stimulation by lead of the enzyme that sialylates NCAMs has been reported [75,76]. Lead was found to increase the particulate form and decrease the soluble form of the amyloid  precursor protein APP in hippocampal cells in vitro [77]. The particulate form of APP plays a role as a mediator of cell–cell adhesion and cell interaction with components of the extracellular matrix, and any changes in the partitioning of APP would be expected to produce major effects within the central nervous system, particularly during the period of neural development. Lead has also been suggested to possibly perturb glucocorticoid-mediated events in the central nervous system hormonal target tissues [78]. Glucocorticoid receptors are widespread in neurons and glial cells and are known to modulate glial cell functions such as synthesis of myelin phosphatide precursors by glycerol phosphate dehydrogenase, amidation of the neurotransmitter glutamate, and detoxification of ammonia by glutamine synthetase. Tonner and Heiman [78] found that addition of lead acetate to C6 glioma cells in vitro resulted in a significant reduction of the binding affinity of glucocorticoids for cytosolic receptors. High-affinity cytosolic lead-binding proteins have been identified in the brain (and kidneys) of rats. These high-affinity zinc- and lead-binding proteins are thought to moderate lead inhibition of -aminolevulinic acid hydratase (ALAD) through lead chelation and zinc donation, and to translocate lead to the nucleus, where it may influence gene expression. Similar lead-binding proteins have been demonstrated in human brain (and kidney and liver) cytosol. The rat brain leadbinding protein appears to be richly acidic, is not the same as that found in the kidney, and is developmentally regulated. Only traces of this protein were detectible in neonatal rats, increasing to adult levels within 2 weeks, with a concomitant increase in resistance to lead-induced encephalopathy. The development of this resistance is thought to be related to the formation of lead–protein inclusion bodies in the astroglia, which sequester lead. Studies in astroglial cultures Met. Ions Life Sci. 1, 395–425 (2006)

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provide supporting evidence that astroglia develop the ability to sequester lead as they mature. In addition, a small amount of lead can bind to metallothionein, which is also present in the astroglia [71,79–83], but this does not appear to be a primary intercellular storage depot.

3.2 Neurotoxicity Studies in Humans 3.2.1 Neurological Signs and Symptoms in Adults Lead is a well-known neurotoxin (Figure 1). The most severe neurological effect of lead in adults is lead encephalopathy which occurs at high blood

Blood Lead (µg/dL) CHILDREN

ADULTS

>300 Encephalopathy-dullness, irritability, poor attention span, headache, muscular tremor, loss of memory, and hallucinations, delirium, convulsions, paralysis, coma, and death

Encephalopathyhyperirritability, ataxia, convulsions, stupor, coma and death 100

Malaise, forgetfulness, irritability, lethargy, headache, fatigue, dizziness, weakness, paresthesia (occupational) 80 Peripheral neuropathy

40 Visual evoked potentials BAEP and PREP latency Nerve conduction velocity Neurobehavioral, IQ

Hand-eye coordination, reaction time, visual motor performance, hand dexterity, oculomotor function, memory, cognitive performance, and learning ability; increased rates of depression, confusion, anger, fatigue, tension (occupational) Nerve conduction velocity

20

<10

Psychomotor speed, manual dexterity, sustained attention, mental flexibility (non-occupational)

Figure 1. Summary of neurological effects of lead and associated blood lead levels observed in children and adults. (g/dL = micrograms/deciliter; BAEP = brainstem auditory evoked potential; PREP = pattern-reversal visual evoked potential). Met. Ions Life Sci. 1, 395–425 (2006)

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lead (PbB) levels (e.g. 120 −>300 g/dL). Early symptoms include dullness, irritability, poor attention span, headache, muscular tremor, loss of memory, and hallucinations. These symptoms may then worsen to delirium, convulsions, paralysis, coma, and death [84]. Neurotoxicity from occupational exposures have been reported in case reports and small cohort studies. These include malaise, forgetfulness, irritability, lethargy, headache, fatigue, dizziness, weakness, and paresthesia at PbB levels that range from approximately 40–120 g/dL [85].

3.2.2 Behavioral Function in Adults Neurobehavioral effects have been observed in workers at PbB levels of 40–80 g/dL. These include deficits in hand–eye coordination, reaction time, visual motor performance, hand dexterity; impaired oculomotor function, memory, cognitive performance, and learning ability; and increased rates of depression, confusion, anger, fatigue, and tension [85]. A meta-analysis of 22 studies, including many of the above-cited studies, concluded that neurobehavioral effects are ‘obvious’ at a PbB level of ∼40 g/dL [86]. Another meta-analysis by Goodman et al. [87] rejected some of the studies used by Meyer-Baron and Seeber [86] and concluded that the data were inconsistent and did not support neurobehavioral deficits at levels below 70 g/dL. Neurobehavioral effects in nonoccupational cohorts were studied by Muldoon et al. [88]. The cohort consisted of 325 rural dwellers and 205 urban dwellers with geometric mean PbB concentrations of 45 g/dL and 54 g/dL, respectively range = 1–21 g/dL. Rural-dwelling women with blood levels ≥8 g/dL performed significantly worst in tests of psychomotor speed, manual dexterity, sustained attention, and mental flexibility than did women with PbB concentrations ≤3 g/dL. A study of 141 men mean PbB = 55 g/dL found that subjects with higher levels of PbB recalled and defined fewer words, identified fewer line-drawn objects, and required more time to attain the same level of accuracy on a perceptual comparison test than did men with the lower levels of PbB [89]. The inconsistency of the PbB levels at which neurobehavioral effects have been observed is due to limitations and methodological differences in the various studies. For example, Lindgren et al. [90] found no association between current PbB levels in lead workers and neuropsychological variables, but did find an effect on performance when measured against a cumulative dose estimate.

3.2.3 Peripheral Nerve Function in Adults Lead affects peripheral nerve function by reducing the conduction velocity of electrically stimulated nerves. Decreased nerve conduction velocities (NCVs) Met. Ions Life Sci. 1, 395–425 (2006)

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were seen in the median and ulnar nerves of newly employed high-exposure workers after 1 year of exposure and in the motor NCV of the median nerve of this group after 2 or 4 years of exposure [91]. PbB levels were 30–48 g/dL. Similar effects were noted by Rosen and Chesney [92] and Triebig et al. [93] in workers at >70 g/dL. Chia et al. [94] measured NCV in a group of 72 male workers from a lead battery manufacturing factory and 82 unexposed referents and observed an association between NCV and PbB levels at ≥40 g/dL. Evidence is suggestive that these effects are transient [95].

3.2.4 Neurological Signs and Symptoms in Children The signs and symptoms of high-level exposure to lead in children are similar to those in adults. Children, however, are more susceptible in that effects are noted at lower PbB levels. An extensive compilation of pediatric patients identified encephalopathy at exposure levels in the range of 90–800 g/dL [96]; signs include hyperirritability, ataxia, convulsions, stupor, and coma. The distribution of PbB levels associated with death (mean, 327 g/dL) was virtually the same as for levels associated with encephalopathy. Histopathological findings in fatal cases of lead encephalopathy in children include cerebral edema, altered capillaries, and perivascular glial proliferation. Nonfatal cases are at greater risk for neurological and cognitive impairments. A report of 19 cases of acute encephalopathy in infants identified mean PbB levels of 745 g/dL (range, 497–331 g/dL) following use of traditional medicines containing lead (surma, Bint al Thahab) [97]. Seven of the infants had PbB levels ≤70 g/dL

3.2.5 Behavioral Function in Children Several cross-sectional studies of asymptomatic children with relatively high lead body burdens were published in the 1970s that identified a pattern of IQ and neuropsychologic deficits at PbB levels of 40–60 g/dL [85]. Studies conducted since that time resulted in the identification of effects at increasingly lower and lower PbB levels. This prompted a public health response by the Centers for Disease Control and Prevention (CDC), ultimately lowering the PbB level of concern in children to 10 g/dL (Figure 2). The landmark study by Needleman [98], of 158 first- and second-grade children, provided evidence for the association of full-scale IQ deficits of approximately 4 points and other neurobehavioral defects with tooth dentin lead values that exceed 20–30 ppm PbB = 30–50 g/dL [45]. A subset n = 132 of this cohort was reexamined 11 years later as young adults (mean age, 18.4 years) [99]. Impairment of neurobehavioral function was still found to be related to the lead Met. Ions Life Sci. 1, 395–425 (2006)

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45 40 35 30 25 20 15 10 5 0 1970

1975

1985

1991

Year

Figure 2. Changes in blood lead levels considered elevated by the Centers for Disease Control and Prevention (CDC), by year.

content of teeth shed at the ages of 6 and 7 years. These included lower class standing in high school, increased absenteeism, lower grammatical-reasoning scores, lower vocabulary, poorer hand–eye coordination, longer reaction times, and slower finger tapping. Many of the early cross-sectional studies showed neurobehavioral and other deficits at mean PbB levels of >15 g/dL. In the years following, several prospective studies were published that used improved analytical techniques for measuring PbB, and large numbers of subjects, and controlled for several potentially confounding factors. These studies identified neurological effects associated with increasingly lower PbB levels in children exposed prenatally and/or postnatally to lead. The Port Pirie, Australia, prospective study followed cohorts of children (from birth to 11–13 years of age) born to mothers living in the vicinity of a large lead smelting operation in Port Pirie, Australia. Mean cord PbB was 10 g/dL and peaked at about 25 g/dL at age 2 range = 10–30 g/dL. Developmental effects were observed as determined by scores for the Bayley Mental Developmental Index (MDI) at age 2 and again at age 4 using the McCarthy Scales of Children’s Abilities [100,101]. At age 7, an inverse relationship existed between lead exposure (lifetime PbB or bone lead) and IQ (Wechsler Intelligence Scale) and visual–motor performance [102,103]. The cognitive effects observed from exposure during the first 7 years of life persisted to the age of 11–13 [104,105]. An additional recent analysis showed a significant relationship between exposure to lead and later childhood emotional and behavioral problems [106]. The Yugoslavia prospective study evaluated children born to women from two towns in Kosovo, Yugoslavia [107,108]. PbB ranged from 22 g/dL at birth to 40 g/dL at age 4. Prenatal and postnatal exposures that occurred at any time during the first 7 years were independently associated with small decrements in later IQ scores [108–111]. PbB at age 3 was associated with Met. Ions Life Sci. 1, 395–425 (2006)

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small increases in behavioral problems as measured by the Child Behavior Checklist [112]. At 54 months of age, PbB was significantly associated with poorer fine motor and visual motor function as measured by the Bruininks–Oseretsky Test of Motor Proficiency and the Beery Developmental Test of Visual–Motor Integration [113]. Gross motor coordination was unaffected. The Mexico City prospective study evaluated children born to mothers residing in Mexico City [114]. Mean PbB concentrations were 101 g/dL at 6–18 months, 97 g/dL at 24–36 months, and 84 g/dL at 42–54 months of age. Increasing PbB concentrations at 24–36 months, were associated with significant declines in McCarthy General Cognitive Index (GCI) at 48 months p < 005; increasing PbB concentrations at 42–54 months were associated with decreased GCI at 54 months p < 005. Significant associations were also found between PbB concentration and brainstem auditory-evoked responses in 9- to 39-day-old infants and children at 67 months of age [115,116]. The Rochester study examined associations between low-level lead exposure and performance on intelligence tests at the age of 3 and 5 years in a population that included children whose PbB concentrations were below 10 g/dL [117]. An increase in lifetime average PbB concentration of 1 g/dL was associated with a decrease in IQ of 0.46 IQ points (95% CI, −075 to −015). Similar findings were obtained when the children were tested at 3 and 5 years of age. Evaluation of the Boston cohort showed similar results. Cord PbB was inversely associated with MDI scores at 6, 12, 18, and 24 months of age [118]. PbB was correlated with deficits in GCI scores 5 years of age, and reduced IQ at age 10 [119,120]. In a cohort of inner-city children from Cincinnati, PbB levels at 5 and 6 years of age were inversely associated with IQ when tested at about 6.5 years of age. Average lifetime PbB levels in excess of 20 g/dL were associated with deficits in Performance IQ [121]. Lifetime PbB levels in excess of 10 g/dL were associated with deficits in fine motor skills at age 6 [122]. A multivariate linear regression analysis of associations between PbB concentrations and cognitive abilities was conducted in a cross-sectional study of 4,853 US children from the Third National Health and Nutrition Examination Survey (NHANES III, 1988 to 1994) [123]. An inverse relationship was found between arithmetic and reading skills and PbB levels under 5 g/dL.

3.2.6 Electrophysiological Evidence of Neurotoxicity in Children Electrophysiological studies have provided evidence suggestive of effects on central nervous system function at PbB levels lower than 30 g/dL, but findings have been inconsistent. These include associations between PbB levels and visual evoked potentials [124], brainstem auditory evoked potential (BAEP) Met. Ions Life Sci. 1, 395–425 (2006)

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latency [116,125,126], and pattern-reversal visual evoked potential (PREP) latency [125,126].

3.2.7 Peripheral Nerve Function in Children Effects of lead on peripheral nerve function have been documented in children. Frank peripheral neuropathy has been observed in children at PbB levels of 60–136 g/dL [127]. NCV studies have indicated an inverse correlation between peroneal NCV and PbB levels over a range of 13–97 g/dL in children living near a smelter in Kellogg, Idaho [128]. A threshold for NCV at PbB levels of 20–30 g/dL was observed by regression analysis [129]. Gross and fine motor functioning were associated with lead exposure as measured by the several subsets of the Bruininks–Oseretsky Test of Motor Proficiency (BOTMP) [122].

3.3 Neurotoxicity Studies in Animals Literature on the neurological effects of lead in animals is extensive and comparable to the effects observed in human studies. Animals appear to be affected generally at the same PbB levels. Effects include impaired motor function and reflexes in rats [130], decreased NCV in rats [131] and impaired behavior/learning in rats and monkeys (see ATSDR [85] for a more complete review). Several histopathological studies have shown a variety of adverse effects, including reductions or delays in the development of the hippocampus or other hippocampal changes [132], reductions or delays in the development of the cerebral cortex [133], reductions in the number and size of axons in the optic nerve of mice [134], and demyelination of peripheral nerves [135]. Cytoarchitectural changes have also been noted in limited studies of the eyes of monkeys chronically exposed to lead beginning at or shortly after birth [136]. Neurochemical changes have been observed in the brains of rats exposed both pre- and postnatally to lead [137]. These include decreased brain norepinephrine and -aminobutyric acid (GABA) levels, decreased glutamic acid decarboxylase (GAD) activity, increased brain glutamate, glutamine + asparagine, tyrosine levels, and monoamino oxygenase (MAO) activity. Kala and Jadhav [138] reported increases and decreases in the levels of several neurotransmitters in the rat brain. Other studies have found that lead disrupts the dopaminergic systems in rats [139–141]. Monkeys (Macaca fascicularis) treated for a lifetime with 0.5 mg lead/kg/day exhibited a slight decrease in vibration sensitivity (increased threshold to stimuli) when tested at the age of 18 years [142] and increased thresholds for pure tones [143] consistent with results reported in humans [144]. Lilienthal and Winneke [145] reported increased latencies of waves of brain stem auditory-evoked potentials Met. Ions Life Sci. 1, 395–425 (2006)

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until about 10 years of age in monkeys exposed in utero, with no improvement observed 18 months after exposure ceased.

