Role of Proteases in the Pathophysiology of Neurodegenerative Diseases
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Role of Proteases in the Pathophysiology of Neurodegenerative Diseases
Role of Proteases in the Pathophysiology of Neurodegenerative Diseases Edited by
Abel Lajtha Nathan S. Kline Institute for Psychiatric Research Orangeburg, New York
and
Naren L. Banik Medical University of South Carolina Charleston, South Carolina
Kluwer Academic Publishers New York, Boston, Dordrecht, London, Moscow
eBook ISBN: Print ISBN:
0-306-46847-6 0-306-46579-5
©2002 Kluwer Academic Publishers New York, Boston, Dordrecht, London, Moscow
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No part of this eBook may be reproduced or transmitted in any form or by any means, electronic, mechanical, recording, or otherwise, without written consent from the Publisher
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CONTRIBUTORS Carmela R. Abraham Boston University School of Medicine Boston, Massachusetts 02118 M. Azuma Research Laboratories Senju Pharmaceutical Co. Ltd. Kobe, 6.51-2241 Japan Naren L. Banik Department of Neurology Medical University of South Carolina Charleston, S. C. 29425 Raymond T. Bartus Alkermes, Inc. Cambridge, MA 02139 Martin J. Berg Center for Neurochemistry New York University Nathan S. Kline Institute for Psychiatric Research Orangeburg, NY 10962 Dieter Brömme Mount Sinai School of Medicine Department of Genetics New York, NY 10029 Sic L. Chan Laboratory of Neurosciences National Institute on Aging Baltimore, MD 21224 Jinyang Cong Muscle Biology Group University of Arizona Tucson, Arizona 85721
M.L. Cuzner Department of Neurochemistry Institute of Neurology University College London WClN 3BG, U.K. Dylan R. Edwards School of Biological Sciences University of East Anglia Norwich, Norfolk NR4 7TJ, England Dwaine F. Emerich Alkermes, Inc. Cambridge, MA 02139 Lawrence F. Eng Pathology Research Service Veterans Administration Hospital Palo Alto, CA 94304 Maria E. Figueiredo-Pereira Department of Biological Sciences Hunter College of the City University of New York New York, NY 10021 Peter A. Forsyth Oncology & Clinical Neurosciences University of Calgary and Department of Medicine Tom Baker Cancer Centre Calgary, Alberta T2N IN4, Canada C. Fukiage Research Laboratories Senju Pharmaceutical Co. Ltd. Kobe 651-2241 Japan Darrel E. Goll Muscle Biology Group University of Arizona Tucson, Arizona 85721 v
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Contributors
Gregor Guncar Jozef Stefan Institute Biochemistry and Molecular Biology Ljubljana SI-1000, Slovenia Edward L. Hogan Department of Neurology Medical University of South Carolina Charleston, SC 29425 Vivian Y.H. Hook Departments of Medicine and Neurosciences University of California, San Diego La Jolla, California 92093-0822 Janko Kos Jozef Stefan Institute Biochemistry and Molecular Biology and Krka, d.d. R&D Division Dept. of Biochemical Research and Drug Design Ljubljana SI-1000, Slovenia Marc A. LaFleur School of Biological Sciences University of East Anglia Norwich, Norfolk NR4 7TJ, England K.J. Lampi Department of Oral Molecular Biology School of Dentistry Oregon Health Sciences University Portland, OR 97201 Hahn-Jun Lee Laboratory for Proteolytic Neuroscience RIKEN Brain Science Institute Saitama, 351 -0198, Japan Hongqi Li Muscle Biology Group University of Arizona Tucson, Arizona 85721 H. Ma Department of Oral Molecular Biology School of Dentistry Oregon Health Sciences University Portland, OR 97201
Neville Marks Center for Neurochemistry Department of Psychiatry New York University Nathan S. Kline Institute for Psychiatric Research Orangeburg, NY 10962 Mark P. Mattson Laboratory of Neurosciences National Institute on Aging Baltimore, MD 21224 Denise C. Matzelle Department of Neurology Medical University of South Carolina Charleston, SC 29425 Michael A. Moskowitz Stroke and Neurovascular Regulation Laboratory Neurology and Neurosurgery Service Massachusetts General Hospital Harvard Medical School Charlestown, MA 02129 Suzana Petanceska Department of Psychiatry New York University Nathan S. Kline Institute for Psychiatric Research Orangeburg, NY 10962 Swapan K. Ray Department of Neurology Medical University of South Carolina Charleston, SC 29425 Patricia Rockwell Department of Biological Sciences Hunter College of the City University of New York New York, NY 10021 Takaomi C Saido Laboratory for Proteolytic Neuroscience RIKEN Brain Science Institute Saitama, 351 -0198, Japan
Contributors
Jörg B. Schulz Neurodegeneration Laboratory Department of Neurology University of Tübingen D-72076 Tübingen, Germany T.R. Shearer Department of Oral Molecular Biology School of Dentistry Oregon Health Sciences University Portland, OR 97201 Donald C. Shields Department of Neurology Medical University of South Carolina Charleston, SC 29425 M. Shih Department of Oral Molecular Biology School of Dentistry Oregon Health Sciences University Portland, OR 97201
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Valery F. Thompson Muscle Biology Group University of Arizona Tucson, Arizona 85721 Boris Turk Jozef Stefan Institute Biochemistry and Molecular Biology Ljubljana SI-1000, Slovenia Vito Turk Jozef Stefan Institute Biochemistry and Molecular Biology Ljubljana SI-1000, Slovenia Kevin K.W. Wang Department of Neuroscience Therapeutics Parke-Davis Pharmaceutical Research A Division of Warner-Lambert Company Ann Arbor, MI 481 05
Franchot Slot Boston University School of Medicine Boston, Massachusetts 02118
Gloria G. Wilford Department of Neurology Medical University of South Carolina Charleston, SC 29425
Marion Smith Department of Neurology Stanford University School of Medicine and Veterans Administration Medical Center Palo Alto, CA 94304
V.W. Yong Oncology & Clinical Neurosciences University of Calgary and Department of Medicine Tom Baker Cancer Centre Calgary, Alberta T2N IN4, Canada
Koichi Suzuki Tokyo Metropolitan Institute of Gerontology Tokyo, 173-0015 Japan
PREFACE Researchers seeking problems that offer more hope of success often avoid subjects that seem to be difficult to approach experimentally, or subjects for which experimental results are difficult to interpret. The breakdown part of protein turnover in vivo, particularly in nervous tissue, was such a subject in the past – it was difficult to measure and difficult to explore the mechanisms involved. For factors that influence protein metabolism, it was thought that protein content, function, and distribution are controlled only by the synthetic mechanisms that can supply the needed specificity and response to stimuli. The role of breakdown was thought to be only a general metabolic digestion, elimination of excess polypeptides. We now know that the role of breakdown is much more complex: it has multiple functions, it is coupled to turnover, and it can affect protein composition, function, and synthesis. In addition to eliminating abnormal proteins, breakdown has many modulatory functions: it serves to activate and inactivate enzymes, modulate membrane function, alter receptor channel properties, affect transcription and cell cycle, form active peptides, and much more. The hydrolysis of peptide bonds often involves multiple steps, many enzymes, and cycles (such as ubiquination), and often requires the activity of enzyme complexes. Their activation, modification, and inactivation can thus play an important role in biological functions, with numerous families of proteases participating. The specific role of each remains to be elucidated. It seems that at least some of the proteolytic processes in the brain differ from those in other organs. Enzymes involved in neuropeptide metabolism, responsible for the formation and subsequently the inactivation of physiologically active peptides, for example, have a function specific to the brain. Other findings, such as the stability of brain proteins in severe malnutrition when most body proteins are greatly diminished, also indicate controls of protein metabolism in the brain that differ from such controls in other organs. The present book focuses on the role of proteases in pathological changes in the brain, an aspect of proteolysis that has recently gained increased importance and interest. Proteolytic enzymes have been implicated in the degeneration and destruction of tissue in a number of degenerative CNS diseases and trauma, and direct evidence of their involvement has been established. Among the first lysosomal proteinases (cathepsins) and uncharacterized neutral proteinases to be studied were in the tissues of patients with multiple sclerosis, and in animals with experimental demyelinating diseases. These studies suggested that cathepsins and carboxypeptidases and several neutral proteinases play a role in neurodegenerative diseases. Recent evidence suggests that neutral proteinases, including calpain (a calcium-activated neutral proteinase), calcium-independent metalloproteinase, multicatalytic proteinase complex or proteosome, and matrix metalloproteinase, are involved in autoimmune and other demyelinating diseases, such as multiple sclerosis, optic neuritis, amyotrophic lateral sclerosis, stroke, muscular dystrophy, and Alzheimer’s disease, also in ischemia, oxidative stress, and CNS trauma. The
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participation of both lysosomal and extra-lysosomal enzymes in tissue degeneration is noted in a growing number of diseases. Now that many of the enzymes are well characterized (and we can expect the characterization of many more), factors influencing their activities, such as inhibitors of specific enzymes, gain importance because they have great therapeutic potential in a number of diseases, for which some are already in clinical use. Currently, information on protease activity changes in the various neurodegenerative conditions is scattered. We hope that the excellent contributions of the authors of this volume will be helpful for investigators interested in the mechanisms of proteolysis in neurodegenerative diseases and in developing therapy for their damage. It is no longer necessary to think that research in this subject is difficult to interpret. The future for studies in this subject is bright. We would like to thank our authors for agreeing to contribute to this book, and for their excellent discussions of this interesting and important field. In their thoughtful summary of what has been achieved to date, they point out not only the significance of the findings so far, but also the yet unresolved problems, thereby indicating future tasks and approaches. The recent rapid expansion of our knowledge in this area should give us confidence and hope for further important advances in our understanding of the mechanisms involved, and in the use of our knowledge for improvements in therapy. Our thanks are also due to Ms. Denise Matzelle and Ms. Susan Foldi for their patience and efforts in putting this book into its final form. We are also grateful to Peter Sís, a fine artist who is always very imaginative and creative, for kindly agreeing to contribute for the cover of the book his drawing symbolizing our quest for knowledge. Naren L. Banik Abel Lajtha
CONTENTS 1.