4 MERCURY 4.1 Toxicokinetics and Mechanism of Action 4.1.1 Toxicokinetics The extent of absorption, the residence time in the body before metabolism, and the nature and degree of neurotoxicity are all dependent upon the specific species of mercury. In the case of metallic (liquid, elemental) mercury, only the inhalation route has proven to be biologically relevant in most instances. Less than 0.01% is typically absorbed through the gastrointestinal (GI) tract [146] compared with up to 80% of inhaled mercury vapor [147,148]. Gastrointestinal absorption of mercury varies greatly among the various forms of mercury, with the organomercurials being more extensively absorbed >95% than the inorganic forms of mercury ∼10% [146,149]. Organomercurials can be absorbed to some extent through the intact skin. Once in the blood, the half-life of metallic mercury is relatively short (∼3 days for a single exposure), as it quickly partitions to other body compartments. Inorganic mercury ions are relatively unavailable to the CNS and typically enter the kidneys readily for elimination in the urine. The residence time of alkyl mercurials in the blood is considerably longer, with approximately 90% found in the red blood cells [150]. The lipophilic nature of alkyl mercury compounds facilitates uniform distribution to all tissues. As a result, blood levels are a good indicator of tissue concentrations independent of dose, with tissue concentrations tending to remain constant relative to blood levels [151,152]. Methylmercury (MeHg), ethylmercury (EtHg), and phenylmercury compounds cross the blood– brain and placental barriers and accumulate in the brain and in the fetus. The distribution of organic mercury is believed to involve complexes with proteins in the body. MeHg associates with water-soluble molecules (e.g., proteins) or thiol-containing amino acids because of the high affinity of the methylmercuric cation CH3 Hg+  for the sulfhydryl groups − SH [153]. The transport of MeHg to the brain after subcutaneous injection appears to be closely linked to thiol-containing amino acids [154]. The MeHg cation can bind to the thiol group of the amino acid cysteine, forming a complex in which the valence bonds link the mercury atom to adjacent iron and sulfur atoms creating a chemical structure similar to that of the essential amino acid methionine [155], thus facilitating its passage across the blood–brain barrier. The uptake of MeHg by the brain is inhibited by the presence of other amino acids such as leucine, methionine, phenylalanine, and other large, neutral amino acids. In the brain, Met. Ions Life Sci. 1, 395–425 (2006)

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MeHg is demethylated to inorganic mercury, which is much more slowly eliminated due to the relative impermeability of the blood–brain barrier to inorganic mercury [156]. Although less is known of the distribution of EtHg than of MeHg, the ethyl group is more electrophilic than is the methyl group, making it more susceptible to cleavage of the carbon–mercury bond by hydroxyl radicals produced by the P450 enzyme system, and suggesting a shorter half-life of EtHg in blood. Unlike MeHg, EtHg is not believed to be helped to cross the blood–brain barrier via an active transport mechanism, and passage of EtHg across the blood–brain barrier is hindered by its larger size and faster decomposition than is MeHg [157,158]. Phenylmercury is less likely to permeate the placental and blood–brain barriers than is MeHg [159]. Metallic mercury is oxidized to the divalent inorganic cation primarily in the red blood cells and lungs. Mercuric mercury compounds can, in turn, be reduced to the metallic form and released as exhaled metallic mercury vapor. In the case of alkyl mercurials, the organomercurial is conjugated with bile salts by glutathione in the liver, and then excreted into the gall bladder bound to low molecular weight, nonprotein sulfhydryl compounds [160,161]. Small amounts of MeHg can also be converted to inorganic mercury in the intestine [162,163]. Most of the MeHg entering the gut, however, is reabsorbed through the intestinal wall and becomes resident in this enterohepatic circulation. When delivered to the tissues, MeHg can be converted to inorganic mercury Hg2+  [164–166]. The aryl-mercurial phenylmercury is rapidly metabolized to inorganic mercury [152]. Urinary excretion accounts for almost all of the absorbed elemental mercury and phenylmercury. Because absorption of inorganic mercury ions is comparatively poor, most of an ingested dose of inorganic mercury compounds is eliminated in the feces, however, high acute doses increase the percentage of excretion via the urine [147,167–169]. The overall half-life of metallic and other inorganic forms of mercury in the body averages approximately 2 months [147,163], with a range of ∼30–90 days, depending on the duration and magnitude of exposure [147,170,171]. The elimination of inorganic mercury from the blood and brain is thought to be a biphasic process, having an initial rapid phase, during which the decline in the body burden is associated with high levels of mercury being cleared from tissues. This is followed by a slower phase of mercury clearance from the same tissues [171,170]. For MeHg, elimination rates vary with dose [172,173]. Elimination generally follows first-order kinetics, with excretion directly proportional to body burden, and is independent of the route of administration [173]. Furthermore, duration of exposure may affect the excretion process of mercury. Biliary excretion is the predominant excretory pathway for MeHg and EtHg, with fecal elimination ultimately accounting for the majority of the absorbed dose [174–176]. MeHg is also eliminated from the body in breast milk [177,178]. Phenylmercury compounds Met. Ions Life Sci. 1, 395–425 (2006)

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are also eliminated primarily in the feces during the first few days after intravenous dosing as a result of biliary secretion and subsequent concentration in the gastrointestinal tract [179]. The initial excretion of phenylmercury represents primarily the parent compound [180]. Several days after exposure, however, elimination is primarily in the urine, which contains predominantly inorganic mercury [181].

4.1.2 Mechanisms of Toxicity Proximal mechanisms of toxicity include the inactivation of various enzymes, structural proteins, and/or transport processes [182]. High-affinity binding of the divalent mercuric ion to thiol or sulfhydryl groups of proteins, with subsequent redox potential of the mercury–thiol complex, is believed to be the underlying mechanism for the biologic activity of mercury [183–186]. Increased formation of reactive oxygen species in the brain following MeHg administration has been associated with neuronal degeneration [187–191]. Through alterations in intracellular thiol status, mercury can promote oxidative stress, lipid peroxidation, and mitochondrial dysfunction [192,193]. Mercury-induced alterations in mitochondrial calcium homeostasis may exacerbate Hg2+ -induced oxidative stress [193]. Thimerosal, whose active ingredient is EtHg ∼49%, has been shown to be a versatile sulfhydryl reagent, a mobilizer of intracellular calcium, and a modulator of cell function [194]. It is widely considered that organic mercury exerts its toxic effects on the nervous system and other organs by being metabolized to inorganic mercury, because both forms elicit very similar histopathologic changes. The activation of cell cycle regulatory genes may also be a mechanism by which MeHg interferes with the cell cycle in both adult and developing organisms [195]. It has been reported that altering intracellular calcium levels in brain capillary endothelial cells has a direct effect on blood–brain barrier permeability and transport [196]. Calcium channels in certain nerve fibers have a high affinity for the plant alkaloid ryanodine [197]. Thimerosal reacts with specific cysteine residues on ryanodine receptors (RyR), contributing to either activation or inhibition of the channel, depending on the domain and particular class of cysteine associated with that receptor [198]. Targets of MeHg include neural cell adhesion molecules, which are sialoglyco-conjugates whose proper temporal and spatial expression is important at all stages of neurodevelopment, especially during the formation of synapses [199]. Neuronal cytoskeleton degeneration of cerebellar granule cells following in vitro exposure to MeHg and disruption in the integrity and number of microtubules and neurofilaments in myelinated fibers of optic nerves of rats have also been reported [200,201]. Thimerosal/EtHg has also been shown to inactivate sodium channels in dorsal root ganglion neurons in a dose-dependent fashion, contributing to diminished neuronal activity [202]. Met. Ions Life Sci. 1, 395–425 (2006)

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4.2 Neurotoxic Effects in Humans Although exposure to significant levels of both organic and inorganic forms of mercury can result in neurologic, respiratory, renal, reproductive, immunologic, dermatologic, and a variety of other effects, neurologic effects are the most prominent feature of excessive exposure to metallic and organic mercury in most cases. These include restlessness, memory loss, headaches, anxiety, agitation, irritability, fatigue, confusion, insomnia, mood lability, erythism, irrational behavior, weakness, tremors, polyneuropathy [203]. Both clinical and behavioral effects in humans have been observed following inhalation of metallic mercury vapor and organic mercury compounds; ingestion or dermal application of inorganic mercury-containing medicinal products such as teething powders, ointments, and laxatives; and ingestion or dermal exposure to organic mercury-containing pesticides or ingestion of contaminated seafood. Specific neurologic symptoms include tremors, emotional lability, insomnia, memory loss, muscle weakness, fasciculations, headaches, polyneuropathy, and performance deficits in tests of cognitive and motor function. Although improvement has been observed upon removal of persons from the source of exposure, some changes may be irreversible. Autopsy findings of degenerative changes in the brains of poisoned patients exposed to mercury support the functional changes observed [204–208]. Epidemic poisonings in Japan and Iraq conclusively demonstrated the neurotoxicity of methylmercury and ethylmercury. The first reported widespread outbreak of neurologic disorders associated with methylmercury involved the ingestion of contaminated fish in the Minamata area of Japan [209]. The neurologic syndrome observed in this poisoning incident was characterized by a long list of symptoms, including paresthesia (prickling, tingling sensation in the extremities); impaired peripheral vision, hearing, taste, and smell; slurred speech; unsteadiness of gait and limbs; muscle weakness; irritability; memory loss; depression; and sleeping difficulties [209,210]. In another methylmercury poisoning incident, the ingestion of bread prepared from wheat and other cereals that had been treated with a methylmercury fungicide in 1971–1972 resulted in more than 6000 patients requiring hospitalization and more than 500 deaths, usually due to central nervous system failure. Autopsy results from several persons who died following ingestion of the contaminated seed grain in Iraq showed neuronal degeneration and glial proliferation in the cortical and cerebellar gray matter and basal ganglia [207]. Granule cells in the brain were variably affected, and Purkinje, basket, and stellate cells were severely affected. Sural nerves removed from two women with neurotoxicity associated with the Minamata incident also showed evidence of peripheral nerve degeneration [205]. Epidemics of neurologic disorders similar to those described above were reported in Iraq in 1956 and 1960 [211,212] as the result of eating flour made from seed grain contaminated with ethylmercury. Effects in affected individuals Met. Ions Life Sci. 1, 395–425 (2006)

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included inability to walk, cerebellar ataxia, speech difficulties, paraplegia, spasticity, abnormal reflexes, restriction of visual fields or blindness, tremors, paresthesia, insomnia, confusion, hallucinations, excitement, and loss of consciousness. In a separate incident, neurotoxic effects seen in two boys who ingested meat contaminated with ethylmercuric chloride included gait disturbance, ataxia, dysarthria, dysphagia, aphonia, hyperreactive tendon reflexes, hypotonia, spasticity, mydriasis, horizontal nystagmus, agitation, and coma. Autopsy revealed nerve cell loss and glial proliferation in the cerebral cortex, demyelination, granule cell loss in the cerebellum, and motor neuron loss in the ventral horns of the spinal cord [204]. Hyperreflexia, muscle rigidity and/or myoclonic, jerky, choreoathetotic movements and/or sensorimotor polyneuropathy were observed in individuals who had ingested food contaminated with MeHg or had ingested extremely high amounts of thimerosal [206,213–216]. In a fatal case of acute dimethylmercury dermal exposure, dysmetria, dystaxic handwriting, wide-based gait, and impaired speech were observed [217]. Similar symptomatology was reported in two fatal occupational exposures to diethylmercury about 100 years ago. Most of what is known about the neurodevelopmental effects of organomercurials comes from massive poisoning incidents in Japan and Iraq, which provided evidence that the developing nervous system of the fetus is highly sensitive to mercury toxicity [207,210,211,218,219]. In a 1955 mercury poisoning outbreak in Minamata, Japan, severe brain damage was observed in 22 infants whose mothers had ingested fish contaminated with MeHg during pregnancy [219]. The effects described in the Minamata outbreak included mental retardation, retention of primitive reflexes; cerebellar symptoms, dysarthria, hyperkinesias, hypersalivation, dysmyelination of the pyramidal tracts, an abnormal neuronal cytoarchitecture, and atrophy and hypoplasia of the cerebral cortex, corpus callosum, and granule cell layer of the cerebellum. The widespread damage has been suggested to involve derangement of basic developmental processes such as neuronal migration and neuronal cell division [220–222]. Severe brain damage was also reported in infants of mothers poisoned with EtHg in Iraq in 1956, 1960, and 1971–1972 [211]. Delays in walking and talking were associated with lower maternal exposures during pregnancy than were mental retardation and seizures [218,223]. More recent studies have focused on the effects of chronic, low level exposure to MeHg through fish consumption. Two prospective epidemiologic studies of seafood-eating populations in the Seychelles [224–226] and Faroe Islands [227,228] have focused on the investigation of more subtle neurobehavioral or neuropsychological effects in children exposed in utero. In the Faroes, extensive neuropsychologic testing conducted on more than 900 children (approximately 7 years of age) of mothers who had consumed mercurycontaminated seafood during pregnancy indicated mercury-related dysfunction in Met. Ions Life Sci. 1, 395–425 (2006)

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the specific domains of language, attention, memory, and visuospatial and motor function [227,228]. These researchers reported that the greatest susceptibility to MeHg neurotoxicity occurs during late fetal development. In the Seychelles longitudinal epidemiologic study, more than 700 mother– infant pairs have, to date, been followed and tested from parturition through 108 months of age [224–226]. No effects considered attributable to MeHg were observed in either clinical/neurodevelopmental evaluation of the children or the results of a broad battery of neurobehavioral tests at 6, 19, 29, 66, and 107 months of age [224–226].

4.3 Neurotoxic Effects in Experimental Animals The neurotoxicity of metallic and inorganic mercury in experimental animals is similar to that seen in humans, and is manifested as functional, behavioral, and morphological changes, as well as alterations in brain chemistry. Effects include changes in cholinergic transmission at the neuromuscular junction, GABA receptor activity and number, and the activities of enzymes involved in cholinergic, adrenergic, dopaminergic, and seratonergic synthesis and/or catabolism [203]. Single dosages of MeHg also produced blood–brain barrier dysfunction in rats [229]. Sensory effects seen in monkeys include slight constriction of visual fields and age-related differences in both spatial and temporal visual function in animals exposed to MeHg in utero or postpartum for 7 years. Additional effects include delayed decrements in auditory and visual function in monkeys exposed to MeHg in utero through maternal treatment, and impaired spatial vision, clumsiness, decreased fine motor performance, impaired high-frequency hearing, and delayed insensitivity to touch in monkeys fed MeHg for 3–7 years from birth [230–233]. Neonatal monkeys receiving MeHg in infant formula exhibited stumbling and falling before termination of exposure [234]. Despite cessation of exposure, abnormalities in several reflexes, blindness, abnormal behavior, and coma developed. Histopathologic analyses showed diffuse degeneration in the cerebral cortex, cerebellum, basal ganglia, thalamus, amygdala, and lateral geniculate nuclei. Monkeys exposed to MeHg orally exhibited tremors and visual field impairment [235]. Slight tremors and decreased sucking responses, followed by claw-like grasp, gross motor incoordination, and apparent blindness, were also observed in monkeys administered MeHg daily for 4 months [233]. MeHg fed to rodents in high dosages caused posterior paralysis and sensory neuropathy, characterized by cerebral and cerebellar necrosis and degeneration of dorsal root ganglionic nerves and sciatic nerve [236–238]. At the ultrastructural level, deposition has been shown to occur within lysosomes of target glial cells. Typical neurotoxic signs observed in rats exposed to MeHg include muscle spasms, gait disturbances, flailing, and hindlimb crossing [239–243]. Histopathological examination of the nervous systems of affected rats has shown Met. Ions Life Sci. 1, 395–425 (2006)

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degenerative changes in cerebellar granule cells, dorsal root ganglia [240], and peripheral nerves [205,244,245]. Degenerative changes in the cerebellum and the cerebral cortex have been seen in cats administered chronic, low-dose dietary MeHg. Whereas cats fed doses of MeHg for 2 years exhibited delayed impairment of motor activity, diminished sensitivity to pain at low doses, and ataxia, alterations in gait, motor incoordination, muscle weakness, changes in temperament, and convulsions, as well as neuronal degeneration in the motor, sensory, auditory, and occipital cortices, and cerebellar granule cell degeneration at higher doses [246–248]. In rabbits, MeHg caused widespread neuronal degenerative changes in cervical ganglion cells, cerebellum, and cerebral cortex, without accompanying behavioral changes, and in rodents, degenerative changes in dorsal root fibers and cerebellum [249–254].

5 CONCLUSIONS As demonstrated in this chapter, the neurotoxicity of lead and mercury is well established. Numerous studies have shown that these substances can induce neurological and neurobehavioral effects in humans and animals. Data are also available on the neurotoxicity of cadmium. These metals have a potential to exhibit the effects mostly by interfering with neurotransmitters on the nerve synapses and by disrupting metabolic processes in the cells. Concerns regarding inductions of neurodevelopmental effects in small children are well justified from the public health viewpoint. In environmental exposure scenarios, humans are exposed to mixtures of chemicals, and the possibility of co-exposure to cadmium, lead, and mercury is likely. Because all three target the neurological system, joint toxic action is plausible. Measures to decrease levels of these chemicals in the environment (e.g., use of unleaded gasoline) or to decrease intake (e.g., fish-eating advisories for contaminated fish) are designed to lower the health risk of the general public from environmental exposures.

ABBREVIATIONS ALAD ATSDR APP BAEP BOTMP CDC CI CNS EtHg GABA

-aminolevulinic acid hyratase Agency for Toxic Substances and Disease Registry amyloid  precursor protein brainstem auditory evoked potential Bruininks–Oseretsky Test of Motor Proficiency Centers for Disease Control and Prevention cognitive index central nervous system ethylmercury compounds -aminobutyric acid Met. Ions Life Sci. 1, 395–425 (2006)

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GAD GCI GI GSH GSSG i.p. IQ MAO MDI MeHg NCAMs NCV NHANES III NMDA PbB PREP RyR − SH

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glutamic acid decarboxylase McCarthy General Cognitive Index gastrointestinal tract reduced glutathione oxidized glutathione intraperitoneal intelligence quotient monoamino oxygenase Bayley Mental Developmental Index methylmercury compounds neuronal cell adhesion molecules nerve conduction velocity Third National Health and Nutrition Examination Survey N -methyl-d-aspartate blood lead level pattern-reversal visual evoked potential ryanodine receptor sulfhydryl group

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15 Neurodegenerative Diseases and Metal Ions. A Concluding Overview Dorothea Strozyk1 and Ashley I. Bush23 1

Department of Neurology, Albert Einstein College of Medicine, Kennedy Center, Suite 220, Pelham Parkway South, Bronx, NY 10461, USA <[email protected] > 2

3

Laboratory for Oxidation Biology, Genetics and Aging Research Unit, Harvard Medical School, Massachusetts General Hospital, Building 114, 16th Street, Charlestown, MA 02129, USA

Oxidation Disorders Laboratory, Mental Health Research Institute of Victoria, and Department of Pathology, The University of Melbourne, 155 Oak Street, Parkville, Victoria 3052, Australia

1 INTRODUCTION 2 THE MEDICINAL CHEMISTRY OF METAL-CENTERED BRAIN DISORDERS 3 OTHER NEUROLOGICAL DISORDERS THAT MAY INVOLVE METALS 3.1 Polyneuropathy/Myelopathy 3.2 Multiple Sclerosis 3.3 Macular Degeneration 3.4 Progressive Supranuclear Palsy, Multisystem Atrophy, and Huntington’s Disease 3.5 Restless Legs Syndrome and Tardive Dyskinesia 4 CONCLUSION AND OUTLOOK ACKNOWLEDGMENTS ABBREVIATIONS AND DEFINITIONS REFERENCES

Metal Ions in Life Sciences, Volume 1 © 2006 John Wiley & Sons, Ltd

Edited by Astrid Sigel, Helmut Sigel and Roland K. O. Sigel

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1 INTRODUCTION The appreciation of the role of metallobiology in neuroscience and in neurology is increasing rapidly. In Chapter 1 of this volume we organized the metal-related neurological disorders into: (i) those caused by a defect in metal ion transport or homeostasis; (ii) those caused by toxicological exposure to metal; and (iii) those caused or associated with metalloprotein aggregation. While this scheme has some organizational value, its value for medical practice will be determined by its ability to guide the practitioner to practical therapeutics. While truly specific and potent metal-centered pharmaceuticals for these diseases currently lie ahead of us, there is progress being made. Additionally, there are other neurological conditions that have not been covered in detail in this volume (see below) which may have aspects of their pathophysiology related to metallochemistry that may also be amenable to metalcentered pharmacological approaches.

2 THE MEDICINAL CHEMISTRY OF METAL-CENTERED BRAIN DISORDERS Simplistically, metal binding drugs, or chelators, would be the first class of compounds considered for the disorders listed in this volume and in Table 1 of Chapter 1. However, the main medicinal chelators (e.g., penicillamine, trientine, desferrioxamine) have limitations that make them unsuitable for the treatment of brain disease. These are, failure to cross the blood–brain barrier (BBB), mode of delivery and lack of specificity for metal ion or metalloprotein target [1]. One of the earliest clinical trials to target metals in neurogenerative diseases was the use of desferrioxamine (DFO) in Alzheimer’s disease (AD), which illustrates some of the difficulties. In this trial patients were given painful intramuscular injections twice daily, 5 days per week, for 24 months. This treatment regime resulted in a modest, but significant reduction in the rate of decline of daily living skills when compared to control patients [2]. DFO, like many chelators, is highly charged and therefore disadvantaged in both crossing the BBB and in enriching in the hydrophobic amyloid target in AD. This problem may explain why d-penicillamine was also subtle in its clinical effects (despite improving oxidative markers) in treating AD subjects, although in this particular study the placebo control group did not deteriorate making it impossible to tell whether the drug had arrested the rate of deterioration [3]. Drugs like penicillamine, trientine, ethylenediaminetetracetic acid (EDTA), and desferrioxamine are useful in the treatment of systemic disorders of toxicological metal exposure or genetic disorders (e.g., Wilson’s disease) where metals accumulate in tissue. These drugs are the agents utilized in ‘chelation therapy’, a term associated with the removal of bulk metals such as in Wilson’s disease (Cu) Met. Ions Life Sci. 1, 427–435 (2006)

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and -thalassemia (Fe). The mechanism of action of traditional chelators is that metal is sequestered peripherally and cleared by excretion. However, because of their inability to enter the brain, these drugs may not be effective in remedying metal accumulations in protein aggregation diseases (e.g., Alzheimer’s disease or Parkinson’s disease) or where metals accumulate in a neuronal subcellular compartment in the brain (e.g., iron in mitochondria in Friedreich’s ataxia). For these disorders, considerably more sophisticated metal-centered drugs than chelators will be required. Over the last five years promising landmarks have been achieved in the development of novel metal-centered drugs that condition the association of biometals with proteins in the central nervous system, and promote the repartitioning of metal collections toward normality. Although these new drugs are technically chemical chelators, it would be a misnomer to label them as such since they do not necessarily remove metals from brain tissue (i.e., they are not medicinal chelators). Therefore, we have coined the term ‘metallotropic’ agent to decribe this novel drug class. One approach is to design molecules that stereochemically hinder the pathogenic metal or metal binding site of target metalloprotein for the metal ions, i.e., a metal–protein attenuating compound (MPAC). The intention of the MPAC is to disrupt an abnormal metal–protein interaction to achieve a subtle repartitioning of metals and a subsequent normalization of metal distribution. Once the toxic cycle is inhibited, endogenous clearance processes can cope more effectively with the accumulated protein. In light of the interactions between A and metals the potential use of MPACs has been most extensively investigated in treating AD. A hydrophobic moderate metal chelator clioquinol (CQ, 5-chloro-7-iodo-8-hydroxyquinoline) that is capable of crossing the BBB is a prototypic MPAC. CQ is able to bind a range of metal ions with moderate affinity, i.e., in the nM range. CQ was given orally in a blinded study to Tg2576 transgenic mice [4]. The results showed a 49% decrease in brain A burden compared with nontreated controls after 9 weeks, with no evidence of toxicity, and the general health and body weight parameters were more stable in the treated animals. Treatment with CQ did not lead to a systematic decrease in metal levels, most likely due to the drug’s moderate binding affinities. Interestingly, in the same study, TETA (=trien, trientine), a hydrophilic high-affinity metal chelator that is incapable of crossing the BBB, and has been used to treat Wilson’s disease, did not inhibit A deposition, indicating that systemic depletion of metal-ions (‘chelation therapy’) is unlikely to be an effective therapeutic strategy for the treatment of AD. Similar benefits to CQ in decreasing brain pathology in APP transgenic mice were reported utilizing a different lipophilic chelator, DP-109 [5]. The success of CQ in the transgenic mouse trials encouraged the testing of this molecule in clinical trials. The effects of oral CQ treatment in a randomized, double-blind, placebo-controlled pilot phase 2 clinical trial of moderately severe AD patients were evaluated [6]. The effect of treatment was statistically Met. Ions Life Sci. 1, 427–435 (2006)

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significant in preventing cognitive deterioration over 36 weeks in the moreseverely affected patients (baseline ADAS-cog ≥ 25). There was also a significant decline in plasma A42 in the CQ group compared with an increase in the placebo group. Parkinson’s disease (PD) is another potential indication for metallotropic drugs [7–10]. The success of CQ as a potential approach for AD encouraged the testing of this molecule in an animal model of PD. The 1-methyl-4phenyl-1,2,3,6-tetrapyridine (MPTP) model of PD is a toxicity paradigm, where Parkinson’s like symptoms are induced by a toxin that targets the dopaminergic pathways, and cells with high neuromelanin content are more susceptible to the toxin [11]. The toxicity induced by MPTP was inhibited by CQ and by overexpression of the iron binding protein ferritin [12]. These results are consistent with 8-hydroxyquinolines being higher affinity ligands for iron than dopamine [13]. Other drugs that can be considered for the treatment of metal-centered neurological disorders include antioxidants, which limit the downstream consequences of metal-centered radical generation. There are numerous reports of antioxidants benefitting preclinical models for neurodegenerative diseases, e.g., N -acetyl cysteine for mSOD1 transgenic mouse model of amyotrophic lateral sclerosis (ALS) [14], curcumin for the treatment of the APP transgenic mouse model of AD [15]. However, there are no compelling clinical trial data as yet that indicate that such antioxidants may be a useful primary treatment for these disorders.

3 OTHER NEUROLOGICAL DISORDERS THAT MAY INVOLVE METALS 3.1 Polyneuropathy/Myelopathy Recently low copper levels and/or excess zinc have been reported to be associated with myelopolyneuropathy and pancytopenia [16]. Subsequent reports described copper deficiency as possibly causing a myelopolyneuropathy syndrome that resembles the subacute combined degeneration of the cord associated with vitamin B12 deficiency [17].

3.2 Multiple Sclerosis There is growing interest in the possibility that abnormal metal metabolism may either contribute or be a pathophysiological consequence of multiple sclerosis (MS). There are several recent descriptive reports. One report on cerebrospinal fluid (CSF) metal content in MS subjects compared to controls revealed that manganese levels were significantly decreased ∼ − 40%; copper levels were Met. Ions Life Sci. 1, 427–435 (2006)

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significantly elevated ∼ + 30%, and there were no significant differences between the CSF zinc levels of MS and control patients [18]. The physiological basis for the differences in manganese and copper concentrations between MS patients and controls is unknown, but could be related to alterations in the manganese-containing enzyme glutamine synthetase and the copper-containing enzyme cytochrome oxidase. Toxicological exposure to heavy metals has been considered in the pathogenesis of MS, but no consistent data has been reported to support that possibility [19]. Abnormal metabolism of brain iron and iron-binding proteins has been implicated in MS [20]. Iron-chelating agents have been reported to be beneficial in the animal model for MS, experimental autoimmune encephalitis (EAE) [21]. Similarly, stress-activated pathways induce expression of matrix metalloproteinases (MMPs), leading to inflammatory responses and pathological damages involved in the etiology of multiple sclerosis. A recent report described a novel compound, N -acetylcysteine amide, that crosses the BBB, chelates Cu(II), and prevents ROS-induced activation of JNK, p38, and MMP-9. In EAE, oral administration of N -acetylcysteine amide reduced pathological signs, inflammation, MMP-9 activity, and protected axons from demyelination [22].

3.3 Macular Degeneration For many years, there has been interest in the role of zinc deficiency in the etiology of macular degeneration [21], a major cause of blindness in the aged. However, oxidative stress had also been considered as a more likely explanation for the pathology of the disease [23]. The Age-Related Eye Disease Study was a large scale study to determine the value of treatment with a combination of vitamins C and E, -carotene, zinc, and copper on the outcome of age-related macular degeneration. It reported favorable results [24], which have been very influential in subsequent clinical practice. However, it is still not known whether the zinc or the antioxidant components in the supplement are responsible for the beneficial effects.

3.4 Progressive Supranuclear Palsy, Multisystem Atrophy, and Huntington’s Disease While there is scant literature on the subject of metals and these neurological disorders, changes in metal levels have been reported. One comprehensive survey of metals in brain tissue from basal ganglia disorders reported that total iron content was increased in substantia nigra in PD, progressive supranuclear palsy (PSP) and multisystem atrophy (MSA), but not in Huntington’s disease (HD) [25]. Total iron levels in the striatum were increased in PSP, MSA, and HD, but not in PD. There were no consistent alterations of manganese levels Met. Ions Life Sci. 1, 427–435 (2006)

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in basal ganglia structures in any of the diseases studied. Copper levels were decreased in the substantia nigra in PD, and were elevated in the putamen and possibly substantia nigra in HD. Zinc levels were only increased in PD, in substantia nigra, caudate nucleus, and lateral putamen. Levels of the iron binding protein ferritin were measured in the same patient groups. Increased iron levels in basal ganglia were generally associated with normal or elevated levels of ferritin. The exception was PD where there was a generalized reduction in brain ferritin immunoreactivity, even in the substantia nigra. The authors conclude [25] that an increase in total iron content appears to be a response to neurodegeneration in affected basal ganglia regions in a number of movement disorders. Increased ferric iron in basal ganglia has been confirmed in PSP, where it can be visualized by magnetic resonance imaging (MRI) [26].