2.
THE ROLE OF PROTEOLYTIC ENZYMES IN AUTOIMMUNE DEMYELINATING DISEASES: AN UPDATE Marion Smith.. .......................................................................
1
PROTEASES IN DEMYELINATION M.L. Cuzner...........................................................................
5
3.
CALCIUM ACTIVATED NEUTRAL PROTEINASE IN DEMYELINATING DISEASES Donald C. Shields and Naren L. Banik.. ......................................... 25
4.
PAPAIN-LIKE CYSTEINE PROTEASES AND THEIR IMPLICATIONS IN NEURODEGENERATIVE DISEASES Dieter Brömme and Suzana Petanceska.. ........................................ 47
5.
THE ROLE OF THE CALPAIN SYSTEM IN NEUROMUSCULAR DISEASE Darrel E. Goll, Valery F. Thompson, Hongqi Li, and Jinyang Cong.. ................................................................................. 63
6.
THE ROLE OF CALPAIN PROTEOLYSIS IN CEREBRAL ISCHEMIA Dwaine F. Emerich and Raymond T. Bartus.. .................................. 75
7.
CALPAIN ISOFORMS IN THE EYE T.R. Shearer, H. Ma, M. Shih, K.J. Lampi, C. Fukiage, and M. Azuma.. ......................................................................
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8.
METALLOENDOPEPTIDASE EC 3.4.24.15 IN NEURODEGENERATION Carmela R. Abraham and Franchot Slot. ........................................ 101
9.
CYSTEINE PROTEASES, SYNAPTIC DEGENERATION AND NEURODEGENERATIVE DISORDERS Mark P. Mattson and Sic L. Chan.. ..............................................
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THE UBIQUITIN/PROTEASOME PATHWAY IN NEUROLOGICAL DISORDERS Maria E. Figueiredo-Pereira and Patricia Rockwell.. .........................
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10.
11.
AMYLOID (TACE, BACE) AND PRESENILIN PROTEASES ASSOCIATED WITH ALZHEIMER'S DISEASE Neville Marks and Martin J. Berg.. ............................................... 155
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12.
13.
Contents
CASPASES IN NEURODEGENERATION Jörg B. Schulz and Michael A. Moskowitz.. ....................................
179
THERAPEUTIC APPROACHES WITH PROTEASE INHIBITORS IN NEURODEGENERATIVE AND NEUROLOGICAL DISEASES Kevin K. W. Wang.. ..................................................................
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14.
PATHOPHYSIOLOGY OF CENTRAL NERVOUS SYSTEM TRAUMA: PROTEOLYTIC MECHANISMS AND RELATED THERAPEUTIC APPROACHES Swapan K. Ray, Denise C. Matzelle, Gloria G. Wilford, Lawrence F. Eng, Edward L. Hogan, and Naren L. Banik.. ......................................... 199
15.
LYSOSOMAL CYSTEINE PROTEASES AND THEIR PROTEIN INHIBITORS Vito Turk, Janko Kos, Gregor Guncar, and Boris Turk.. ...................... 227
16.
PROTEASES AND THEIR INHIBITORS IN GLIOMAS Peter A. Forsyth, Dylan R. Edwards, Marc A. LuFleur, and V. W. Yong......................................................................
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17.
PROTEOLYSIS OF MUTANT GENE PRODUCTS ARE KEY MECHANISMS IN NEURODEGENERATIVE DISEASES Vivian Y.H. Hook ................................................................... 269
18.
MAMMALIAN PROTEINASE GENES Hahn-Jun Lee, Koichi Suzuki, and Takaomi C. Saido..........................
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AUTHOR INDEX .................................................................
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SUBJECT INDEX .................................................................
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Role of Proteases in the Pathophysiology of Neurodegenerative Diseases
THE ROLE OF PROTEOLYTIC ENZYMES IN AUTOIMMUNE DEMYELINATING DISEASES: AN UPDATE
Marion Smith Department of Neurology Stanford University School of Medicine and Veterans Administration Medical Center Palo Alto, CA 94304
Twenty-three years ago a review by this author (MES) summarized the evidence for the participation of proteolytic enzymes in myelin destruction in experimental allergic (autoimmune) encephalomyelitis (EAE)1. Because EAE was, as now, considered to be an animal model for multiple sclerosis (MS), the review described investigations up to that time, pointing to the involvement of proteolytic enzymes in EAE lesions and by analogy, in MS plaques. Since then, due to intensive investigations of proteolytic enzymes in these demyelinating conditions, it has become even more apparent that tissue destruction in EAE and MS, as well as in other degenerative diseases is dependent on proteolytic enzymes. These enzymes are contained in inflammatory cells such as macrophages, neutrophils, and lymphocytes that utilize proteases for their invasive mechanisms, as well as for tissue destruction. At the turn of a new century it is instructive to compare our former ideas with those of the present state of knowledge attained by the contributions of many investigators. One problem in comparing our present understanding of enzymic mechanisms with those of the past is that the nomenclature of the proteases has changed, with further purification and investigation of their properties. Thus, the calcium-activated neutral protease, originally described by Guroff 2 is now recognized as “calpain”, which exists in several forms. New proteinase families have been discovered, including the caspases, and the matrix-metalloproteinases, which encompass some of the proteases formerly known as collagenase, elastase, and gelatinase. Although the lesion of the EAE animal was described in detail by both light and electronmicroscopy in the 1960s, the use of immuno-methods and markers has allowed a much better delineation of the kinds of cells present, the timing of their invasion, their state of activation, as well as some insight into their function. In addition to the lymphocytes and small monocytes formerly mentioned, several classes of lymphocytes and activated macrophages have been identified, and microglia have been recognized as resident macrophages in the central nervous system. Microglia and macrophages are notable for their content of proteases, and their capability as secretors of proteases, including plasminogen activator 3,4, cysteine proteases 5 , calpain 6, and metalloproteinase-9 (gelatinase B)7. The latter has been shown to be augmented when microglia are activated in vitro8, and undoubtedly other proteases are similarly increased in activated phagocytes. In addition, a number of these enzymes appear to occur in greater than normal amounts in activated lymphocytes and astrocytes. Myelin proteins were formerly thought to be relatively few in number, but since 1978, many more myelin constituents have been identified. Some are enzymes, especially those involved in lipid metabolism, and may give rise to signaling molecules9. Furthermore, new structural proteins have been detected, including the myelin oligodenRole of Proteases in the Pathophysiology of Neurodegenerative Diseases, edited by Lajtha and Banik. Kluwer Academic/Plenum Publishers, New York, 2001.
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drocyte glycoprotein (MOG) 10, and another basic protein, the myelin-associated oligodendrocytic basic protein (MOBP)11. Myelin basic protein and the myelin-associated glycoprotein, have both been shown to be susceptible to degradation by a number of proteases, and depleted in MS 12, while even proteolipid protein which is insoluble in water, is slowly degraded by calpain13. Other constituents of myelin may be vulnerable to enzymatic destruction, and some of the newly found active myelin constituents such as the signalling molecules, when injured, may cause metabolic collapse of the oligodendrocyte-myelin axis. In former years the ease of destruction of myelin basic protein in vitro by the acid protease cathepsin D was most emphasized (reviewed by Berlet)14. This lysosomal enzyme is probably not secreted, but may be a major effector of intracellular myelin degradation after phagocytosis of myelin in conjunction with other cathepsins such as B and L. Before myelin can be ingested, however, it must be disrupted into smaller fragments to facilitate its ingestion. This may be accomplished by several mechanisms, such as complement15, 16 and/or extracellular neutral proteases secreted by activated phagocytic cells. Lampert 17 first described areas of “vesicular degeneration” in demyelinating lesions of animals with EAE, and similar disruptive lesions have been noted in areas of the CNS in MS18, viruses19, or by various neurotoxic substances. Phagocytic cells may secrete these enzymes in the vicinity of the myelin sheath to disrupt the lamellae and to peel away the layers in MS and EAE. Traumatic damage, as in spinal cord injury, may result in an influx of calcium 20, which can activate calpain, thus causing vesicular myelin degradation. Therefore, both neutral and acidic proteolytic enzymes may be involved in myelin destruction, the former for disruption of myelin lamellae enabling phagocytic cells to ingest the droplets, then the acidic lysosomal cathepsins internally complete the protein digestion, while the myelin lipids are esterified or hydrolyzed. Evidence exists for the participation of a number of proteolytic enzymes in myelin destruction by autoimmune reactions, viral infection, and trauma. Most frequently mentioned are metalloproteinases 21, plasminogen activator 22, calpain 23, and the lysosomal cathepsins including cathepsin D, B, and L. Another proteolytic enzyme family, the caspases, may also be involved in cellular destruction of lymphocytes, phagocytic cells and oligodendroglia as activators of the apoptotic mechanisms shown to accompany EAE and MS 24. In this chapter and others, these enzymes and their roles in tissue destruction will be documented in detail. The 1978 review concluded “Further studies on proteinases and their role in disease will be of importance in devising a rationale for treatment. Many proteinase inhibitors have been identified, and it is possible that such inhibitors may be useful to intervene in the course of degenerative CNS diseases of myelin.” As of today, although protease inhibitors are standard treatment for other diseases, it is not clear whether these substances may be beneficial for MS. A number of these inhibitors appear to suppress EAE, including pepstatin, for cathepsin D25, inhibitors of plasminogen act ivator 26, neutral proteases such as leupeptin27 and others28. More recent work has suggested that metalloproteinase inhibitors may be useful as therapy for MS 29,30. We are further along than in 1978 in working out a treatment for MS with proteolytic inhibitors, but progress has been slow. As the newer aspects of these enzymes are described in this volume, the authors will undoubtedly point out possible new inhibitors as candidates for further investigation.