3.5 Restless Legs Syndrome and Tardive Dyskinesia Restless legs syndrome (RLS) is an idiopathic movement disorder of uncertain etiology, where the patient feels an uncontrollable compulsion to move their lower limb, disturbing sleep. Tardive dyskinesia is an irreversible adverse effect of antipsychotic medication treatment and affects axial movements, muscles of the face and limbs. Both disorders are thought to reflect abnormalities of the basal ganglia. A growing literature has implicated abnormal iron metabolism in the basal ganglia in their pathogeneses. Blood ferritin and iron levels are decreased in RLS [27] and decreased brain iron levels can be visualized by MRI [28]. RLS responds to treatment with iron supplements [29]. However, there is no evidence that iron deficiency alone can cause RLS. Rather growing evidence suggests that iron uptake into the basal ganglia, which is mediated by the transferrin receptor, is diminished in the syndrome [30,31]. The association between iron or metal status and tardive dyskinesia is less clear. Antipsychotic drugs promote the uptake of iron into synapses, and so may increase the ROS burden on the neuron [32]. However, clinical studies have found contradictory evidence of peripheral iron dysregulation in antipsychoticrelated abnormal movement disorders [33,34].

4 CONCLUSION AND OUTLOOK The brain is an exceptional tissue in that it constitutively concentrates biometals (Cu, Zn, Fe, and Mn) for its functional metabolism. This, together with its obligate requirement for oxygen, appears to make the central nervous system vulnerable to a host of metal-centered pathologies. The importance of this emerging Met. Ions Life Sci. 1, 427–435 (2006)

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aspect of biochemistry is that we are rapidly developing novel drugs and classes of drugs that manipulate metal-centered neuropathology far more precisely and elegantly than mere chelation therapy. Metallochemistry has the potential to offer innumerable novel protein and other medicinal chemistry targets. Within the next five years, the first pivotal trial data will emerge on the utility of metallotropic drugs in the treatment of Alzheimer’s disease and Parkinson’s disease. If these studies show efficacy, this realm of neurochemistry becomes of major research interest.

ACKNOWLEDGMENTS Supported by funding from the Australian Reseach Council, the National Health and Medical Research Council and the Alzheimer’s Association.

ABBREVIATIONS AND DEFINITIONS AD ADAS-cog ALS APP A BBB CQ CSF DFO EAE EDTA HD MMP MPAC MPTP MRI MS MSA PD PSP RLS ROS TETA trientine

Alzheimer’s disease Alzheimer’s disease assessment scale, cognitive subset amyotrophic lateral sclerosis amyloid precursor protein amyloid  blood–brain barrier clioquinol, 5-chloro-7-iodo-8-hydroxyquinoline cerebrospinal fluid desferrioxamine experimental autoimmune encephalitis ethylenediamine-N,N,N ,N  -tetraacetic acid Huntington’s disease matrix metalloproteinase metal-protein attenuating compound 1-methyl-4-phenyl-1,2,3,6-tetrapyridine magnetic resonance imaging multiple sclerosis multisystem atrophy Parkinson’s disease progressive supranuclear palsy restless legs syndrome reactive oxygen species triethylenetetramine (=trien) triethylenetetramine (=trien) Met. Ions Life Sci. 1, 427–435 (2006)

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Met. Ions Life Sci. 1, 427–435 (2006)

Subject Index

A AAS, see Atomic absorption spectroscopy Absorption of cadmium, 397 lead, 400 mercury, 409 A peptide, 2–4, 50, 62, 116, 165, 166, 195, 251, 253, 256, 289, 309, 429, 430 aggregates, 77–80, 117, 253, 254, 256, 259, 269, 377 aluminum binding, 377, 381 as antioxidant, 254 brain deposition, 328 conformational change, 256 human, 77–80 hypothetical model for the metallobiology in Alzheimer’s disease, 255 imidazole nitrogens, 78 metal ion interactions, 76–80, 377 mouse, 77, 117, 257, 381 rat, 77, 79, 80 structure, 79, 80 Aceruloplasminemia, 4, 130, 262, 263 Acetylacetonate aluminum complex, 384

Metal Ions in Life Sciences, Volume 1 © 2006 John Wiley & Sons, Ltd

Acetylcholine, 324 esterase, 382 synthesis, see Biosynthesis Acidity constants of phosphate, 375 zinc-aminofluorescein, 349 zinpyr complex, 345, 346 Aconitase, 2, 4, 230, 235–237, 299 Active site (of) Cu,Zn superoxide dismutase, 187 redox potentials of, 12 Adenosine 5 -monophosphate, see AMP Adenosine 5 -triphosphate, see ATP Adrenaline, 228, 291, 292, 310, 311 vanadyl complex, 311 AEDANS, see 5-((((-2-Iodoacetyl)amino)-ethyl)-amino)-naphthalene-1sulfonic acid Affinity constants, see Stability constants Aging, 135, 228, 241–243, 382 aluminum accumulation, 383 amyotrophic lateral sclerosis, 185 brain diseases (see also individual names), 151–169 copper accumulation, 255 iron accumulation, 239, 244, 255 magnesium depletion, 383 myelin breakdown, 159, 162, 169 oxidative damage, 185 Albumin(s), 208, 210, 219 aluminum interaction, 376, 379, 380 cadmium binding, 397

Edited by Astrid Sigel, Helmut Sigel and Roland K. O. Sigel

438

Alkali metal ions (see also individual names), 355, 356 Alkaline earth ions (see also individual names), 355, 356 Aluminum (including +III state) (in) 26 Al, 377 A peptide, 377, 381 absorption, 378 accumulation, 4 acetylacetonate, 384 acetylcholinesterase, 382 Alzheimer’s disease, see Alzheimer’s disease animal studies, see Animal studies ATP binding, 375, 376, 383 biogeochemical cycle, 375 biological fluids, 373 biospeciation, 375 blood-brain barrier transfer, see Blood-brain barrier brain, see Brain calmodulin interaction, 376 carbonic anhydrase, 382 central nervous system, 380, 383 chemical properties, 372–374 citrate complex, see Citrate DNA binding, 375 fluoro complex, 375 food, 372 history, 372 hydroxide, 377, 385 hydroxo complex, 374, 377 hydroxy carboxylate complex, 376 hypothesis of Alzheimer’s disease, 385 lactate, 380, 381 macromolecular complexes, 376, 377 metabolism, see Metabolism monoamine oxidase, 382 mononuclear ions, 374, 375 neurodegenerative diseases, see Neurodegenerative diseases neurofibrillary tangles, 377 neuromelanin, 132 occupational exposure, 383 oligonuclear complex, 374 outersphere coordination in neurofibrillary tangles, 377

SUBJECT INDEX

[Aluminum (including +III state) (in)] Parkinson’s disease, 128 phosphate complex, 375 serum, see Serum silicate, 375, 378 speciation, 378–380 stability constants, 383 tartrate, see Tartrate ternary complex, 379, 380 tissues, see Tissues toxicology, 373, 380–384 transferrins, see Transferrin(s) transport, see Transport Alzheimer’s disease, 2–5, 62, 76–82, 126, 127, 135, 153–155, 169, 180, 185, 193, 195, 241, 243, 250–260, 269, 299, 308, 309, 323, 377, 428–430 acetylcholinesterase, 382 aluminum hypothesis, 385 alumnium in, 384–387 amyloidosis, 250  amyloid peptide, see A peptide brain iron, 130, 131, 154, 160–163, 165, 167, 168 characterization, 115 familial, 284 Hirano bodies, 141 histopathology, 250 hypothetical model for the metallobiology of A, 255 misfolded proteins, 10 myelin breakdown, 159 myelin integrity, 162 neuritic (amyloid) plaques, 77, 156, 251, 253, 255, 257, 325, 385 neurofibrillary tangles, 141, 156, 165, 251, 256, 289, 380, 381, 385 oxidative damage, see Oxidative damage oxidative stress, 117 pathology, 118, 327 rabbit model, see Rabbit reactive oxygen species, see Reactive oxygen species role of metal ions, 115–121, 165, 166 sporadic, 284 tau proteins, see Tau proteins therapy, 328

SUBJECT INDEX

Amine oxidase Cu2+ , 211 Amino acids, see also individual names artificial, 334 -Aminobutyric acid, 134, 300, 327, 408, 414 neurons, 155 synthesis, see Biosynthesis 2-Amino-3-(3-hydroxy-5-methyl-4isoaxazolyl)propionate receptor, 324, 327 -Aminolevulinic acid hydratase, 402 synthase, 235 o-Aminophenol-N,N,O-triacetic acid, 343, 354 AMP aluminum complex, 383 Amphibia prion protein homologues, 91, 92 -amyloid, see A peptide Amyloid plaques (see also Senile plaques), 117, 254, 256, 325 aggregation, 327 deposition, 115 reduction, 118, 119 Amyloid precursor protein, 62, 165, 251, 256, 269, 309, 383, 385, 402 antioxidant activity, 116 copper binding domain, 81, 116, 120 cRNA, 258 domains, 116 effects of copper, 121, 259 human, 81, 116 metal ion interactions, 76–82, 115–121 mRNA, 257, 258 overexpression, 120 overproduction, 255 proteolytic processing, 252–254 redox properties, 81, 116 Amyotrophic lateral sclerosis, 3, 4, 127, 290, 384, 430 age-related, 185 Bunina bodies, 62 familial, see Familial amyotrophic lateral sclerosis pathogenesis, 184 sporadic, 180, 181, 185, 287

439

[12]aneN4 , see Cyclen Anemia microcytic, 373, 384 Animal studies (of) (see also individual species) aluminum toxicology, 380–384 cadmium neurotoxicity, 400, 409 lead neurotoxicity, 408, 409 mercury neurotoxicity, 414, 415 multiple sclerosis, 431 Parkinson’s disease, 265–270, 301, 430 transgenic, 45 Antacids aluminum in, 385 Antagonistic action, see Interdependencies Antibiotics (see also individual names), 118 Antibodies anti-prion protein, 100 transferrin receptor, 246, 249 Antioxidant(s) (see also individual names), 256, 301, 305, 399, 430 amyloid precursor protein, 116 A peptide, 254 Cu,Zn superoxide dismutase, 180 prion protein as, see Prion protein vitamin E, see Vitamin E Apolipoprotein E, 381 Apomorphine, 304 Apoptosis (see also Cell, death), 246, 247, 249, 250, 309, 341 APP, see Amyloid precursor protein Ascorbate (or ascorbic acid), 139, 431 as reductant, 131 Aspartic acid synthesis, see Biosynthesis Association constants, see Dissociation constants and Stability constants Astrocytes, 101, 106, 109, 385 copper uptake, 97, 98, 100 iron in, 131, 134, 240, 242, 243, 262, 300 Ataxias Friedreich’s, see Friedreich’s ataxia spinocerebellar, 286, 290 Atomic absorption spectroscopy, 131 flameless, 307

440

SUBJECT INDEX

ATP, 216, 217, 265, 267, 373, 385 aluminum binding, 375, 376, 383 -dependent enzymatic activities, 382 -dependent proton pump, 232 hydrolysis, 240 metabolism, see Matabolism synthesis, see Biosynthesis ATPases Ca2+ -, 215 copper-transporting, see Copper-transporting ATPases heavy metal, 212, 213 Menkes disease, see Copper-transporting ATPases Na+ ,K+ -, 382 P-type, 117, 213 rat, see Rat Wilson’s disease, see Copper-transporting ATPases Atrophy multisystem, 127, 131 Azide, 187 Azurin, 94

B BAL, see 2,3-Dimercaptopropanol Battery lead-acid, 396, 405 Benzimidazole zinc complex, 356 Benzothiazole, 356 Benzoxazole, 356 Biliverdin, 230 Binding constants, see Dissociation constants and Stability constants Bint al Thahab, 405 Biosynthesis of acetylcholine, 383 -aminobutyric acid, 383 aspartate, 383 ATP, 302 coenzyme A, 260 dopamine, 245 ferritin, 234, 236, 238, 249 glutamate, 383 heme, 235, 265 neurotransmitters, 228

Birds prion protein homologues, 91, 92 Bis(o-aminophenoxy)ethane-N,N,N,N tetraacetic acid, 349, 354 Blood (see also Serum) lead levels, 401, 403–408 mercury in, 409 zinc in, 324 Blood-brain barrier (transfer of), 2, 3, 428, 429, 431 aluminum, 376, 380, 381, 387 Alzheimer’s disease patients, 119 copper, 119 iron, 126, 133, 136, 239, 262, 268, 299 mercury, 409–411, 414 Parkinson’s disease patients, 249, 265, 266 verapamil, 134 zinc, 324 Bond(s) C–Hg, 410 disulfide, see Disulfide bond hydrogen, see Hydrogen bonds Zn–S, 326 Bone lead in, 404 Bovine spongiform encephalopathy, 90, 91 Brain (containing) aluminum, 377, 381, 383, 385, 387 blood barrier, see Blood-brain barrier calcium, 323 copper accumulation, 254–256 copper, 2–4, 118–120, 165, 166, 185, 210, 211, 217, 323 damage, 400 development, 154–159, 239–245 disease phenotypes, 154–159 function, 239–245 iron accumulation, 2–4, 126, 129–137, 159–169, 185, 228, 229, 239–245, 254–256, 260–270, 298–306, 323, 324, 431, 432 iron distribution, 242 iron loading, 265–270, 296 lead, 402 magnesium, 2, 324 manganese, 107, 306–308 mercury accumulation, 409

SUBJECT INDEX

441

[Brain (containing)] metallothioneins, 325 myelination, 153–159 nitric oxide, 135 rat, see Rat reactive oxygen species, see Reactive oxygen species zinc, 2, 118, 165, 166, 256, 257, 323–328 British anti-Lewisite, see 2,3-Dimercaptopropanol BSE, see Bovine spongiform encephalopathy Bunina bodies, 62

C Cadmium(II) (element and ion) (in) absorption, see Absorption accumulation, 4, 399 albumin, 397 animal studies, 398–400 excretion, 398 free, 398 hair, 399 interdependency with other metal ions, see Interdependencies mechanism of action, 398, 399 metallothionein, 397, 398 nephrotoxicity, see Nephrotoxicity neuromelanin, 304 neurotoxicity, see Neurotoxicity toxicokinetics, 397, 398 Calbindin aluminum interaction, 376 Calcineurin, 323 Calcium(II) (element and ion) (in), 376, 383 ATPase, see ATPases brain, see Brain cellular, 340, 343–347, 351–355, 357, 358 central nervous system, see Central nervous system channels, see Calcium channels cytosolic, 301 depletion, 387 extracellular, 398

[Calcium(II) (element and ion) (in)] fluorescent probes, 338, 349–352, 354–359 homeostasis, see Homeostasis in neuronal signal transduction, 323 interdependency with other metal ions, see Interdependencies signalling, 387 Calcium channels, 254, 327, 411 blocker, 127 voltage-dependent, 324, 327, 401 Calmodulins, 323, 327, 398 aluminum interaction, 376 lead interaction, 401 Camelot disaster, 283 Carbonic anhydrase aluminum in, 382 aryl sulfonamide binding, 341 mutants, 335 zinc in, 325, 335, 336 Carbon monoxide, 42, 44, 45, 230 Carotene , 431 Caspases, 247 Cat (studies with) aluminum, 380 mercury, 415 Parkinson’s disease, 265 Catalase(s), 3, 140, 301, 398 iron, 229 Cataract, 4 Catechin green tea, 267 Catechol(ate)(s) (and residues) 3,5-di-tert-butylpyro-, 306 Fe(III) complex, see Iron(III) oxidized, 248 Catecholamines (see also individual names), 141 aluminum binding, 373, 375 Cu(II) complexes, 292, 308, 309 in vitro chemistry, 291–297 interplay with Fe(II/III), 293–295 interplay with metal ions in neurological diseases, 291–312 Ni(II) complexes, 309 oxidation, 291, 313 vanadium complexes, 309–311

442

Cattle bovine spongiform encephalopathy, see Bovine spongiform encephalopathy CD, see Circular dichroism Cell(s) CHO, 119 death, 238, 245, 247, 265, 266, 298, 301, 303, 327 dopaminergic, 265 HeLa, 357 neuronal, see Neuron Center for Disease Control and Prevention, 405, 406 Central nervous system (containing), 305, 310, 383, 429 acetylcholinesterase, 382 aluminum, 380, 383 calcium, 323 copper levels, 117, 118, 121, 384 disorders, 228 function of metal ions, 322 genetic degenerative diseases, 166 iron accumulation, 129, 384 lead effects, 402 manganese exposure, 306 metallothioneins, 325 prion proteins, 90 rat, see Rat -synuclein expression, 245 zinc, 117, 324, 326, 340 Ceruloplasmin, 4, 120, 136, 208, 210, 220, 232, 248, 262, 308 Channels calcium, see Calcium channel cation-selective, 254 ion, see Ion channels potassium, 322 sodium, 411 Chaperone(s) copper, 117, 120, 184, 185, 189, 196, 208, 216 cytoplasmic, 245 heat-shock, 246, 247 metallo-, 2, 3, 260 protein folding, 10, 46 Charge transfer internal, 354 photoinduced, 354

SUBJECT INDEX

Chelators (see also individual names), 428, 429 aluminum, 377 calcium, 349, 350, 354 copper, 211, 217, 270, 431 iron, 131, 138, 245, 249, 266–270, 305, 431 magnesium, 354 therapy, 250, 268, 428, 429 zinc, 327, 342, 343 Chicken prion proteins, see Prion proteins Children exposure to lead, 405–408 prospective studies, 406, 407 5-Chloro-7-iodo-8-hydroxyquinoline, see Clioquinol Cholesterol, 3, 254 diet, 118 Chromatography affinity, 100, 104 fast liquid, 379 Chromium (in) interdependency with other metal ions, see Interdependencies neuromelanin, 138, 304 Chromophores, 15 benzofuran, 355 fura, 355 naphthalimide, 352 Chromosome mutations, 290 Chronic manganese intoxication, 307 Chronic wasting disease, 90, 92 Chymotrypsin, 381 Cigarettes cadmium exposure, 396 Cinnarizine, 127 Circular dichroism studies of A peptide, 78 copper-transporting ATPases, 214 Cu-prion protein, 76, 95 cytochrome b, 39 cytochrome c, 19, 24, 26, 28, 36–38, 42, 43 far-UV, 19, 24, 28, 36 neurofibrillary tangles, 377 -synuclein, 46, 47

SUBJECT INDEX

Citrate (or citric acid), 293, 297, 372, 373 aluminum, 378–380 cycle, 261 Clioquinol, 118, 119, 270, 328, 429, 430 Clusters 4Fe–4S, 230, 236, 237 hydroxo-bridged aluminum, 377 iron–sulfur, 265 metal–thiolate, 337 oxyhydroxide Fe(III), 241 Cobalt (different oxidation states) (in) neuromelanin, 304 transport, see Transport Cobalt(II), 351, 383 autoxidation of dopamine, 312 Cobalt(III) cytochrome c, see Cytochrome c porphyrin, 37 Coenzyme A, 260 biosynthetic pathway, 260 deficiency, 260 Collagen maturation, 230 Coordination sphere of copper in A peptide, 259 Cu,Zn superoxide dismutase, 187, 188 neurofibrillary tangles, 377 Copper (different oxidation states) (in) 67 Cu, 97, 100 accumulation, 309 and reactive oxygen species, 62 brain, see Brain central nervous system, see Central nervous system chaperones, see Chaperones chelators, see Chelators and individual names coordination sphere in A peptide, 259 deficiency, see Deficiency excretion, 208, 210 homeostasis, see Homeostasis Huntington’s disease, 432 intracellular trafficking, 120 -mediated oxidative damage, see Oxidative damage metabolism, see Metabolism multiple sclerosis, 430, 431

443

[Copper (different oxidation states) (in)] neuromelanin, 138, 304 oxidative damage, see Oxidative damage Parkinson’s disease, 128 serum, see Serum transport between neurons and astrocytes, 100 transport, see Transport treatment of macular degeneration, 431 Copper(I) (in), 75, 312 amyloid precursor protein coordination, 82, 117 copper-transporting ATPase, 214 Cu,Zn superoxide dismutase, 181, 184 glutathione complex, 208 Copper(II) (binding to), 208 amyloid precursor protein, 80–82, 116, 120 A peptide, 77–79 catecholamines, 292, 308, 309, 312 Cu,Zn superoxide dismutase, 181, 184, 187 multi-histidine binding, 70 nitrilotriacetate, 118 octapeptide repeat fragment, see Octarepeat fragment of prion proteins prion proteins, 63–76, 92–101 reduction, 64, 75, 116, 119 stability constants of complexes, 64 tyrosine binding, 78 Copper(III), 75 Copper transport (in) biological, 208, 209 disorders, 207–221 genes, 212, 213 Menkes disease, 207–221 pathways, 209 Wilson’s disease, 207–221 Copper-transporting ATPases, 4, 92, 117, 212 Ag+ binding, 216 ATP-binding domain, 217 cellular trafficking, 214 conformational changes, 214–216 Cu(I), 214 Cu–Cu interaction, 214 functional studies, 214, 215, 217

444

[Copper-transporting ATPases] Menkes disease, 208, 213, 216, 217 mutation, 217 structure, 213, 214, 216, 217 Wilson’s disease, 213–217 Copper-transporting receptor, 97 Cornea copper accumulation, 210 Corticobasal degeneration, 284, 290 Coumarin fluorophore, 329, 333, 341–343, 357 Creutzfeldt-Jakob disease, 3, 5, 92, 107, 108, 127 familial, 288 misfolded proteins, 10 oxidative damage, 108 sporadic, 287 variant, 89, 90, 288 cRNA amyloid precursor protein, 258 Cuprizone, 92, 93, 118 Curcumin, 270, 399, 430 Cu,Zn superoxide dismutase, 308, 398 active site channel, 187 activity in transgenic mice, 117 aggregation, 183, 185–187, 191, 193, 196–198 and amyloid precursor protein, 119–121 and familial amyotrophic lateral sclerosis, 179–198 as antioxidant, 180 aspartate residue, 187, 188 coordination sphere, 187, 188 Cu(I), 181, 184 Cu(II), 181, 184, 187 dimeric structure, 182 dimerization, 190 dissociation, 196 enzymatic activity, 181 histidine residues, 187, 188 imidazolate bridge, 187, 188 insect, 191 metal-binding sites, 191–194 metal-deficient, 194, 195 misfolded, 198 monomeric, 194–198 mutations, 180–185, 188–198 negative design, 189, 190

SUBJECT INDEX

[Cu,Zn superoxide dismutase] oxidative damage, see Oxidative damage pathogenic proteins, 183–187, 190–198 self-association, 183, 190, 198 self-inactivation, 186, 187 self-oxidation, 195 structural features, 187–194 toxicity, see Toxicity wild-type, 185–191, 196–198 yeast, 188, 191, 195, 196 zinc in, 187, 188 Cyanide complexes, 187 iron, 131 Cyclen dansylamidoethyl-, 341, 342 fluorescein complex, 350, 351 lanthanide complexes, 343, 344 Cysteine (and residues) N -acetyl-, 430, 431 AEDANS-modified, 21–26, 31–35, 37, 38 S-nitroso-, 359 nucleophillic attack, 296, 313 Cytochrome a, 230 Cytochromes b, 230 b561 , 231 b562 , 39–42 duodenal, 231 folding kinetics, 41 iron in, 39–45 redox potential, see Redox potentials Cytochromes c, 14, 19, 230 AEDANS-labeled, 21–26, 31–35, 37, 38 c , 40–45 c556 , 42–45 c-b562 , 42–45 Co(III)-substituted, 35–39 compact structure, 33 conformational states, 17 correctly folded, 32 electron transfer kinetics, 19 equine, 28, 31, 32, 37 Fe(II/III), 28, 29, 37–45 fluorophore-labeled, 21–25 folding landscapes, 28–39 hydration, 19 iso-1, 21–26, 31–35

SUBJECT INDEX

445

[Cytochromes c] misfolded, 41 molten globule, 24–26 rate constants, see Rate constants redox potential, see Redox potentials refolding, 33, 41, 45 site-directed mutagenesis, 21 solvent exposure, 20 tuna, 36 unfolded, 21–24, 36 yeast, 21–26, 31–37 zinc, 17–20, 27–31 Cytochrome c oxidase, 2, 208, 211, 229, 249, 308, 309, 431 Cytokine, 238 Cytotoxicity of protein aggregates, 46

D Dansyl, see 5-(Dimethylamino)-1naphthalenesulfonyl Databases of proteins PDB, see Protein Data Bank Deer chronic wasting disease, 90, 92 Deferiprone, 250, 267, 268 Deferrioxamine, see Desferrioxamine Deficiency of copper, 92, 120, 121, 211, 430 iron, 231, 266 vitamin B12 , 430 zinc, 431 Dehydrogenases glucose-6-phosphate, 398 glycerol phosphate, 402 Dementia, 127, 153 brain iron, 130 familial British/Danish, 288 frontotemporal, 256, 284, 290 vascular, 243 with Lewy bodies, 140, 165, 167, 169, 285, 289, 299 Dendritic plaques, 289 Dentatorubropallidoluysian atrophy, 287, 290, 299 Deprotonation constants, see Acidity constants

Desferal (see also Desferrioxamine), 267 Desferrioxamine, 138, 237, 250, 263, 267, 268, 270, 301, 304, 377, 428 B, see Desferal Desferrithiocein, 250, 267, 268 Diabetes, 262 Dialysis chronic, 384, 385 hemo-, 384 Diethylenetriamine-N,N,N,N,Npentaacetate, Eu(III) complex, 343 Gd(III) complex, 344 Tb(III) complex, 343 Dihydrobenzothiazines, 296, 302 3,4-Dihydroxyphenylalanine, 127, 228, 291, 292, 295, 303, 310 oxidation, 295 synthesis, 228 treatment of Parkinson’s disease, 303 2,3-Dimercaptopropanol treatment of Wilson’s disease, 217, 218 5-(Dimethylamino)-1naphthalenesulfonyl fluorophore, 329, 333, 342 Dioxygen (see also Oxygen) and catecholamines, 295, 296, 310–313 storage, 230 transport, 230 Diphenylthiocarbazone, see Dithizone Di-(2-picolyl)amine, 342, 344–346, 349–351, 355 Diseases (see also individual names) age-related, see Aging Alzheimer’s, see Alzheimer’s disease brain, see Brain Menkes, see Menkes disease neurodegenerative, see Neurodegenerative diseases Parkinsons’s, see Parkinson’s disease Pick’s, see Pick’s disease Wilson’s, see Wilson’s disease Dissociations constants, see also Stability constants benzazole probes, 356 Cu-prion protein complex, 75, 76 cyclen-zinc complex, 341–343 fluorescein complex, 349, 350, 352

446

[Dissociations constants] fura fluorophores, 355 indo gluorophores, 355 metallothionein-zinc complex, 325 Newport Green dye, 351 zinc-fluorescent sensors, 329, 333, 337 zin-naphthopyr, 358 zinpyr-zinc complex, 345–347, 357 Disulfide bonds, 182, 188, 189, 196, 197, 326, 333 Dithizone, 328 Divalent metal transporter 1, 231–233, 235, 238, 248, 249, 267 DNA aluminum binding, 375 damage, 139, 140, 301, 399 repair, 139 Dog aluminum studies, 380 Parkinson’s disease studies, 265 Dopa, see 3,4-Dihydroxyphenylalanine Dopamine, 127, 132, 136, 137, 139, 141, 228, 245, 247, 249, 291, 292, 296, 297, 301, 304, 400, 408, 430 and lead, 402 and manganese, 306 biosynthesis, see Biosynthesis 5-cysteinyl-, 296 5-glutathionyl-, 297 6-hydroxy-, see 6-Hydroxydopamine -hydroxylase, 211, 308 metal ion effect on autoxidation, 311, 312 oxidation, 302, 306 Dopaminochrome, 292, 293, 302 leuco-, 292, 293 Dopaminoquinone, 293, 296, 297, 302 Down syndrome, 121 Drinking water containing aluminum, 385 cadmium, 400 lead, 396 Drosophila, 246 Drug (see also individual names) -induced parkinsonism, 127 DTPA, see DiethylenetriamineN,N,N ,N,N-pentaacetate

SUBJECT INDEX

Dyes (see also individual names) fluorescence, see Fluorophores and individual names Newport Green, 351, 353 Texas Red Dextran, 353 zinc-sensitive, 351, 353

E EDTA, see Ethylenediamine-N,N,N,N tetraacetate Electrode normal hydrogen, 13, 17 Electron paramagnetic resonance, see EPR Electron spin resonance, see EPR Electron transfer (in) bimolecular quenching, 18, 19 cytochrome c, 14, 15, 17–20, 27–31 effect of folding, 15 irreversible photochemical reagents, 14 kinetics, 14–16, 27–30 mitochondrial, 301 outersphere, 294, 295 photoinduced, see Photoinduced electron transfer quenching, 27, 340 rate constant, 28–30, 39 triggered protein folding, 11–15, 39 Electrospray ionization mass spectrometry studies of aluminum citrate complex, 379 Elk chronic wasting disease, 90, 92 Encephalopathies dialysis, 384, 387 lead-induced, 402, 403, 405 with neuroreserpin inclusions, 288 Endocytosis, 65, 66 transferrin-mediated, 232, 233 Energy transfer dipole–dipole, 15 fluorescence, see Fluorescence resonance energy transfer quenching, 44