REFERENCES 1. M.E. Smith, The role of proteolytic enzymes in demyelination in experimental encephalomyelitis., Neurochem. Res. 2:233 (1977). 2. G. Guroff, A neutral, calcium-activated proteinase from the soluble fraction of rat brain, J. Biol. Chem. 239:149 (1964). 3. W. Cammer, B.R. Bloom, W.T. Norton, and S. Gordon, Degradation of basic protein in myelin by neutral proteases secreted by stimulated macrophages: A possible mechanism of inflammatory demyelination, Proc. Natl. Acad. Sci. U.S.A. 75:1554 (1978). 4. K. Nakajima, N. Tsuzaki, M. Shimojo, M. Hamanoue, and L. Kosaka, Microglia isolated from rat brain secrete a urokinase-type plasminogen activator, Brain Res. 577:285(1992).
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5. R.B. Banati, G. Rothe, G. Valet, and G.W. Kreutzberg, Detection of lysosomal cysteine proteinases in microglia: Flow cytometric measurement and histochemical localization of Cathepsin B. and L, Glia 7:183 (1993). 6. D.C. Shields and N.L. Banik, Pathophysiological role of calpain in experimental demyelination, J. Neurosci. Res. 55:553 (1999). 7. T. Yamada, Y. Yoshiyama, H. Sato, M. Seiki, A.Shinagawa, and M. Takahashi, White matter microglia produce membrane-type matrix metalloprotease, an activator of gelatinase A, in human brain tissues, Acta Neuropathol. 90:421 (1995). 8. P.E. Gottschall, X. Yu, and B. Bing, Increased production of gelatinase B (metalloproteinase-9) and interleukin-6 by activated rat microglia in culture, J. Neurosci. Res. 42:335 (1995). 9. J.N. Larocca, A. Cervone, and R.W. Ledeen, Stimulation of phosphoinositide hydrolysis in myelin by muscarinic agonist and potassium, Brain Res. 436:357 (1984). 10. C. Linington, M. Webb, and P.L. Woodhams, A novel myelin-associated glycoprotein defined by a mouse monoclonal antibody, J. Neuroimmunol. 6:387 (1984). 11. Y. Yamamoto, R. Mizuno, T. Nishimura, Y. Ogawa, H. Yoshikawa, H. Fujimura, E. Adashi, T. Kishimoto, T. Yanagahara, and S. Sakoda, Cloning and expression of the myelin associated oligodendrocytic basic protein. A novel basic protein constituting the central nervous system myelin, J. Biol. Chem. 269:31725 (1994). 12. Y. Itoyama, N.H. Sternberger, H.DeF. Webster, R.H. Quarles, S.R. Cohen, and E.P. Richardson, Immunocytochemical observations on the distribution of myelin-associated glycoprotein and myelin basic protein in multiple sclerosis lesions, Ann. Neurol. 7:167 (1980). 13. N.L. Banik, D. Lobo-Matzelle, G. Gantt-Wlford, and E.L. Hogan, Calpain, A catabolic mediator in spinal cord trauma, in: Neurodegenerative Diseases, G. Fiscum, ed., Plenum Press, New York (1996). 14. H.H. Berlet, Degradation of myelin proteins by proteinases, in: Myelin, Biology and Chemistry, R. Martenson, ed., CRC Press, Ann Arbor (1992). 15. W. Cammer, C.F. Brosnan, C. Basile, B.R. Bloom, and W.T. Norton, Complement potentiates the degradation of myelin proteins by plasmin: Implications for a mechanism of inflammatory demyelination, Brain Res. 364:91 (1986). 16. P. Vanguri, C.L. Koski, B. Silverman, and M.L. Shin, Complement activation by isolated myelin. Activation of the classical pathway in the absence of myelin-specific antibodies, Proc. Natl. Acad. Sci. U.S.A. 79:3290 (1982). 17. P. Lampert, Electron microscopic studies on ordinary and hyperacute experimental allergic encephalomyelitis. Acta Neuropathol. 9:99 (1967). 18. H. Lassmann, H. Budka, and G. Schnaberth, Inflammatory demyelinating polyradiculitis in a patient with multiple sclerosis, Arch. Neurol. 38: 99 (1981). 19. M.C. Dal Canto, and H.L. Lipton, Primary demyelination in Theiler’s virus infection. An ultrastructural study, Lab. Invest. 33:626 (1975). 20. J.D. Ballentine, Spinal cord trauma. In search of the granular axoplasm and vesicular myelin. J. Neuropathol. Expt. Neurol. 47:77 (1988). 21. B.C. Keiseier, T. Seifert, G. Giovannoni, and H.-P. Hartung, Matrix metalloproteinases in inflammatory demyelination. Targets for treatment, Neurology 53:20 (1999). 22. M.L. Cuzner, and G. Opdenakker, Plasminogen activators and matrix metalloproteases, mediators of extracellular proteolysis in inflammatory demyelination of the central nervous system, J. Neuroimmunol. 94:1 (1999). 23. D.C. Shields and N.L. Banik, Upregulation of calpain activity and expression in experimental allergic encephalomyelitis: a putative role for calpain in demyelination, Brain Res. 794:68 (1998). 24. R. Furlan, G. Martino, F. Galbiati, P.L. Poliani, S. Smiroldo, A. Bergami, G. Desina, G. Comi, R. Flavell, M.S. Su, and L. Adorini, Calpase-1 regulates the inflam- matory process leading to autoimmune demyelination, J. Immunol. 163:2403 (1999). 25. D.H. Boehme, H. Usezawa, G. Hashim, and N. Marks, Treatment of experimental allergic encephalomyelitis with an inhibitor of cathepsin D (Pepstatin), Neurochem. Res. 3: 185 (1978). 26. C.F. Brosnan, W. Cammer, W.T. Norton, and B.R. Bloom, Proteinase inhibitors supress the development of experimental allergic encephalomyelitis, Nature 285:235 (1980). 27. Y. Nagai, Suppression of demyelination in acute EAE: New strategies for the therapy of EAE and MS. In: Proc. Asian Multiple Sclerosis Workshop, Y. Kurawa, and L.T. Kurland, eds., Kyushu University Press, Fukuoka, Japan (1982). 28. M.E. Smith, and L.A. Amaducci, Observations on the effects of protease inhibitors on the suppression of experimental allergic encephalomyelitis, Neurochem. Res. 7:541 (1982). 29. K. Gijbels, R.E. Galardy, and L. Steinman, Reversal of experimental autoimmune encephalomyelitis with a hydroxamate inhibitor of matrix metalloprotase, J. Clin. Invest. 94:2177 (1994).
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30. W. Liedtke, B. Cannella, R.J. Mazzaccaro, J.M. Clements, K.M. Miller, K.W. Wucherpfennig, A.J.H. Gearing, and C.S. Raine, Effective treatment of models of multiple sclerosis by matrix metalloproteinase inhibitors, Ann. Neurol. 44:35 (1998).
PROTEASES IN DEMYELINATION
M.L. Cuzner Department of Neurochemistry Institute of Neurology University College London WC1N 3BG, U.K.
INTRODUCTION In both peripheral (PNS) and central (CNS) nervous systems a final common pathway of myelin breakdown is followed regardless of the mechanisms initiating demyelination. Myelin proteins and lipids are vulnerable to proteolytic and lipolytic action, following separation or splitting of the tightly compacted membrane lamellae. The hydrolytic agents are enzymes present in serum, secreted by inflammatory cells or lysosomal in origin acting on phagocytosed myelin. The spectra of enzymes include the endopeptidases, serine, carboxyl and thiol proteinases, the metalloproteinases, and a number of exopeptidases. This chapter will attempt to enumerate the enzymes most closely involved with myelin metabolism and breakdown and their sources, to highlight the relative vulnerability of the different myelin proteins and delineate the processes of demyelination.
PROTEASES IN METABOLIC PROCESSING OF MYELIN In all tissues proteases play an important role in protein turnover, in unmasking active sites of enzymes from proenzymes, in the regulation of intracellular protein concentration and in post translational modifications of newly synthesized proteins1. Additionally in the nervous system they have an important role in controlling the release of active neuropeptides and their inactivation at effector sites. Proteolysis is irreversible, unlike other post translational events and can be highly specific. Most proteinases are synthesized as precursors, with little or no proteolytic activity and can regulate their own activation. Thus large amounts of precursor can be present constitutively for activation on demand, Where there are proteinases, there are physiological inhibitors. The initiation of proteolysis is due to endopeptidase activity, located both in lysosomes (cathepsins acting
Role of Proteases in the Pathophysiology of Neurodegenerative Diseases, edited by Lajtha and Banik. Kluwer Academic/Plenum Publishers, New York, 2001.
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at acid pH) and in the cytosol (mostly neutral proteinases). The final stage is the production of dipeptides which can then be cleaved to amino acids. However limited proteolysis is more frequent as, unlike prokaryotes that regulate cellular proteins via gene expression, eukaryotes do so more often by post translational modification. As cells in the adult CNS are generally post mitotic this is of central importance during the different stages of brain development. Myelin is an abundant, relatively metabolically stable membrane of the PNS and CNS2. The lipid composition of both myelins is similar and although they have only one protein in common (Table 1) their molecular architecture varies minimally. The selectivity of myelin loss in immunopathological conditions must be ascribed to fundamental differences in antigenic specificity of the proteins. There are also differences in the structural integrity of the two sheaths. The structure of the CNS depends on the presence of myelin basic protein (MBP), linking the cytoplasmic faces at the major dense line whereas that of the PNS appears to require the Po glycoprotein which spans the bilayer of the compacted lamellae3. Table 1. Protein composition of myelin (%)
Long-term metabolic studies designed to assess the turnover and catabolic rate of myelin proteins have highlighted three features of myelin metabolism4. Molecules entering myelin appear both to have half-lives comparable to those in other membranes and to exhibit long term metabolic stability. Myelin proteins leave the membrane at different rates, indicating that myelin is not degraded as a unit and lipids and proteins radiolabeled during myelination appear more stable than the same components labelled in the adult. Rapid turnover may represent exchange or proteolysis occurring shortly after deposition of a newly synthesized protein before lamellar compaction or localization in an outer cytoplasmic loop or at a nodal region. Slow turnover is probably representative of
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7
compacted myelin, when exposure to enzymes and metabolic pools occurs only at a limited number of sequestered sites. Turnover rates for individual myelin proteins vary, for example higher -molecular weight proteins localized near cytoplasmic loops or in the periaxonal space are accessible to intrinsic proteases. In contrast MBP of CNS myelin sequestered on the cytoplasmic face of the membrane is protected from proteolysis. Some proteins, such as proteolipid protein (PLP), are very stable in the membrane moving only slowly to areas where they would be exposed to degradation. Matrix metalloproteinases (MMPs), key effectors of extracellular matrix remodelling and regulators of neurite extension, have recently been reported to implement oligodendrocyte process extension during the initial phase of myelinations5. Specifically oligodendrocytes are found to utilize MMP-9 to encourage process extension in vitro along an astrocyte-derived extracellular matrix. In support of this data is the observation that a temporal increase in MMP-9 expression in murine white matter parallels signposts of myelination in vivo.