SUBJECT INDEX

447

(-)-Epigallactocatechin-3-gallate, 267–269 Epinephrine, see Adrenaline nor-, see Noraderenaline EPR studies of A peptide, 78, 259 melanin, 306 neuromelanin, 138, 248 prion protein, 95, 96 -synuclein, 46 Escherichia coli cytochrome b562 , 42 prion protein, 101 ESI-MS, see Electrospray ionization mass spectrometry ESR, see EPR Ethylenediamine-N,N,N,N -tetraacetate, 101, 428 Ethylmercury, 409–413 Europium(III) (see also Lanthanides) cyclen complex, 343 diethylenetriamine-N,N,N,N,Npentaacetic acid, 343 Excitotoxicity, 128, 157–159, 162, 164, 169, 254, 301, 327 kainin acid, 247

F FALS, see Familial amyotrophic lateral sclerosis Familial amyotrophic lateral sclerosis, 62, 179–198, 287, 290, 309, 323 and Cu,Zn superoxide dismutase, see Cu,Zn superoxide dismutase Cu-Zn superoxide dismutase mutations, 180–185, 188–198 etiology, 194 molecular mechanisms, 181–187 Faroe Islands contaminated seafood, 413 FDRI, see Field-dependent relaxation rate method Fenton chemistry, 102, 248, 256, 266, 309 (-like) reaction, 62, 136, 139, 141, 295, 296, 301, 302, 306

Ferredoxins (see also individual names) yeast, 265 Ferrets aluminum studies, 380 Ferrihydrite, 234 Ferritin(s), 126, 132, 134–137, 159, 230, 231, 233, 236–240, 243, 269, 295, 298–300, 302–305, 430, 432 and 6-hydroxydopamine, see 6-Hydroxydopamine as biomarker, 167, 168 biosynthesis, see Biosynthesis in vivo iron measurement, 166–168 iron release, 162, 267, 296 mammalian, 234 mRNA, 135, 161, 234–236, 258 receptor, 241 subunits, 234, 239, 241, 242, 248 Ferrocene, 267 3,5,5-trimethyl hexanoyl, 266 Ferroportin, see Iron regulated gene protein 1 Ferrozine, 131 Fetus mercury toxicity, 409, 413, 414 Fibrils, 245 A, 165, 253 Lewy body, 140 prion protein, 74, 90, 106 -synuclein, 45, 141 Field-dependent relaxation rate method ferritin iron, 166–168 Filaments amyloid-like, 193, 194 Fish mercury-contaminated, 397, 412–414 prion protein homologues, 92 Flunarizine, 127 Fluorescein, 333, 344 amino-, 348, 349, 355 Ca2+ -responsive, 349–352 2 ,7 -dichloro-, 344, 351 quinoline-, 353 Fluorescence assay, 75, 76 decay rates, 16, 22, 31 donor–acceptor distance, 15, 16, 22, 23, 25, 31–33, 48

448

[Fluorescence] donor–acceptor pair, 15, 21–23, 46, 49 energy transfer, see Fluorescence resonance energy transfer microscopy, 344 tryptophan, 46–48 Fluorescence polarization time-resolved, 47 Fluorescence resonance energy transfer, 15, 16, 21 calcium probes, 338 decay kinetics, 15, 16, 21, 24–26, 31–35, 37, 38, 46–50 distance measurements, 15, 16, 22, 23, 25, 37, 38, 46, 50 zinc probes, 333–338 Fluorescent probes for zinc, 323, 328–334 adapted calcium probes, 349–352, 354–359 adapted magnesium probes, 354–359 artificial amino acids, 334 benzazole, 355, 356 fluorescein/rhodamine, 351 lanthanide-containing, 343, 344 list of, 330–323 materials-based, 353, 354 nucleic acids, 338, 339 peptides, 329, 333, 334 proteins, 335–338 quinolines, 338–341, 345 quinones, 358, 359 rhodafluor, 351 sulfonamides, 335 zinc-aminofluorescein, 348 zinpyr, 344–346 Fluorides, 187, 378 Fluorophores (see also individual names) coumarin, see Coumarin coumazin-1, 357 dansyl, see 5-(Dimethylamino)-1naphthalene sulfonyl fura, 354, 355 indo, 354, 355 naphthalene diimide, 338 Newport Green dye, 351, 353, 354 probes for zinc sensing, see Fluorescencent probes for zinc

SUBJECT INDEX

[Fluorophores] stains for Pb2+ , 338 Texas Red Dextran, 353, 354 Food (containing), 78 aluminum, 372 cadmium, 396 mercury, 397 sea-, see Seafood Food and Drug Administration of the United States, 197 Formation constants, see Stability constants Fossil fuel burning, 396 Frataxin, 4, 130, 263–265 mRNA, 263 mutations, 264 structure, 264 Free energy protein folding, 36 Friedreich’s ataxia, 4, 130, 263–265, 286, 429 Fungicides methylmercury in, 412

G Gadolinium(III) (see also Lanthanides) diethylenetriamine-N,N,N,N,Npentaacetate, 344 Gastrointestinal tract (absorption of) iron, 230–232 lead, 400 mercury, 409, 411 Genes copper transport, see Copper transport Menkes disease, 212, 213, 220 Wilson’s disease, 212 Genetic diseases, see individual names Gerstmann-Sträussler-Scheinker syndrome, 90 Glucocorticoid receptor, 402 Glucose metabolism, see Metabolism 6-phosphate dehydrogenase, 398 Glutamate (or glutamic acid) abnormal transport, 183 decarboxylase, 408

SUBJECT INDEX

449

[Glutamate (or glutamic acid)] synthesis, see Biosynthesis toxicity, 157 Glutamine synthetase, 401, 402, 431 -Glutamyl transpeptidase, 296, 297 Glutathione, 4, 139, 140, 250, 296, 297, 301, 302, 399 Cu(I) complex, 208 peroxidase, see Peroxidases reductase, 398 Glycine carbonyl oxygen interactions, 73 Cu(II) complex, 98, 99 Glycoproteins, 91 P-, 133, 134 Glycosylases DNA repair, 139 Guanidine hydrochloride, 16, 18–20, 22, 23, 28–31, 36–39, 41–43, 45 Guanosine 8-hydroxydeoxy-, 301

H Hair cadmium in, 399 kinky hair syndrome, 211 lead in, 399 Hallervorden–Spatz syndrome, 4, 129 iron accumulation, 166, 260, 261 Hamster aluminum studies, 380 Chinese, 119 models of transmissible spongiform encephalopathies, 90 ovary cells, 119 prion protein, see Prion proteins scrapie, 109 Syrian, 64 Hemagglutinins, 254 Heme(s) biosynthesis, see Biosynthesis oxygenase, 140, 230, 231, 242, 256 proteins, 229 unfolded, 14 Hemerythrin, 230 Hemochromatosis genetic, 130, 248

Hemodialysis (see also Dialysis), 384 Hemoglobin, 2, 230 lead in, 401 Hemosiderin, 126, 167, 233, 234, 243, 248 Heparin, 116 Hepatitis, 210 Hepatocytes copper accumulation, 210 iron uptake, 232 Hephaestin, 231, 232 Hirano bodies, 141 Histidine (and residues) (see also Imidazole) -copper, 70, 211, 219, 220 Cu,Zn superoxide dismutase, see Cu,Zn superoxide dismutase 2-oxo-, 186 Homeostasis (of) (see also Metabolism) calcium, 128, 301, 411 copper, 117–121 iron, 126, 135, 137, 163, 229, 234–238, 242, 249, 305 metal ion, 80 prooxidant-antioxidant, 62 transition metal ions, 166 zinc, 325, 327 Human amyloid precursor protein, 81 A peptide, see A peptide diseases, see individual names neurotoxicity studies, 399, 403–408, 412–414 prion protein, see Prion protein Huntingtin, 152, 155, 157, 159, 163, 164, 166, 289 Huntington’s disease, 62, 180, 185, 260, 286, 289, 290, 299, 431, 432 aggregates, 289, 290 brain iron, 130, 153, 159–169 copper levels, 432 in vivo assessment of iron, 151–169 mouse model, see Mice myelin breakdown, 158, 160–162 myelin integrity, 162 oxidative damage, 157 Hydrogen bond benzazole probes, 356 Cu,Zn superoxide dismutase, 188

450

SUBJECT INDEX

[Hydrogen bond] protein folding, 17, 27 quinolines, 340 Hydrogen peroxide, 3, 105, 117, 137, 139, 181, 183, 184, 186, 210, 237, 247, 248, 255, 267, 295, 301, 302, 306 formation, 141 8-Hydroxydeoxyguanosine, 301 6-Hydroxydopamine (in), 291, 294, 296 and ferritin, 297 Fe(III) complex, 293, 294, 313 hypothetical mechanism of toxicity, 267 Parkinson’s disease, 133, 137, 265, 267, 268, 301–303, 306 Hydroxylases dopamine -, 211, 308 tyrosine, 228, 245 Hydoxyl radical, 3, 117, 135, 139, 141, 183, 184, 186, 210, 238, 245, 255, 256, 266, 267, 296, 301, 302, 410 Hydroxypyridin-4-ones (see also individual names) 1,2-dimethyl-3-, see Deferiprone 8-Hydroxy-5-(N,Ndimethylsulfonamido)-2methylquinoline, 334 8-Hydroxyquinoline, 430 chromophore, 334, 340, 341 Hyperphosphatemia, 385

I ICP-MS, see Inductively coupled plasma-mass spectrometry Imidazole (and moieties) (see also Histidine) Cu,Zn superoxide dismutase, see Cu,Zn superoxide dismutase heme iron, 31 Iminodiacetic acid phenyl-, 352, 354 Inductively coupled plasma-mass spectrometry, 299 Cu,Zn superoxide dismutase, 191 Influenza virus, 254

Infrared spectroscopic studies of neurofibrillary tangles, 377 neuromelanin-iron complex, 304 In-gel assay prion protein, 102 Insect Cu,Zn superoxide dismutase, 191 Insomnia fatal familial, 90 Interdependencies (between metal ions) cadmium–calcium, 397 cadmium–chromium, 397 cadmium–iron, 397 cadmium–magnesium, 397 cadmium–zinc, 397 calcium–lead, 401 copper–zinc, 219 Interferons , 238 , 238 Interleukins, 238, 258 5-((((2-Iodoacetyl)-amino)-ethyl)-amino)naphthalene-1-sulfonic acid as cytochrome c label, see Cytochromes c fluorescence decay kinetics, 21–25, 31–35 structure, 21 Ion channels (see also Channels) agonist-gated, 326 glutamate-gated, 351 voltage-gated, 326, 352 Ionization constants, see Acidity constants Ionophores (see also individual names) dansyl, 341 IR, see Infrared spectroscopic studies Iraq ethylmercury poisoning, 413 IREG1, see Iron regulated gene protein 1 Iron (different oxidation states) (in) absorption from the gastrointestinal tract, 230–232 and reactive oxygen species, 62 blood-brain barrier transfer, see Blood-brain barrier brain, see Brain -catalyzed free radical generation, 233

SUBJECT INDEX

[Iron (different oxidation states) (in)] central nervous system, see Central nervous system deficiency, see Deficiency depletion, 236 essential roles, 126 ‘free’, 233 Hallervorden–Spatz syndrome, see Hallervorden–Spatz syndrome homeostasis, see Homeostasis human biology, 229, 230 -induced oxidative damage, see Oxidative damage inorganic chemistry, 229, 230 interaction with prion protein, 105 interdependency with other metal ions, see Interdependencies intracellular utilization, 232–234 intracellular, 135, 137 labile pool, see Labile iron pool liver, see Liver -mediated toxicity, 162 metabolism, see Metabolism neurodegenrative diseases, see Neurodegenerative diseases neuromelanin, 138 oligodendrocytes, see Oligodendrocytes oxyhydroxide, 243, 304 Parkinson’s disease, see Parkinson’s disease storage in macrophages, see Macrophages –sulfur bond, 42 –sulfur cluster, 230, 236, 237, 265 transport, 230–238 Iron(II) (in) cytochrome b, 39–45 cytochrome b562 , 39, 40, 42 cytochrome c, see Cytochrome c cytochrome c , 41, 42, 44 cytochrome c556 , 44 cytochrome c-b562 , 44 interplay with catecholamines, 293–295, 312 neurodegenerative diseases (see also Neurodegenerative diseases), 227–279

451

[Iron(II) (in)] oxidation, 232 reactive oxygen species formation, 139 Iron(III) (in), 310, 376 amyloid binding, 377 brain, 132, 134–137, 305 catecholamines, 291, 293–295, 312, 313 cytochromes c, see Cytochromes c neurodegenerative diseases, 227–279 oxyhydroxide cluster, 241 reductase, 231, 232 reduction, 131 Iron regulated gene protein 1 (see also Metal transporter protein), 231, 232, 235, 262, 263 Iron regulatory element, 135, 165, 235–237, 249, 257–259 mRNA, 299 Iron regulatory proteins, 135, 230, 235, 237, 238, 248, 249, 258, 259, 267, 269 Iron–sulfur proteins (see also individual names), 229, 230 non-, 229 Ischemia cerebral, 327 Isotope effects, 17–19

J Japan Minamata poisoning, see Minamata

K Kainate, 324 Kayser–Fleischer rings, 210 Kennedy disease, 286 Kidney cadmium toxicity, 397, 399 copper accumulation, 210, 217 lead in, 402 mercury excretion, 409 Kinases pantothenate, 4, 260 protein, 401 Src, 327

452

SUBJECT INDEX

Kinky hair syndrome, see Menkes disease Kuru, 90

L Labile iron pool, 233, 234, 236, 248 free radical production, 238, 239 Lactate aluminum(III), 380, 381 Lactoferrin, 299 receptor, 250, 265 LAMMA, see Laser microprobe mass analysis Lanthanides(III) (see also individual elements) cyclen complexes, 343, 344 luminescent probes, 343 Laser excitation, 43 Laser microprobe mass analysis, 132 Lead (different oxidation states) (in) and children, 405–408 animal studies, 402, 408, 409 -based paints, 396 battery, see Battery blood, see Blood bone, 404 brain, 402 drinking water, see Drinking water hair, 399 half-life, 401 hemoglobin, 401 interdependency with other metal ions, see Interdependencies mechanism of action, 401–403 monoamine oxidase, 408 neurological effects, 403–405 neuromelanin, 304 neurotoxicity, see Neurotoxicity occupational exposure, 404 Parkinson’s disease, 128 teeth, 405, 406 toxicokinetics, 400, 401 Lead(II) accumulation, 4 fluorescent stains, 338 Leucodopaminochrome, 292, 293 Levinthal’s paradox, 26

Levodopa, see 3,4-Dihydroxyphenylalanine Lewy body (in) dementia, see Dementia Parkinson’s disease, 45, 46, 62, 127, 131, 136, 140, 141, 244, 245, 247, 285, 289, 298, 300, 303, 305, 306 Lipid(s), 305 peroxidation, 3, 108, 137–140, 210, 238, 247, 301, 399, 411 Liver cadmium in, 397 copper in, 211, 217 disease, 210 iron accumulation, 263 lead in, 402 Luminescence (see also Fluoroscence) lanthanide(III) probes, 343 Lysyl oxidase, 211, 218, 220

M Machado-Joseph atrophy, 286 Macaca fascicularis, 408 Macroconstants, see Acidity constants and Stability constants Macrophages iron storage, 234, 235, 250, 260, 262, 263 zinc in, 355 Macular degeneration, 431 treatment, 431 Magnesium(II) (element and ion) (in), 376, 382, 383 brain, see Brain cellular, 340, 343–347, 351, 353–355, 357, 358 interdependency with other metal ions, see Interdependencies Magnetic resonance imaging brain, 132, 141, 158, 243, 299, 302, 307 ferritin iron, 161, 166–168, 432 field-dependent relaxation rate, 166–168 myelin breakdown, 162, 169 Malondialdehyde, 268

SUBJECT INDEX

Maltose binding protein, 336 conformational change, 336 mutants, 337 Mammalian prion diseases, 91 prion proteins, see Prion proteins Manganese (different oxidation states) (in) and reactive oxygen species, 62 binding to prion protein, 104–106 brain, see Brain metabolism, see Metabolism multiple sclerosis, 431 neurodegenrative diseases, see Neurodegenerative diseases neuromelanin, see Neuromelanin neurotoxicity, 307 occupational exposure, 306, 308 Parkinson’s disease, 128, 306–308 pathophysiology, 308 superoxide dismutase, see Superoxide dismutase transport, see Transport Manganese(II), 383 and dopamine, 306, 312 and melanin, 306 carbonyl oxygen interaction, 73 Manganese(III), 306 Manganism, 306 Mass spectrometry Cu(II)-prion protein complex, 75 Matrix metalloproteinases, 431 tissue inhibitor, 4 Maximum-entropy analysis, 22 Melanin, 228, 267, 292, 295, 296, 300, 302, 304, 313 and manganese(II), 306 neuro-, see Neuromelanin sulfur content, 296, 297 Menkes disease, 4, 308, 309 ATPase, 216, 217 clinical features, 211 copper transport, see Copper transport copper-histidine administration, 211, 219, 220 gene, 212, 213, 220 kinky hair syndrome, 211 treatment, 219, 220

453

Mercury (different oxidation states) (in) absorption, see Absorption alkyl compounds, 409 animal studies, 411, 414, 415 blood, 409 ethyl-, 409–413 excretion, 409–411 fish, see Fish food, see Food mechanism of toxicity, 411–414 methyl-, see Methylmercury neuromelanin, 304 Parkinson’s disease, 128 phenyl-, 409, 410 seafood, see Seafood toxicokinetics, 409–411 vapor, 409 Mercury(II) accumulation, 4 Metabolism (of) (see also Homeostasis and Transport) aluminum, 377, 378 ATP, 383 copper, 107 dopamine, 250, 267, 268 glucose, 383 iron, 128–130, 230–239, 262, 263, 267, 268 manganese, 107 mitochondrial, 299 transition metal ions, 165, 166 Metal ions (see also individual names) (in) Alzheimer’s disease, 115–121 amyloid precursor protein, 76–82, 115–121 interplay with catecholamines, 291–312 Metallothioneins, 185, 208, 210, 219, 325, 326 cadmium binding, 397, 398 function, 325 mutant, 337 nitric oxide interaction, 338 recombinants human, 337, 338 Methionine oxidation, 105 p-Methoxy-N,N -dimethylaniline, 14

454

N -Methyl-D-aspartate, 159, 164, 326, 402 receptor, 157, 254, 324 Methylmercury, 409–415 epidemic poinsonings, 412 excretion, 410 fungicide, 412 1-Methyl-4-phenylpyridinium in Parkinson’s disease, 265, 266 1-Methyl-4-phenyl-1,2,3,6tetrahydropyridine (in) hypothetical mechanism of toxicity, 266 Parkinson’s disease, 265–268, 302, 304, 430 Mice aceruloplasminemic, 263 aluminum studies, 380 APP knockin, 260 APP knockout, 5, 117–119, 260, 429, 430 APP transgenic, 5, 429, 430 atransferrinemic, 232 A peptide, see A peptide cadmium studies, 400 frataxin-deficient, 265 H-ferritin knockout, 248 IRP-1 knockout, 268 lacking IRP-2, 249, 268 model of Huntington’s disease, 153 models of transmissible spongiform encephalopathies, 90 mSOD1 transgenic, 430 parkin-deficient, 247 Parkinson’s disease studies, 265, 267 prion protein knockout, 91, 101, 208, 209 prion protein, see Prion protein RML-infected, 107, 108 scrapie, see Scrapie SOD1 knockout, 183 TG2576, 277, 381, 429 transcription factor, 338 transgenic, 90, 100, 101, 117–120, 183–185, 195, 196, 198, 257, 289 ZnT-3 knockout, 325 Micelles (see also Vesicles) sodium dodecyl sulfate, 47–49

SUBJECT INDEX

Microglia, 242, 243, 248, 255, 260, 385 ferritin in, 239, 240, 263, 300 Microperoxidase-8 zinc, 17, 19 Microscopy cryoelectron transmission, 132 fluorescence, 344 Minamata poisoning, 283, 412, 413 Mining, 396 Mitochondria(l) dysfunction, 139, 298, 301, 308 electron transfer, see Electron transfer metabolism, see Metabolism Molecular dynamics simulation, 17 Monkey (see also individual names) (studies of) aluminum, 380 lead, 408, 409 mercury studies, 414 Parkinson’s disease, 265, 267 Monoamine oxidases and aluminum, 382 and lead, 408 Parkinson’s disease, 127, 136, 139, 265–267, 301 Morphine apo-, 304 Mössbauer spectroscopy of iron in brain, 131, 138, 304 Motoneuron diseases (see also individual names), 287, 290 Mouse, see Mice MRI, see Magnetic resonance imaging mRNA, 91, 254 amyloid precursor protein, 257, 258 ferritin, 135, 161, 234–236, 258 frataxin, 263 iron regulatory element, 299 transferrin receptor, 235, 241 translation, 235 Multiple sclerosis (levels of) copper, 430, 431 manganese, 430, 431 zinc, 431 Multiple system atrophy, 285, 289, 298, 431 Papp-Lantos bodies, 289

SUBJECT INDEX

455

Mutations, 46 carbonic anhydrase, 335 chromosome, 290 copper-transporting ATPases, 217 Cu,Zn superoxide dismutase, 180–185, 188–198 frataxin, 264 maltose-binding protein, 337 metalliothionein, 337 parkin, 246, 247 point, 45 -synuclein, 245, 247 Myelin, 167 breakdown, 156–163, 168, 169 dysfunction, 157, 159 integrity, 163 postmortem stain, 158 Myelopathy, 430 Myoglobin, 230 Zn(II)-substituted, 28

N NAD+ radical, see Radicals NADH, 14, 41, 43 autofluorescence, 344 Naphthalene diimide, 338 National Health and Nutrition Examination Survey, 407 Nephrotoxicity of cadmium, 398 desferrithiocein, 250 Nerve(s) (see also Central nervous system) conduction velocity, 405, 408 damage, 400 effects of lead on function, 404, 405 growth factor, 101 non-adrenergic, non-cholinergic, 425, 426, 439, 444 Neuritic plaques (see also Senile plaques) Alzheimer’s disease, see Alzheimer’s disease Parkinson’s disease, 45 Neurodegenerative diseases (see also individual names) aluminum in, 371–388

[Neurodegenerative diseases] classification, 284–288 general descriptions, 283–290 interplay between catecholamines and metal ions, 291–312 iron in, 126–142, 151–169, 227–270 lesions, 156 manganese in, 306–308 movement disorders, 129, 130 overview, 427–432 pantothenate kinase-associated, 129 production of cytotoxins, 302 role of zinc, 322–358 tauopathies, see Tauopathies Neuroferritinopathy, 129, 260 Neurofibrillary tangles (in), 115, 375, 380, 381, 385, 387 aluminum(III) accumulation, 377 aluminum(III) outersphere coordination, 377 Alzheimer’s disease, see Alzheimer’s disease Neurofilament dysfunction, 183 Neuroleptics, 127 Neuromelanin (binding to), 127, 241, 242, 248, 250, 296, 313, 430 aggregates, 304 aluminum, 132 cadmium, 304 chromium, 138, 304 cobalt, 304 copper, 138, 304 degradation, 296 granules, 240, 304 iron, 132, 136–139, 298, 300, 302–305 lead, 304 manganese, 304 mercury, 304 -protein interaction, 306 zinc, 138, 304 Neuron(al) accumulation of toxic metal ions, 4 -aminobutyric acid, 134 CA3 pyramidal, 324, 327 catecholaminergic, 132, 248, 303 cell death, 91, 128, 136, 139, 140, 384, 385

456

[Neuron(al)] cellular machinery, 180 copper uptake, 97, 98, 100 damage, 139, 140 degeneration, 115, 185 degradation, 373 dopaminergic, 45, 49, 127, 133–135, 137, 139, 140, 240, 243–250, 262, 268, 299, 300, 303, 304, 413 dystrophic, 385 ferritin, 239, 240 glutamatergic, 324 glutaminergic, 324 iron in, 131, 242, 243, 263 motor, 183–185, 195, 198 noradrenergic, 137, 240, 243 pigmented, 132, 137, 304 postsynaptic, 327 zinc accumulation, 324 zinc trafficking, 326 Neurotoxicity of cadmium, 396–400 lead, 400–408 mercury, 409–415 Neurotransmitters (see also individual names), 137, 291, 324, 326, 408 metal ions as, 322 release, 401 synthesis, 228 Nickel (different oxidation states) prion protein binding, 106 transport, see Transport Nickel(II), 351, 383 catecholamine complex, 309, 312 Nicotinamide adenine dinucleotide, see NAD+ Nicotinamide adenine dinucleotide (reduced), see NADH Nitric oxide, 139, 184, 237, 238, 327, 358, 359 and zinc trafficking, 326 in brain, 135 metallothionein interaction, 338 Nitrilotriacetate copper-, 118 Nitrite, 135 peroxy-, 135, 139, 183, 184, 238 Nitrogen monoxide, see Nitric oxide

SUBJECT INDEX

NMR (studies of) 27 Al, 375 13 C, 305, 375 1 H, 36, 375, 379 Ag+ , 216 aluminum citrate, 379 A peptide, 78, 259 ATPase, 214, 216 Co-cytochrome c, 36 multinuclear, 377 neuromelanin, 305 prion proteins, 63, 64, 69, 72, 94, 95 pulsed H/D exchange, 36 -synuclein, 46, 47 Noradrenaline, 291, 294, 408 exocytosis, 401 oxidation, 294, 313 Norepinephrine, see Noradrenaline NTA, see Nitrilotriacetate Nuclear magnetic resonance, see NMR Nucleic acids, 338, 339 DNA, see DNA RNA, see RNA Nucleophilic attack by cysteine, 296, 313

O Octapeptide repeat of prion protein, 92, 98, 99, 102, 103 Cu(II) binding, 64–70, 73, 74, 93–97 structure, 64–70 Oligodendrocytes, 153, 155 development, 156 overproduction, 157, 159 iron in, 159–162, 168, 239, 241, 242 in old age, 159–162, 168, 242 vulnerability, 162 Osteomalacia, 373, 384 Oxidases (see also individual names) l-amino acid, 301 glycollate, 301 lysyl, 211, 218, 220 monoamine, see Monoamine oxidases xanthine, 301 Oxidative damage (in) Cu,Zn superoxide dismutase, 195–198 copper-mediated, 183, 210

SUBJECT INDEX

457

[Oxidative damage (in)] Creutzfeldt-Jakob disease patients, 108 Alzheimer’s disease, 254, 256 iron-induced, 134, 165, 169, 269 radicals, 135 Parkinson’s disease, 139–141, 298, 304 Huntington’s disease, 157 oligodendrocytes, 162 Oxidative stress, 62, 101, 108, 117, 132, 137, 139–141, 162, 184, 185, 228, 242, 244, 248–251, 260, 265–267, 298, 300–303, 308, 309, 325, 384, 398, 411, 431 Oxine, see 8-Hydroxyquinoline Oxygen (see also Dioxygen) radicals, see Radicals Oxygenases heme, see Heme(s)

P Pantothenate, 260, 261 kinase, 4, 260 Parkin, 127, 245–247, 249, 289 mutant, 246, 247 Parkinson’s disease, 3, 4, 45–50, 62, 153, 154, 169, 180, 185, 193, 195, 241, 243, 283, 296, 297, 323, 384, 429, 430 aluminum in, 128 animal models, 133, 134, 139, 265–270, 301, 302, 430 autosomal recessive juvenile, 246, 247 brain iron, 130–133, 163, 164, 168, 169, 244–250, 260–270, 298–306, 431, 432 clinical features, 126 copper levels, 432 different types, 127 drug-induced, 127 etiology, 49, 128, 139, 289 familial, 127, 128, 140, 285 genetic disorders, 128–130 Guam, 127 6-hydroxydopamine in, see 6-Hydroxydopamine iron overload, 139–141, 153, 167, 250, 304 juvenile, 285

[Parkinson’s disease] lead in, 128 Lewy body, see Lewy body manganese in, 128, 306–308 misfolded proteins, 10 misregulation of iron metabolism, 128–130 monoamine oxidase, see Monoamine oxidase neuromelanin in, see Neuromelanin occupational metal exposure, 128 oxidative damage, see Oxidative damage pathogenesis, 46, 49, 126–142 pathophysiology, 164 reactive oxygen species, see Reactive oxygen species role of iron, 126–142, 165, 166 sources of increased iron, 133–138, 299 sporadic, 249, 285 symptoms, 45 synonyms, 126, 127 -synuclein, see -Synuclein toxin-induced, 127 treatment with l-dopa, 303 zinc levels, 432 d-Penicillamine, 428 treatment of scrapie, 105 treatment of Wilson’s disease, 218, 219 Peroxidases, 229 glutathione, 3, 140, 246, 249, 265, 301 Peroxidation lipid, see Lipid Peroxide(s), 230, 247 hydrogen, see Hydrogen peroxide Phenyliminodiacetic acid, 352, 354 Phosphate aluminum complex, 375, 379, 380, 387 Phosphatidylcholine, 383 Phosphatidylinositol glycosyl-, 98, 262 Photoinduced electron transfer coumarins, 341–343 lanthanide-containing zinc probes, 343, 344 zinc-aminofluorescein, 348 zinpyr, 344–348, 357 Pick’s disease, 127, 256, 284, 290