PROTEASES IN DEMYELINATION IN VITRO The relative metabolic stability of myelin might be expected to impart a significant degree of protection from proteolytic attack. However, there are a number of pathological conditions, which are characterized by demyelination, perpetrated generally by the proteolytic and lipolytic enzymes of phagocytic cells. While useful information can be gained from in vitro assays of proteolytic activity the potential enzymatic capacity may not bear a direct relationship to activity in vivo. Regulatory factors in vivo include cellular or subcellular compartmentation of proteases, their stereochemical and stoichiometric links with substrate, inhibitory and feedback signals and the ionic environment, including pH and specific metal activation 1 . Most tissue homogenates have proteolytic activity with pH peaks in the acid and neutral ranges. Brain is no exception and the inflammatory cells that constitute the hallmark of inflammatory demyelination in the CNS and PNS are also rich in proteases6. The best studied and most widely distributed acid (carboxy1) proteinase in the CNS is cathepsin D, a lysosomal enzyme with a pH optimum of 3.57; the stability however of brain lysosomes under normal circumstances limits autolytic action of lysosomal cathepsins in the CNS. A soluble, neutral Ca++-activated proteinase can be extracted from brain and is considered to be identical to that prepared from extraneural tissue, notably from cardiac and skeletal muscle8. A lysosomal thiol proteinase, cathepsin B9, and the intracellular exopeptidase carboxypeptidase A, have also been reported to be present in normal brain tissue10. MMPs and plasminogen activators (PAs) are constitutively expressed in CNS brain homogenates11. These enzymes all play a role in homeostasis in the nervous system, but the exceptional stability of brain lysosomes and the overall tightly compacted nature of the myelin sheath would not normally expose this membrane structure to high levels of proteolytic activity. Nonetheless myelin-associated neutral proteases have been reported12-14, and once myelin lamellae are disrupted due to entry of serum components or cellular infiltration following blood brain barrier (BBB) leakage, the local environment alters significantly and myelin proteins become vulnerable to attack. There is convincing evidence that electrostatic interactions between basic membrane proteins and acidic phospholipids, can result in changes of conformation resulting in increased stability with respect to proteolytic enzymes15. The constituent phospholipids of isolated myelin are hydrolyzed by crude snake venom and purified phospholipase A2,
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but the co-operative action of phospholipases and proteolytic enzymes, e.g. trypsin, results in a more extensive loss of the basic protein and proteolipid protein and conversion of myelin phosphoglycerides to the corresponding lysocompounds 16 . When injected under the perineurium of the sciatic nerve, both trypsin and phospholipase produce lesions in the myelin sheath, with lamellar splitting and expansion of the myelin structure as observed in electron micrographs. The product of lecithin hydrolysis, lysolecithin, has been demonstrated to produce extensive demyelination both in vivo and in vitro 17 . Not all myelin proteins are equally vulnerable to proteolysis and these will be individually addressed, initially in in vitro systems.
Myelin basic proteins The open conformation of CNS MBP, which is readily soluble as a purified molecule, predisposes it to the action of proteases. It is rapidly digested into a large number of peptide fragments by trypsin, pepsin, chymotrypsin and pronase, and is susceptible to the action of hydrolytic enzymes found in the brain and in inflammatory cells18. Depending on the incubation conditions up to a dozen peptide fragments can be produced from intact MBP19. Detailed peptide patterns are listed in an accompanying table (Table 2). The major acid protease cathepsin D, hydrolyses purified MBP in a limited manner initially into two components of approximately MW 4.5 and 13.5Kd, followed in time by the appearance of a new antigenic determinant when the larger peptide is further degraded20,21. Although the rate of digestion varies according to the cellular source of the enzyme, the peptide spectrum produced is common to all. Neutral proteolytic activity towards MBP is associated with myelin itself, as well as being present in brain cytoplasmic fractions, and both Ca++-dependent and Ca++-independent activity have been reported13. MBP that dissociates from myelin incubated at neutral pH is hydrolyzed, in a limited fashion, with the appearance of two/three fragments14 . The major neutral proteases of leukocytes are cathepsin G, elastase, collagenase and u-PA22,23 and although the rate of hydrolysis varies according to the cell type, whether macrophages in different states of activation or granulocytes the pattern of proteolytic fragments of MBP produced is similar reflecting a limited sequential hydrolysis24. Metalloprotease activity, which is generally associated with modelling of the extracellular matrix, has been documented in myelin and in both macrophages and microglia25,26. Guinea pig MBP is digested by MMP-9 into 6 major bands on electrophoresis with cleavage sites within encephalitogenic epitopes. Therefore, production of this enzyme in the CNS may constitute an important, if non-specific, pathogenic mechanism for both the disruption of the BBB and of the myelin sheath11. As all the enzymes are secreted as precursors and activation is initiated by plasmin, itself generated through proteolytic activation by PAs, regulation of the PA-MMP cascade of activity is stringent. There are at least 6 isoforms of MBP, whose relative susceptibilities to proteolysis have not been explored, although this could represent another level of control in the developing CNS. The P1 basic protein of PNS myelin is identical to the 18Kd MBP from CNS myelin while the tryptic peptide map of the 14Kd P2 protein is unique18. The P2 protein does however have similarities to the CNS MBP, in that it is highly basic, easily extracted with acid and digestible by the same proteolytic enzymes.
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Proteolipid proteins The hydrophobicity of the transmembrane PLP renders the molecule more resistant to enzymatic cleavage than MBP, but this resistance is not absolute and there are in vitro conditions, although somewhat unphysiological, under which the protein is digested. Crude PLP and the delipidated apoprotein are digested 10% and 40% respectively by trypsin, producing a peptide map with 17-18 ninhydrin-positive spots27. Elastase was found to be the only other protease capable of digesting crude PLP, but oxidized PLP and apoprotein were also susceptible to digestion by chymotrypsin and thermolysin. These observations highlight the way in which the microenvironment or the form in which the myelin proteins are presented to the enzymes will influence proteolysis. The major protein of PNS myelin is the Po protein, a glycoprotein with a 6% by weight carbohydrate content18. Like PLP it is very hydrophobic but trypsin digestion produces a 19Kd glycoprotein component from the 28-30Kd protein, removing the hydrophilic portion that represents approximately 30% of the total protein. The carbohydrate entity can also be released in soluble form by proteolytic digestion.
Wolfgram protein (CNP) The highly conserved Wolfgram protein constitutes two closely spaced molecules of approximately 46 and 48Kd and represents the activity of the myelin specific enzyme 2',3' -cyclic nucleotide 3' phosphodiesterase (CNP)28. CNP has a higher turnover than other myelin proteins, possible due to its asymmetric distribution in myelin and association with tongue processes and paranodal loops but is not as sensitive to proteolytic digestion as MBP. Following spinal cord compression, a progressive decrease in Wolfgram protein over 72 hours was matched by a comparable loss in CNP activity29. However, leukocyte neutral proteases have also been reported to cause degradation of the protein which is cleaved by treatment with elastase at carboxyl residues 149 and 385, the 26Kd elastase fragment retaining its CNP activity30.
Myelin glycoproteins Glycoproteins are quantitatively minor components of the myelin sheath but two well-characterised ones are reputed to play an important role in the molecular architecture and properties of the membrane. Myelin-associated glycoprotein (MAG) with a molecular weight of 100Kd constitutes 1% of myelin protein. A neutral proteinase associated with highly purified CNS myelin selectively degrades MBP and converts MAG to a smaller derivative, dMAG, with a molecular weight less than 100,000Kd12. Incubation of buffered human myelin at 25°C resulted in a conversion of half of the glycoprotein in 30 minutes, whereas degradation of half of the MBP required 18 hours. There was no significant loss of the PLP, the Wolfram doublet or other myelin proteins for up to 18 hr under these conditions. The endogenous proteolytic activity is not affected by protease inhibitors, which indicates a close association with the myelin membranes thus preventing soluble inhibitors from reaching the active site. All of the detectable degradation products of MBP were present in the supernatant, but no intact MAG was detected in the supernatant, and about half of the dMAG remained associated with the particulate fraction after a 30-min incubation.
14,25
MBP dissociated from rat, human and guinea pig myelin is hydrolysed with the production of two major polypeptides 1-73 and 74-170, the formation of which is inhibited by phenanthroline and DTT but not by inhibitors of serine or cysteine proteases
purified myelin
1.1.4Metalloendoprotease
1.2.1 Neutral proteases (proteinase 3, u-PA) (elastase, cathespsin G)10 (collagenase)
24,3 5,84
12
Incubation of rat or human myelin at pH 7.6 results in 50% degradation of MBP and 50% conversion of MAG in 30 minutes to dMAG with a molecular wt 10Kd less, which is stable and remains associated with myelin membrane
integral, myelin-associated
1.1.3 Neutral proteinases
neutrophils (PMN) PMN soluble extract hydrolyzes greater than 50% MBP and Wolfgram protein in isolated myelin. Plasmin degrades up to 70% of MBP in myelin with the appearance macrophage, microglia of 2 major proteolytic fragments. In the absence of plasminogen the decrease in MBP is less (39%). Purified MBP is hydrolyzed by cell homogenates of PMNs and activated macrophages to produce 3 major and 1 minor proteolytic fragment.