458

Pleurodeles waltl, 399 Polyglutamine repeat extension, 286 Huntington’s disease, 289 Polyneuropathy, 430 Porphyrins (see also Heme and individual names) proto-, see Protoporphyrin IX triplet lifetimes, 20 zinc, 17, 19, 20 Positron emission tomography, 133, 307 Potassium channel, 322 –sodium ATPase, 382 transport, 323 Presenilins, 253 mutation, 256 Prion proteins, 3–5, 62, 166 aggregation, 73, 90, 91, 100, 104, 105 as antioxidant, 101–104, 107 -associated brain degeneration, 165 bird, 91, 92 bovine, 64 cellular isoform, 91, 93–102, 107–109 chicken, 70, 71, 101 cleavage, 105 conformational changes, 104, 106 copper uptake, 75, 97–101 Cu(II) interaction, see Copper(II) Cu(II)-mediated neurotoxicity, 74 diseases (see also individual names), 3, 4, 287, 323 EPR studies, 95, 96 fibrils, see Fibrils fish, 92 hamster, 64, 93, 100, 109 human, 63, 71, 74, 94 internalization, 97–99, 103 iron interaction, 105, 106 isolation, 92 loss of function, 107, 108 mammalian, 63–76, 91, 92, 98, 103 manganese binding, 104–106 metal binding sites, 63, 64 metals in, 89–109 murine, 64, 94, 97, 100–103, 107 nickel binding, 106 NMR studies, see NMR

SUBJECT INDEX

[Prion proteins] octapeptide repeat, see Octapeptide repeat of prion proteins oxidation, 106 oxidative chemistry, 73 pathways upon Cu(II) binding, 105 polymerization, 106 protease resistant, 104–106 recombinant, 95, 101, 102, 106 reptile, 91, 92 schematic repesentation, 103 scrapie isoform, 90, 93, 99–101, 104, 106–109 sheep, 63 structures, 63–65, 68, 72, 73, 75 superoxide dismutase activity, 74, 75, 82, 103, 106, 107 tandem repeat, 70 Xenopus, 98 zinc binding, 106 Progressive supranuclear palsy, 127, 131, 256, 284, 290, 298, 431, 432 Proteases (see also individual names) K, 104 metallo-, 98 Proteasomes, 140 20 S, 197, 198 26 S, 246 Protein(s) (see also individual names) aggregation, 2, 3, 45–47, 62, 136, 139–141, 185, 239, 386, 429 carrier, 325 denatured, 16–26 folding, see Protein folding glyco-, see Glycoproteins green fluorescent, 99, 337, 338 heme, see Heme kinase C, 401 -metal interactions, 327, 397, 429 misfolding, 10–50, 62, 185, 385 -protein interactions, 17, 214, 249 tau, see Tau protein unfolded, see Unfolded proteins zinc finger, see Zinc finger proteins zinc transporter, see Zinc(II) Protein Data Bank (files of protein structures) cytochrome b562 , 40 cytochrome c , 40 yeast iso-1 cytochrome c, 21

SUBJECT INDEX

459

Protein folding dynamics, 26–45 energy landscapes, 11, 27, 39 experimental methods, 10–16 four-helix bundles, 39–45 free energy, 36 hydrophobic pockets, 20 kinetics, 42, 43 pathways, 16, 17, 39 redox active, 11 three-helix bundles, 42, 45 Proteinopathies (see also individual names), 169 transition metal metabolism, 165, 166 Proteosome dysfunction, 49 Protonation constants, see Acidity constants Proton transfer, 340 excited-state intramolecular, 356 Protoporphyrin IX, 234, 265

Q Quinoline(s) 8-amino-, 340 fluorescein, 353 8-hydroxy-, see 8-Hydroxyquinoline 6-methoxy-8-p-toluenesulfonamido-, 340, 345 zinc complex, 341 Quinones (see also individual names), 291, 292, 295, 297, 303, 310 and manganese, 306 dopamino-, see Dopaminoquinone naphthoxy-, 358, 359 phenoxynaphthoxy-, 358 semi-, see Semiquinones

R Rabbit aluminum studies, 380, 381 Alzheimer’s disease model, 118, 119 mercury studies, 415 Radical(s) (see also individual names), 62, 128 free, 138–141, 159, 161, 163–165, 238, 239, 298, 301, 304, 309, 310, 399

[Radical(s)] hydroxyl, see Hydroxyl radical NAD, 14, 43 oxygen, 2, 3, 139, 140, 186, 210, 301, 305 scavenger (see also individual names), 139 semiquinone, 295 Raman spectroscopic studies of prion proteins, 95, 96 Rat (studies of) aluminum in brain, 377, 380, 381, 384 Alzheimer’s disease model, 382 ATPase, 214 A peptide, see A peptide cadmium, 398–400 central nervous system, 384 ferrocene-loaded, 267 iron-deficient, 266 lead, 402, 408 mercury, 411, 414 Parkinson’s disease, 133, 134, 139, 265, 267, 301, 302 PC12 tumor, 101 pituitary tumor cells, 342 zinc in brain, 80, 256, 257, 347, 349 Rate constants of electron transfer, see Electron transfer Fe(II)-cytochrome c, 41, 44, 45 ferrocytochrome b562 , 39 protein (re)folding, 43, 45 Reactive nitrogen species, 2, 228, 238, 326 Reactive oxygen species (in), 2, 3, 228, 238, 308, 327, 431, 432 Alzheimer’s disease, 254, 256 brain, 62, 266, 267, 411 Parkinson’s disease, 132, 134, 135, 139, 140, 245, 247, 302 Redox potential of active site, 12 Fe(II)/Fe(III), 229 Fe(II)-cytochrome c , 41 Fe-cytochrome b562 , 39 zinc-cytochrome c, 17 Reductases ferric, 231, 232 glutathione, 398 Reduction potential, see Redox potential Reperfusion injury, 327

460

SUBJECT INDEX

Reptiles prion protein homologues, 91, 92 Reserpine, 127 Respiratory chain inhibition, 266, 267 Restless legs syndrome, 432 Retina(l), 2 degeneration, 262, 309 Retinitis pigmentosa, 129 Rhodafluor sensors, 351 Rhodamine, 344 Ca2+ -responsive sensors, 349–352 Rhodopseudomonas palustris, 40, 42 RNA c, 258 m, see mRNA Rodents (see also individual names) transgenic models, 154 Ruthenium(II) ammine complex, 17–19 tris(2,2 -bipyridyl) complex, 13, 14, 43 Ruthenium(III) ammine complex, 27, 29, 30 tris(2,2 -bipyridyl) complex, 14 Ryanodine, 411

S Saccharomyces cerevisiae (see also Yeast) cytochrome c, 24, 31–37 Schizophrenia, 153 Scrapie, 90–93, 104, 105 hamster, 109 mouse, 100, 107–109 prion protein, see Prion proteins sheep, 107 Seafood mercury in, 397, 413 Secretase -, 251, 253, 254, 259 -, 253, 254 -, 253 Semiquinones, 291, 292, 306, 312 radical, 295

Senile plaques (see also Amyloid plaques), 121, 289, 328, 385 aluminum(III) accumulation, 377 Serum (see also Blood) aluminum speciation, 378–380, 384 copper level, 118, 119, 121, 211 Seychelles contaminated seafood, 413, 414 Shaking palsy, (see also Parkinson’s disease), 127, 244 Sheep copper deficiency, 92 prion protein, see Prion proteins scrapie, see Scrapie sway-back, 92 Silicate aluminum, 375, 378 Silver(I) in Menkes copper-transporting ATPase, 216 Site-directed mutagenesis of cytochrome c, 21 Small-angle X-ray scattering, 46, 49 time-resolved, 38 Smelting, 396 SOD, see Superoxide dismutase Sodium channel, 411 –potassium ATPase, 382 transport, 323 Sodium dodecyl sulfate micelles, 47–49 Soret absorption spectroscopic studies of cytochrome b562 , 39 cytochromes c, 29, 36, 45 Spiperone, 304 Stability constants of (see also Dissociation constants) aluminum complex, 383 Cu(II) complexes, 64 Cu-A peptide, 78 iron-neuromelanin, 138 Steele-Richardson-Olszewski syndrome, see Progressive supranuclear palsy Stopped flow studies of cytochrome c, 33 Sulfonamides (see also individual names) aryl, 341 fluorescent zinc probes, 335, 336

SUBJECT INDEX

461

Sulfur (different oxidation states) (in) iron–sulfur proteins, 229, 230 melanin, 296, 297 –zinc bond, 326 Superoxide, 101, 135, 139, 181, 183, 184, 210, 266 degradation, 102 generation, 3 scavenger, 254 Superoxide dismutases, 94, 136, 140, 208, 211, 248, 290, 301 copper, 2, 4 Cu,Zn (SOD1), see Cu,Zn superoxide dismutase extracellular, 101 manganese (SOD2), 2, 101, 108, 398 prion protein, see Prion proteins Synapses cholinergic, 398 degeneration, 115 formation, 411 Synaptotagmins, 323 I, 401 Synphilin-1, 245–247 Synthesis bio-, see Biosynthesis Synucleins -, see -Synuclein -, 141, 244, 245 -, 141, 244, 245 -Synuclein (in), 3, 4, 45–50, 165, 195 aggregation, 46, 47, 49, 129, 141, 245, 246, 249 conformational change, 46 EPR studies, 46 fibrils, see Fibrils mutants, 245, 247 Parkinson’s disease, 127, 136, 140, 141, 244, 245, 289, 303, 305 wild-type, 244, 245 Synucleinopathies (see also individual names) classification, 285

T Tardive dykinesia, 432 Tartrate aluminum(III), 380

Tauopathies (see also individual diseases), 256, 290, 381 classification, 284 Tau protein, 245, 251, 254, 256, 289, 381 accumulation, 256 hyperphosphorylation, 256, 385, 386 lesions, 256 Tea green, 267 Teeth lead in, 405, 406 Terbium(III) (see also Lanthanides) cyclen complex, 343 DTPA complex, 343 Ternary complexes of aluminum(III), 379, 380 Tetrahydrobiopterin, 230 Tetrathiomolybdate treatment of Wilson’s disease, 218, 219 Thalassemia, 248, 250, 429 Thimerosal, 411, 413 Thiocarbazone diphenyl-, see Dithizone Thioether amyloid precursor protein, 81 heme iron, 31 linkage, 42 Thiols S-nitroso-, 326 Timm’s staining, 328 Tissues aluminum in, 373 Tocopherol, see Vitamin E Toxicity (of) aluminum, 373 cyto-, 166 excito, see Excitotoxicity iron-mediated, 162 nephro-, see Nephrotoxicity SOD1-mediated, 183 Transcription factors, 237, 324, 338 Transferrin(s), 133, 135, 136, 163, 166, 232, 241, 243, 248, 262, 299 aluminum(III) interaction, 373, 376, 379, 380 apo-, 232, 233 cycle, 233

462

[Transferrin(s)] holo-, 233 lacto-, 133 Transferrin receptor, 135, 233, 235–238, 248, 249, 265, 267, 299 antibodies, see Antibodies mRNA, 235, 241 Transglutaminase, 245 Transition metal ions homeostasis, see Homeostasis metabolism, see Metabolism Transmissible spongiform encephalopathies (see also individual names), 4, 62, 89–109, 118 Transport (of) (see also Metabolism) aluminum, 378 cobalt, 231 copper, 100, 231 dioxygen, 230 manganese, 231 nickel, 231 potassium, 323 sodium, 323 zinc, 231 Trien, see Triethylenetetramine Trientine, see Triethylenetetramine Triethylenetetramine, 428 treatment for Wilson’s disease, 218, 429 Trinucleotide repeat, 299 Trypsin, 381 chymo-, see Chymotrypsin Tryptophan Cu(II) reduction, 64 Tubulin, 383 aggregation, 387 Tumor breast, 245 Tumor necrosis factor, 238 Tuna cobalt-cytochrome c, 36 Tyrosinases, 211, 304 Tyrosine (and residues) and Cu2+ binding, 78 hydroxylase, see Hydroxylases nitration, 184 3-nitro-, 46–48

SUBJECT INDEX

U Ubiquitin, 140, 240, 246, 381 –protein ligase, 246, 247 Unfolded proteins properties, 19 Urea, 19, 20 UV-Vis spectrophotometric studies of cytochrome c, 26, 28, 43, 44

V Vanadium(IV) catecholamine oxidation, 309–312 Verapamil radiolabeled, 133, 134 Vesicles (see also Micelles) lipid, 46 phosphatidylcholine, 383 synaptic, 46, 245, 255–257, 325 Virus influenza, 254 Vitamins B12 deficiency, 430 C, see Ascorbate E, 139, 210, 301, 431

W Waste hazardous, 396 Water drinking, see Drinking water Wilson’s disease, 4, 92, 127, 308, 309, 428 clinical features, 210, 211 copper excretion through bile, 208, 210 copper overload, 210 copper transport, see Copper transport copper-transporting ATPases, see Copper-transporting ATPases gene mutations, 212 symptoms, 210 treatment, 217–219, 428, 429

SUBJECT INDEX

463

X XAFS studies of prion proteins, 94 Xanthine oxidase, 301 XAS, see X-ray absorption spectrsocopy Xenopus prion protein, 98 X-ray absorption spectroscopic studies of copper-transporting ATPases, 213, 214 X-ray absorption fine-structure spectroscopy, see XAFS X-ray crystal structure studies of copper-histidine, 220 X-ray fluorescence spectrometry, 138 X-ray microanalysis, 299 energy dispersive, 131, 132 X-ray scattering experiments (of) small-angle, see Small-angle X-ray scattering prion proteins, 93, 94

Y Yeasts (see also Saccharomyces cerevisiae) Cu,Zn superoxide dismutase, see Cu,Zn superoxide dismutase cytochrome c, see Cytochromes c ferredoxin, 265 zinc level, 355

Z Zinc(II) (in), 383 binding to amyloid precursor protein, 80–82 binding to A peptide, 77–80, 377 blood, see Blood brain, see Brain carbonic anhydrase, see Carbonic anhydrase central nervous system, see Central nervous system

[Zinc(II) (in)] cerebrospinal fluid, see Cerebrospinal fluid coordination spheres, 333 Cu,Zn superoxide dismutase, see Cu,Zn superoxide dismutase cytochrome c, see Cytochrome c deficiency, see Deficiency detection methods, 328, 329 excess, 430 fluorescent sensors, see Fluorescent probes for zinc future sensor design, 359 homeostasis, see Homeostasis interdependency with other metal ions, see Interdependencies intracellular, 357, 359 labile (free), 324–328, 336, 340, 343, 349, 350 metalloneurochemistry, 321–359 microperoxidase-8, 17, 19 multiple sclerosis, 431 myoglobin, 28 neurodegenerative diseases, see Neurodegenerative diseases neuromelanin, 138, 304 Parkinson’s disease, 432 pathology, 322–359 physiology, 322–359 porphyrin, see Porphyrins prion protein binding, see Prion proteins probes, see Fluorescent probes for zinc ratiometric detection, 354–359 salts as treatment of Wilson’s disease, 218, 219 shock, 355 staining of amyloid plaques, 257 –sulfur bond, 326 transport, see Transport transporter, 5, 257, 324, 357 treatment of macular degeneration, 431 yeast, 355 Zinc finger proteins, 326, 329, 341 dansylated, 333, 334 Zinpyr sensors (see also Fluorescent probes for zinc), 343–348