19
Sequential hydrolysis of crude MBP occurs with the production of 12 proteolytic fragments in molecular weight range 17.5 to 6Kd. Inhibitor pattern points to activity of cysteine, metallo - and/or serine proteases
acid extract of bovine brain
1.1.2 Neutral proteinases
1.2. Leukocyte - associated
13
Incubation of rat spinal cord myelin at pH 7.6 with or without Ca++ results in 60% or 30% degradation respectively of MBP over 24 hours, whereas incubation with Triton - X 100 leads to preferential loss of PLP (60%) and DM-20.
cytoplasmic myelin-associated
1.1.1. Neutral proteinases Ca++-independent and Ca++-activated
Ref.
Hydrolytic products
Enzyme source
1.1 CNS - associated
1. Neutral proteinases
Table 2. Proteolysis of myelin proteins
plasma
1.3.2.Thrombin
Lysosomes
Lysosomes
2.1 CNS - Associated
2.2 Leukocyte associated
2. Acid proteinase (Cathepsin D)
serum but not plasma
leukocytes microglia
1.3.1. Neutral proteases
1.3 Serum/plasma associated
1.2.2. Metalloproteinases (MMP-2, MMP-9)
The cathepsin activity in PMN and macrophages resembles that of brain, with the appearance of 3 major peptides 1-43,43-88 and 89-169. The ranking of specific activity is macrophage >> PMN > brain
MBP undergoes sequential but limited proteolysis initially with the appearance of two components, 1-42 and 43-169, and subsequently with peptides 1-36 and 43-88, -89, -92 and 89-, 92- 169, resulting in appearance of a new antigenic determinant 2.2.1. Leukocyte associated
Arg-X bonds at a single site of MBP, 95-96 are cleaved under mild conditions increasing to 8 sites under more stringent conditions.
Incubation of MBP in serum results in loss of encephalitogenicity and appearance of fragments similar to those produced by trypsin
Pure MBP is hydrolyzed by MMP-9 with two cleavage sites at residues 92-93 and 116- 117, both of which are within the encephalitogenic epitopes of SJL/J mice and guinea pig respectively
24
20,21
55
54,56
26,37
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The susceptibility of MAG and MBP to cleavage by the endogenous proteinase suggests that in addition to inflammatory processes, auto-degradation may have a role in demyelinating disease. The two proteins were found to break down more rapidly on incubation of multiple sclerosis (MS) myelin in comparison to myelin from control brain12. If periaxonally localized MAG is involved in interactions between myelinated axons or in maintenance of the cytoplasmic collar around myelin sheaths or in the promotion of neurite outgrowth, proteolytic alteration of MAG could disrupt these processes. MAG preparations from the PNS and CNS showed the same peptide maps after digestion with three different proteases18. Myelin oligodendrocyte glycoprotein (MOG) constituting only 0.1% of myelin protein and a member of the immunoglobulin superfamily31 was originally identified by a mouse monoclonal antibody against rat cerebellar glycoproteins, which on intravenous injection greatly augmented CNS-specific demyelination32. MOG localization on the outer surface of the myelin sheath provides an ideal target for hydrolytic attack, but there are no documented studies on its susceptibility to proteolysis or its fate in demyelinating pathologies.
PROTEASES IN DEMYELINATION IN VIVO The biochemical changes in MS and experimental allergic encephalomyelitis (EAE) have been documented extensively6,24. In the demyelinated plaque myelin proteins and lipids are almost completely replaced by the glial fibrillary acidic protein of astrocytic fibrils. In lesions with ongoing demyelination myelin is apparent within macrophages in the hypercellular zone between normal-appearing white matter and the plaque centre and histochemical, immunocytochemical and biochemical analyses show clearly the preferential loss of myelin basic protein and CNP-ase activity in the presence of both acid and neutral proteolytic activity. In the established chronic lesion lysosomal enzyme activity persists and CNP-ase is dramatically decreased. Even in normal-appearing white matter there is evidence of biochemical change, with increases in lysosomal hydrolase activity and a diffuse widespread decrease in myelin proteins, accompanied by gliosis. In the active MS plaque, in which proliferating astrocytes and macrophages are present, an increase is found in a wide spectrum of lysosomal hydrolases, cathepsin D, and acid phosphatase. ß-glucuronidase, arylsulfatase, plasmalogenase, phospholipase A2, and carboxypeptide A and B and in secreted neutral proteases, including PAs and MMPs11 . The finding of increased MMP-9 activity in the CSF in MS33,34 and of the vulnerability of myelin proteins such as MBP to digestion by proteases, notably plasmin35and MMP-936,37 has stimulated work on the cellular pathology of these enzymes in MS38-40. In the demyelinating MS lesion expression of t-PA and u-PA is prominent in foamy macrophages, as is that of components of the MMP cascade (MMP-2 and 9)39. Lymphocytes and macrophages appear strongly positive in the perivascular cuff in active plaques, the staining extending to macrophages and reactive astrocytes throughout the hypercellular zone radiating from the plaque center. u-PA forms a unique combination of enzyme and chemotactic factor upon interaction with its surface receptor u-PAR inducing focal pericellular proteolysis and promoting cell adhesion and migration41. The generation of plasmin, directed to the cell-matrix interface by the action of the C terminus of u-PA42 is a rate-limiting step in the activation of the MMP cascade. MMPs degrade basement membrane and the extracellular matrix and promote extravasation of leucocytes 43, but they
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also have the capacity within the CNS to cleave myelin basic protein (MBP) into fragments retaining encephalitogenicity 36,37,44 . Nine MMPs have been identified by cDNA cloning and sequencing, including forms of collagenase, gelatinase and stromelysin. Two separate but similar cDNAs encode a 72Kd gelatinase A and a 95Kd - gelatinase B and production of the latter by macrophages or neutrophils is regulated by cytokines 45. Serine proteases and MMPs are secreted as inactive precursors together with plasminogen activator inhibitors (PA1) and tissue inhibitors of metalloproteases (TIMPs), ensuring that local activation is stringently controlled 45,46 . Another family of proteinases which now attracts intense research interest are the caspases, cysteine proteases which execute apoptosis47. They are among the most specific of proteases, with an absolute requirement for cleavage after aspartic acid and substrates to date are proteins associated with the nucleus; there is no documented effect on myelin proteins. Based on higher than normal protease activity in areas of gliosis, astrocytes would appear to be an important source of proteolytic enzymes, while enhanced cathepsin A activities in MS brain are apparently associated with macrophages. In a microanalytical study 48 Hirsch found that enzymatic changes were not observed in grossly normal white matter despite the presence of patchy cellular infiltrates and areas of gliosis. The conclusion drawn was that a primary role should not be attributed to lysosomal or cytosolic CNS proteinases in plaque formation and that elevated activity was likely to be the result of inflammatory cell invasion of the tissue. More recent studies appear to contradict this conclusion as immunocytochemical studies suggest that the very early MS lesion consists of focal areas of activated microglia with internalised MBP but without obvious myelin loss around cells, preceding an inflammatory lymphocytic reaction49,50. The functional role of the PA - MMP cascade in cell migration may influence the development of these microglia foci and their progression to hypercellular demyelinating plaques. The increase in proteolytic enzyme activity in the CNS in EAE also appears to be primarily due to cellular infiltration as it is greatest in the hyperacute localized lesions observed in primates with EAE. The increments are greater in respect of cathepsin A than acid proteinase, reflecting the difference in levels of these enzymes between lymph nodes and brain stem, and are not seen until the onset of symptoms which corresponds to the time of cellular infiltration. Both acid and neutral proteinase levels are increased by approximately 130-180 percent, compared to unaffected areas, while cathepsin A is several-fold greater 51. In experimental models of CNS inflammation, in particular EAE, there is in general a correlation between mRNA levels and protein expression of MMPs which is localized to the sites of inflammation 11 . The spectrum of MMPs varies according to the cellular nature of the lesion, reflecting the proportions of neutrophils, lymphocytes and macrophages in the perivasculature and possibly the makeup of the extracellular matrix, although the majority of MMPs have a broad substrate specificity. Of the inhibitors TIMP-1 mirrors most accurately the expression of MMPs at sites of inflammation, the most prominent of which are matrilysin, MMP-9 and metalloelastase.
MECHANISMS OF DEMYELINATION The myelin sheath can be disrupted by soluble mediators from the circulation or
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secreted by inflammatory cells, through activation of enzymes associated with the myelin itself or by phagocytic processes. In autoimmune models of demyelination, such as EAE, mononuclear cell processes can be seen to penetrate between the myelin lamellae near the outer loop and phagocytosed myelin is observed within coated pits and vesicles 52. Vesiculation and swelling of myelin sheaths are also very early features of demyelination in EAE. This begins with a splitting at the intracellular apposition, followed by a curling of the lamellar fragments to form loops or vesicles 53. These are then phagocytosed by what appears to be a receptor-mediated mechanism. However demyelination is not extensive in EAE unless mediated by anti-MOG antibody or through chronic inflammation.
Systemic mediators of myelin damage In general the process of demyelination is preceded by permeabilization of the BBB and blood nerve-barrier (BNB). Serum and plasma contain proenzymes which upon conversion have the capacity to cause disruption of the myelin sheath despite the presence in blood of proteinase inhibitors which inactivate neutral proteinases 54,55. Incubation of isolated myelin with human or rabbit serum resulted in 50% loss of MBP, while no breakdown of other major myelin proteins was observed. A smaller loss of MBP was also observed upon incubation of brain slices with serum 56. Activation of complement may be responsible for myelin breakdown by serum as isolated rat and human myelin consume complement in the absence of specific antibodies and sera heated to inactivate complement only induce myelin swellings 57. Not only is complement activated by isolated rat CNS myelin but the activation proceeds via the terminal component C9 to the formation of the membrane attack complexes of complement58 and myelin is lysed following pore formation in the lamellae52. There is also evidence that tissue culture demyelination can result from nonimmunoglobulin activation of the alternate complement pathway. The demyelinating activity was heat labile at 50°C, which leaves the classic complement pathway intact but inactivates properdin factor B, a crucial component of the alternate pathway59.
Cell-mediated myelinolysis in CNS demyelination The predominant cellular route of myelin breakdown is via macrophages, although polymorphonuclear leukocytes participate in acute haemorrhagic lesions, and Schwann cells are involved in the PNS. Astrocytes probably also contribute, particularly in later stages as they are observed with ingested myelin in MS plaques 60. Activated macrophages possess a wide range of receptors which can effect myelin endocytosis 61. In vitro myelin is efficiently taken up by macrophages in the absence of specific antibody, and this uptake can be inhibited by zymosan, (a bacterial cell wall product), oxidized lipoprotein and by antibodies which block complement receptors, intimating the involvement of the mannose/fructose and scavenger receptors as well as the complement receptor, CR3. In serum-free media opsonization of myelin with a mixture of antibodies directed against individual myelin proteins results in a greater degree of phagocytosis but this can be blocked more efficiently by zymosan than by competing immune complexes. This suggests that Fc-receptor mediated mechanisms may not be the major route for demyelination in vivo. In view of the search for the antigenic specificity of the intrathecally produced immunoglobulins in MS, this is an important consideration. MS lesions are characterised by inflammatory macrophages 62 but as it is difficult to identify the earliest lesion, the mechanisms of demyelination are more difficult to decipher
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15
in the human disease. There is however evidence that macrophages containing material staining with antibodies against MBP epitopes and neoepitopes are detectable in areas of white matter with no apparent myelin loss and where there is no lymphocytic infiltration suggesting a primary role for macrophages in the demyelinating process in MS63. There are few reports of direct T cell-mediated demyelination, the emphasis being placed on recruitment of effector cells by lymphocytes sensitized to brain antigens. Intraocular injections of supernatants from non-brain specific activated T cells in the rabbit only cause retinal fibre demyelination in animals sensitized to spinal cord or in the presence of serum from sensitized animals64. However, damage to myelinated cultures produced by lymphocytes sensitized to peripheral nerve has been observed in vitro65, and in isolated rat optic nerve, MBP specific T lymphocytes alone were capable of blocking action potentials, providing both T cells and optic nerves were HLA compatible 66. When a particle is physically larger than the macrophage or microglia, internalization of myelin cannot occur and "frustrated" phagocytosis or reverse endocytosis results in discharge of lysosomal enzymes. In a system modelling extracellular phagocytosis in the CNS, lysosomal enzyme release is accompanied by production and secretion of lactic acid and a subsequent drop in pH 67. At local pockets of low pH interstitial fluid may penetrate the lamellae, leading to reversible disruption of the myelin ultrastructure. In the model system turbidity changes associated with acidification were consistent with an increase in the size of the multilamellar myelin particles (ie. swelling). Hence proteolytic attack on MBP could be secondary to myelin degeneration and the primary pathological process in inflammatory demyelination a spontaneous disruption of lamellae in response to localized macrophage hyperlactemia. Histochemical analysis of MS brain tissue has shown that lactic dehydrogenase activity is increased in plaques 68.
Cellular sources of proteases Transcriptional and translational control, activation of latent enzymes and specific inhibitors influence to a great extent the cellular distribution of proteinases. An important issue in the biological context is constitutive production versus induction. Some enzymes are constantly produced, although at low levels only. For instance MMP-2 is produced by most cell types in a constitutive way in contrast to MMP-9 which is induced by specific agonists11. Also relevant to the PA-MMP cascade action in vivo, is the ubiquitous presence of plasminogen, regulating plasmin-mediated conversion through the activators u-PA and t-PA. Astrocytes in culture produce u-PA constitutively while t-PA, initially high in the control, is downregulated by proinflammatory cytokines69 . Furthermore, proteinases can be targeted to specific membrane sites by protein sequences in the transmembrane domains of an enzyme as for example with membrane type-MMPs70 which activate MMPs downstream. Secreted proteases such as u-PA may be confined to the edge of migrating cells by specific receptors. Finally, proteinase activity may be localised to extracellular matrix, as in the case of the adamalysin-disintegrin metalloproteases71. Neutrophils are a rich source of proteolytic activity. Human neutrophils are rich in neutral proteinases, the two major components of which are elastase and cathepsin G72. Degranulation of preformed neutrophil MMP-8 and MMP-9 by CXC-chemokines is a fast phenomenon. MBP is highly susceptible to digestion at pH7.6 by neutrophils while there is negligible digestion by acid proteinases of these cells24.
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Macrophages alter their phenotype upon encountering inflammatory or immunologic stimuli becoming phagocytic and enriched in lysosomal hydrolases and secreting the neutral proteinases, elastase, collagenase and u-PA, which can be plasma membrane bound or in a secreted form72,73. The steps leading to activation may be initiated by the interaction of specific receptors with cytokines or components of complement or immunoglobulins. These can be early or late effects, the former including triggering of the respiratory burst and arachidonate metabolism. The late responses which require the generation of cytoplasmic messengers will include protease synthesis. Stimulated production by Fc receptor occupation is only seen when initial secretion rates are low. Complement coated particles bound, but not ingested, stimulated only elastase secretion transiently, which may reflect a release from intracellular stores, rather than stimulating accelerated enzyme synthesis. The acid proteinase, cathepsin D, also plays an important part in the digestion of proteins taken up by macrophages, in the acid environment of the lysosomal system7 . However, while the acid proteinase in guinea pig activated peritoneal macrophages is increased three-fold over that in resting cells, and is much higher than neutral proteinase activity with traditional substrates, the difference in the rate of digestion of purified MBP at neutral and acid pH is not significant24 . The rate of digestion of MBP by brain homogenates is greater at acid pH, possibly due to lysosomal hydrolase activity in astrocytes, and the high molecular weight peptide pattern is similar to that generated by phagocytic cell acid hydrolysis. Conditioned media from microglia has been found to contain an elastase-like protease which hydrolyzes MBP74 and a urokinase-type PA which markedly increases MBP degrading activity in the presence of plasminogen. Secretion of the PA from microglia was enhanced by interleukin-1 and basic fibroblast growth factor. Microglia secrete a wide range of MMPs, including MMPs-2 and 9 upon activation by chemokines and cytokines. Thus, a battery of proteinases at both acid and neutral pH with myelinolytic activity is present in phagocytic cells, while little digestion of myelin proteins is effected by human lymphocytes or products of activated rabbit lymphocytes at either pH75. As a result of its immunogenicity in EAE the processing of MBP by cells and proteolytic enzymes has been the most extensively studied. Nonetheless MBP is the myelin protein with by far the greatest sensitivity to proteolytic action and although all other myelin proteins are susceptible in vitro to hydrolysis they are considerably more resistant to attack in vivo.
PNS demyelination Macrophages also form an important part of the cellular response to peripheral nerve injury76,77. In the PNS during nerve fibre degeneration both Schwann cells and macrophages participate in the process of myelin degeneration. Initially the myelin sheath is interrupted by Schwann cell processes which then fragment into ovoids and ellipsoids. Phagocytic macrophages appear later, and are essential for myelin clearance, as shown elegantly in a study of transected nerve segments in intraperitoneal millipore chambers which regulate the entry of macrophages78. In vivo macrophages enter PNS nerve fibres during Wallerian degeneration and participate in myelin removal in a similar fashion to that in the CNS but at a much more rapid rate. The differences between the CNS and PNS may be reflected in the origin and timing of the signal that attracts the macrophage. They are recruited in significant numbers in the first 3-5 days, following nerve crush, restricted to the
Proteases in Demyelination
17
region containing degenerating axons, and then phagocytose myelin, to become foamy macrophages76 . When pieces of peripheral nerve are placed in chambers within the peritoneal cavity, or when teased fibres are placed in culture, the Schwann cells extrude their myelin through the basement membrane surrounding the fibre and thus allow macrophages to phagocytose myelin78 . A consensus points to macrophages playing the major role in phagocytosing PNS myelin in vivo. Extracellular myelin degradation must also be considered as macrophages recruited outside the nervous system are know to secret potent myelinolytic neutral protease activities. The slow rate of Wallerian degeneration in the CNS has been well documented, but there is little evidence to show why this might be the case. Results suggest that limited recruitment of macrophages after injury might be important because it is these cells that play a major role in myelin removal in the PNS79. However in a mutant mouse, in which Wallerian degeneration and monocyte recruitment are extremely slow the evidence suggests that the gene product of the autosomal dominant mutation affects the nerve per se. Nonetheless the paucity of regeneration may be ascribed to a decline in the synthesis of nerve growth factor which can be demonstrated in culture by the inclusion of macrophages. It is not clear why there should be such limited recruitment in the optic nerve but differences between the BNB and BBB.
PROTEINASE INHIBITORS OF INFLAMMATORY DEMYELINATION From the evidence that increased neutral and acid proteolytic activities are associated with demyelinating lesions in the CNS and PNS, probably originating from the macrophages in the cellular infiltrate, proteinase inhibitors have been tested both in the experimental model, EAE, and in MS. Proteolytic enzymes may be necessary at several steps of the sensitization process in the development of EAE including the activation of the immunologic process, the invasion of cells into the brain parenchyma, as well as the final dissolution of myelin. The first reports in the literature indicating some degree of success with proteolytic inhibitors in suppressing EAE showed that pepstatin, an inhibitor of acid proteinase will suppress the clinical signs as well as the incidence of lesions of EAE in the Lewis rats80 . In another report amino caproic acid, an inhibitor of plasminogen activator, given in large amounts suppressed both the paralysis and lesions of EAE and in Lewis rats 81 . A large series of experiments to identify proteolytic inhibitors and their effects on EAE have been carried out in two laboratories, those of Marion Smith82 and Celia The inhibitors tested were pepstatin, aprotinin, B r o s n a n 83. trans-4-(aminomethyl)-cyclohexanecarboxylic acid (AMCA), e-amino-n-caproic acid (EACA), nitrophenyl p-guanidinobenzoate (NPGB), leupeptin and antipain. Pepstatin is a peptide which has been found to inhibit pepsin and cathepsin D at very low concentrations. Aprotinin (trasylol), inhibits elastase, trypsin, chymotrypsin, and plasmin, but not plasminogen activator. Leupeptin inhibits various neutral proteases including trypsin, plasmin, papain, and cathepsin B; antipain, inhibits trypsin, papain, and cathepsins A and B. AMCA, EACA, and NPGB inhibit a variety of neutral proteases including plasminogen activators. Some inhibitors of proteases, particularly those active at neutral pH were effective in inhibiting the clinical symptoms of EAE and in some instances in decreasing the incidence and severity of CNS lesions. Three of the most effective AMCA, EACA, and NPGB act on the plasminogen activators system, a pointer to its
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importance in the demyelinating process. Several enzyme systems are probably involved at various steps in the sensitization and demyelinative processes in EAE as partial protection is also provided by pepstatin. These results may be taken as evidence of a role for proteolytic enzymes in the primary attack on the myelin sheath, notably hydrolysis of basic protein. Despite observed increases in proteolytic enzyme activity in the CSF and inflammatory lesions of MS patients with active disease84 , no matching increase in the circulating inhibitors of neutral proteinases, a2-macroglobulin and a1 -antitrypsin has been observed 85 . In view of the putative role of plasminogen activator in promoting demyelination, an open trial of EACA was carried out in the U.S. and in Italy but inconclusive results discouraged continuance of this approach to therapy of MS86 . Renewed interest in the role of MMPs in promoting the traffic of leukocytes into the CNS has raised the possibility that inhibitors of these enzymes could modify the inflammatory process in the CNS. The metalloproteinase cascade is highly regulated by cytokines, which may also induce TIMPs 11 . For example IL- 1 can promote local synthesis of proteinases, while IL-6 and TGF-ß both induce TIMP production. Recent efforts to alleviate clinical signs of EAE have focused on synthetic inhibitors of MMPs. One hydroxamate inhibitor, GM6001, was found to suppress the development of EAE or reverse established clinical symptoms respectively, when administered prophylactically or therapeutically 87. A broad spectrum MMP inhibitor, BB- 1101 was effective in reducing the severity of EAE in the Lewis rat88 , and completely blocked the onset of EAE and reversed severe acute disease in the SJL/J mouse89 . Chronic relapsing EAE was also significantly modulated, with clinical improvement accompanied by a reduction in demyelination and glial scarring. The hydroxmate MMP inhibitor Ro31-9790 reduces the severity of EAE induced both by primary sensitization and following transfer of MBP-primed splenocytes in the Lewis rat, in both cases with a good correlation between clinical severity and histopathology 90 . In the actively induced model the beneficial effect was greatest in animals with moderate clinical signs, declining in those with more severe symptoms. MMP inhibitors might be expected to work at two levels - by blocking the extravasation to the CNS and effector properties of lymphocytes and macrophages in the initial stage of inflammation and by limiting the myelin loss in the established lesion. More specifically, the inhibitors appear to prevent degradation of extracellular matrix and basement membrane but seem to have no influence on the priming of MBP-specific T cells, as the course of clinical disease becomes essentially the same in inhibitor-treated as in vehicle-treated animals after cessation of treatment 87 . The clinical benefits of interferon (IFN)ß- 1b in the treatment of MS patients may be due, at least in part, to its ability to reduce the MMP-9 activity of T lymphocytes, resulting in their decreased migration91 . Results demonstrate that IFNß- 1 b treatment in vitro significantly decreases the migration of activated T cells through a fibronectin matrix. Migration of T cells was affected by IFNß-1b concentrations ranging from 10 to 1,000 IU/ml, concentrations which can be reached in serum following the systemic administration of 8 MIU of IFNß-1b in MS patients. All tested MMP inhibitors to date are broad spectrum and do not target individual metalloproteases hence the need to define selective and specific inhibitors of key enzymes.
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CONCLUSION Proteases in demyelination are effector molecules for leukocyte extravasation and in the myelinolytic process. As detailed in earlier sections myelin proteins, in particular basic protein are vulnerable to digestion by proteases acting at both neutral and acid pH, generally by limited sequential hydrolysis. In the case of MBP peptides in the molecular weight range 5- 10K are generated, and when isolated myelin is the substrate these peptides are released into the incubation medium. In contrast, the high molecular weight product of MAG proteolysis remains associated with the myelin membrane. In the inflammatory lesion macrophages are the most common source of these proteases, although neutrophils characterize the very acute demyelinating lesion, precipitated by administration of anti-MOG antibody. The neutral proteases elastase, cathepsin G and MMPs released from neutrophil granules are prime candidates for degrading myelin, on the assumption that BBB leakage has resulted in myelin lamellar splitting. Acid proteolytic activity would not be expected to play a large part initially, but as myelin is phagocytosed local changes in pH following fusion with lysosomes would enhance acid proteolytic activity. Macrophages have little proteolytic activity unless stimulated and the major neutral proteases with the capacity to hydrolyze myelin proteins are in a secreted form and would be predicted to act on myelin proteins extracellularly. In the situation when activated macrophages display increased expression of Fc, complement and scavenger receptors leading to myelin uptake in both a specific and non-specific manner lysosomal catheptic digestion of myelin proteins may supervene. Solubilized MBP would then be released into the cell cytoplasm. A limited amount of lysosomal hydrolase activity would be predicted to occur extracellularly in the presence of local acidic microenvironment. These reactions reflect the end point of the inflammatory demyelinating process and the mechanisms controlling the extent of demyelination in the CNS may be at the level of macrophage activation, whether specific or non-specific. Although myelin proteolysis is considered to be downstream from the priming of inflammation in immune-mediated diseases of the CNS and PNS, the metalloproteinases that influence the modelling of the extracellular matrix at the BBB and control entry of leukocytes into the CNS in the primary stage of immunological events are also capable of hydrolyzing myelin basic protein which could lead to release into the circulation of immunogenic peptides. Although some cytokines, notably tumour necrosis factor, are reported to act directly on myelin92, the major regulation of demyelination will be through the cytokines and other stimuli of macrophage activation, in this case most notably phagocytosis of myelin itself. Hence approaches to inhibiting of myelin breakdown per se are directed at specific inhibition of the proteases effecting demyelination.
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5. J.H. Uhm, N.P. Dooley, L.Y.S. Oh, and V.W. Yong, Oligodendrocytes utilize a matrix metalloproteinase, MMP-9, to extend processes along as astrocyte extracellular matrix, GLIA 22:53-63 (1998). 6. M.L. Cuzner and W.T. Norton, Biochemistry of demyelination, in: Immunopathology of Demyelinating Disease, M.L. Cuzner and H. Wekerle, eds., Brain Pathol. pp. 231-242 (1996). 7. A.J. Barrett, Proteinases in Mammalian Cells and Tissues. Research monographs in Cell and Tissue Physiology. North Holland, Amsterdam (1977). 8. G. Guroff, A neutral calcium activated proteinase from the soluble fraction of rat brain, J. Biol. Chem. 239:149-155 (1964). 9. K.R. Govindjaran, H.C. Rauch, J. Clausen, and E.R. Ginstein, Changes in cathepsins B-1 and D, neutral proteinase and 2'3' -CNP-ase activities in monkey brain with EAE, J. Neurol. Sci. 23:295-306 (1974). 10. D.M. Bowen and A.N. Davison, Cathepsin A in human brain and spleen, Biochem. J. 131:417-419 (1 974). 11. M.L. Cuzner and G. Opdenakker, Plasminogen activators and matrix metalloproteases, mediators of extracellular proteolysis in inflammatory demyelination of the central nervous system, J. Neuroimmunol. 94:1-14 (1999). 12. S. Sato, R.H. Quarles, and R.O. Brady, Susceptibility of the myelin-associated glycoprotein and glycoprotein and basic protein to a neutral protease in highly purified myelin from human and rat brain, J, Neurochem. 39:97-105 (1981). 13. N.L. Banik, W.W. McAlhaney, and E.L. Hogan, Calcium-stimulated proteolysis in myelin: Evidence for a Ca2+ -activated neutral proteinase associated with purified myelin of rat CNS, J. Neurochem. 45:581-588 (1985). 14. P. Glynn, A. Chantry, N. Groome, and M.L. Cuzner, Basic protein dissociating from myelin membranes at physiological ionic strength and pH is cleaved into three major fragments, J. Neurochem. 48:752-759 (1987). 15. R. Schafer and R.M. Franklin, Resistance of the basic membrane and proteins of myelin and bacteriophage PM2 to proteolytic enzymes, Febs Letters 58:265-268 (1975). 16. N.L. Banik, K. Gohil, and A.N. Davison, The action of snake venom, phospholipase A and trypsin on purified myelin in vitro, J. Biochem. 159:273-277 (1976). 17. S.M. Hall and N.A. Gregson, The in vivo and ultrastructural effects of injection of lysophosphatidyl choline into myelinated peripheral nerve fibres of the adult mouse. J. Cell Sci. 9:769-789 (1971). 18. M.B. Lees and S.W. Brostoff, Proteins of myelin, in: Myelin, P. Morell, ed., Plenum, New York pp. 197-224( 1984). 19. H.H. Berlet and H. Ilzenhofer, Sequential limited proteolysis of myelin basic protein by neutral protease activities of bovine brain, J. Neurochem. 45:116-123 (1985). 20. J.N. Whitaker and J.M. Seyer, The sequential limited degradation of bovine myelin basic protein by bovine brain cathepsin D, J. Biol. Chem. 254:6956-6963 (1979). 21. J.N. Whitaker, The appearance of a new antigenic determinant during the degradation of myelin basic protein, J. Neuroimmunol. 2:201-207 (1982). 22. P.M. Starkey, Elastase and cathepsin G, the serine proteinases of human neutrophil leucocytes and spleen, in: Proteinases in Mammalian Cell and Tissues, A.J. Barrett, ed., Elsevier, Holland, pp.57-89 (1977). 23. R. Takemura and Z. Werb, Regulation of elastase and plasminogen activator secretion in resident and inflammatory macrophages by receptors for the Fc domain of immunoglobulin G, J. Exp. Med. 159: 152-166 (1984). 24. D.A.S. Compston, M.L. Cuzner and A.N. Davison, Clinical features and pathophysiology of demyelinating disease, in: Clinical Neurochemistry, H.S. Bachelard, G.C. Lund, and C.D. Marsden, eds., Academic Press, London pp.77-189 (1986). 25. A. Chantry, C. Earl, N. Groome, and P. Glynn, Metalloendoprotease cleavage of 18.2- and 14.1-kilodalton basic proteins dissociating from rodent myelin membranes generates 10.0- and 5.9-kilodalton C-terminal fragments, J. Neurochem. 50:688-694 (1988). 26. A.K. Cross and M.N. Woodroofe, Chemokine modulation of matrix metalloproteinase and TIMP production in adult rat brain microglia and a human microglial cell line in vitro, GLIA 28: 183-189 (1 999). 27. M.B. Lees and D.S. Chan, Proteolytic digestion of bovine brain white matter proteolipid, J. Neurochem. 25:595-600 (1975). 28. T.J. Sprinkle, 2'3'-cyclic nucleotide 3'-phosphodiesterase, an oligodendrocyte-Schwann cell and myelin-associated enzyme of the nervous system, CRC Crit. Rev. Neurobiol. 4:235-301 (1989). 29. N.L. Banik, E.L. Hogan, and C.Y. Hsu, Molecular and anatomical correlates of spinal cord injury, Cent. Nerv. Syst. Trauma 2:99-106 (1985). 30. T. Kurihara, Y. Nishizawa, Y. Takahashi, and S. Odamic. Chemical, immunological and catalytic properties of 2’3'-CNP-ase purified from brain white matter, J. Biochem. 195:153-159 (1981). 31. M.V. Gardinier, P. Amiguet, C. Linington, and J.M. Matthieu, Myelin/oligodendrocyte glycoprotein is a unique member of the immunoglobulin superfamily, J. Neurosci. Res. 33: 177-187 (1992).
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58. B.A. Silverman, D.F. Carney, C.A. Johnston, P. Vanguri, and M.L. Shin, Isolation of membrane attack complex of complement from myelin membranes treated with serum complement, J. Neurochem. 42: 1024-1030 (1984). 59. D.H. Silberberg, M.C. Manning, and A.D. Schreiber, Tissue culture demyelination by normal human serum, Ann. Neurol. 15:575-580 (1984). 60. J.W. Prineas, The neuropathology of multiple sclerosis, in: Handbook of Clinical Neurology, J.C. Koetsier, ed., Elsevier Science Publishing, pp.213-257 (1985). 61. K. Mosley and M.L. Cuzner, Receptor usage in myelin phagocytosis by microglia and macrophages, Neurochem. Res. 54:185(1994). 62. M.L. Cuzner, G.M. Hayes, J. Newcombe, and M.N. Woodroofe, The nature of inflammatory components during demyelination in multiple sclerosis, J. Neuroimmunol. 20:203-209 (1988). 63. H. Li, J. Newcombe, and M.L. Cuzner, Characterisation and distribution of phagocytic macrophages in MS plaques, Neuropathol. Appl. Neurobiol. 19:214-223 (1993). 64. C.F. Brosnan, G.L. Stoner, B.R. Bloom, and H.M. Wisniewski, Studies on demyelination by activated lymphocytes in the rabbit eye. II. Antibody-dependent cell mediated demyelination, J. Immunol. 118:2103-2110 (1977). 65. B.G.W. Arnason, G.F. Winkler, and N.M. Hadler, Cell-mediated demyelination of peripheral nerve in tissue culture, Lab. Invest. 21 : 1 -10 (1969). 66. Y. Yarom, Y. Naparstek, V. Lev-Ram, J. Holoshitz, A. Ben-Nun, and I.R. Cohen, Immunospecific inhibition of nerve conduction by T lymphocytes reactive to basic protein of myelin, Nature 303:246-247 (1983). 67. P.R. Young and A.P. Zygas, Secretion of lactic acid by peritoneal macrophages during extracellular phagocytosis - The possible role of local hyperacidity in inflammatory demyelination, J. Neuroimmunol. 15:295-308 (1986). 68. H.E. Hirsch, P. Duquette, and M.E. Parks, The quantitative histochemistry of multiple sclerosis plaques: acid proteinase and other acid hydrolases, J. Neurochem. 26:505-512 (1976). 69. A. Faber-Elman, R. Miskin, and M. Schwartz, Components of the plasminogen activator system in astrocytes are modulated by tumor necrosis factor-a and interleukin-lb through similar signal transduction pathways, J. Neurochem. 65:1524-1535 (1995). 70. H. Sato, T. Takino, Y. Okada, J. Cao, A. Shinagawa, E. Yamamoto, and M. Seiki, A matrix metalloproteinase expressed on the surface of invasive tumour cells, Nature 370:61-65 (1996). 71. Z. Werb, ECM and cell surface proteolysis: regulating cellular ecology, Cell 91:439-442 (1997). 72. C.A. Owen and E.J. Campbell, The cell biology of leukocyte-mediated proteolysis, J. Leukocyte Biol. 65: 137-150 (1999). 73. C.G. Ragsdale and W.P. Arend, Neutral protease secretion by human monocytes - Effect of surface-bound immune complexes, J. Exp. Med. 149:954-968 (1979). 74. K. Nakajima, N. Tsuzaki, M. Shimojo, M. Hamanoue, and S. Kohsaka, Microglia isolated from rat brain secrete a urokinase-type plasminogen-activator, Brain Res. 577:285-292 (1 992). 75. H.M. Wisniewski, H. Lassmann, C.F. Brosnan, P.D. Mehta, A.A. Lidsky and R.E. Madrid, Multiple sclerosis: Immunological and experimental aspects, in: Recent Advances in Clinical Neurology, W.B. Matthews, G.M. Glack, eds., Churchill, London, pp. 95-125(1982). 76. V.H. Perry, M.C. Brown, and S. Gordon, The macrophage response to central and peripheral nerve injury, J. Exp. Med. 165:1218-1223 (1987). 77. G. Stoll, B.D. Trapp, and J.W. Griffin, Macrophage function during Wallerian degeneration of rat optic nerve: clearance of degenerating myelin, J. Neurosci. 9:2327-2335 (1989). 78. W. Beuche and R.L. Friede, The role of non-resident cells in Wallerian degeneration, J. Neurocytol. 13:767-796 (1984). 79. V.H. Perry and S. Gordon, Macrophages and the nervous system, Int. Rev. of Cytology 125:203-244 (1 99 1). 80. D.H. Boehme, H. Umezawa, G. Hashim, and N. Marks, Treatment of experimental allergic encephalomyelitis with an inhibitor of cathepsin D (pepstatin), Neurochem. Res. 3: 185-194 (1978). 81. W.A. Sibley, S. Kiernat, and S.F. Laguna, Modification of experimental allergic encephalomyelitis with EACA, Neurology 28:102-105 (1978). 82. M.E. Smith, Proteinase inhibitors and the suppression of EAE, in: The Suppression of Experimental Allergic Encephalomyelitis and Multiple Sclerosis, A.N. Davison, M.L. Cuzner, eds., Academic Press, New York, pp.211-226 (1980). 83. C.F. Brosnan, W. Cammer, W.T. Norton, and B.R. Bloom, Proteinase inhibitors suppress the development of EAE, Nature 285:235-238 (1980). 84. M.L. Cuzner, A.N. Davison, and P. Rudge, Proteolytic enzyme activity of blood leucocytes and CSF in multiple sclerosis, Ann. Neurol, 4:337-344 (1978). 85. P. Price and M.L. Cuzner, Proteinase inhibitors in cerebrospinal fluid in MS, J. Neurol. Sci. 42:251-259 (1979). 86. L. Amaducci, C. Arfaioli, R. Capparelli, D. Inzitari, D. Sita, P. Antuono, P. Zaccara, A. Doni, G. Lippi, and G. Leoncini, The clinical use of epsilon-amino caproic acid in MS, in: The Suppression
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CALCIUM ACTIVATED NEUTRAL PROTEINASE IN DEMYELINATING DISEASES
Donald C. Shields and Naren L. Banik Department of Neurology Medical University of South Carolina Charleston, S.C. 29425
INTRODUCTION The myelin sheath functions as an insulator to aid impulse conduction along axons. Myelin is synthesized by oligodendrocytes in the central nervous system (CNS) and by Schwann cells in the peripheral nervous system (PNS). Physical trauma or neurodegenerative disease processes affecting the white matter result in degradation of the myelin sheath with resulting impairment in axonal impulse conduction. In addition to sensory and motor function losses, biochemical changes including release of proteinases and lipases are commonly observed in the white matter or the myelin sheath following an insult. Although the mechanism(s) by which myelinolysis occurs has not been completely elucidated, the release of proteinases is believed to be at least one factor in this process. Various studies have provided evidence of proteolytic enzyme involvement in myelinolysis associated with demyelinating disorders. Myelin protein degradation has been implicated in chemical-induced demyelination, myelin degeneration in experimental allergic encephalomyelitis (EAE), and separation of myelin lamellae with splitting of the intraperiod line in Wallerian degeneration1-4 The incubation of myelin with trypsin in vitro resulted in ultrastructural alterations with a loss of myelin basic protein (MBP) which suggested involvement of proteinase in myelin breakdown5. This finding is firmly established by demonstration 6,7of increased acid proteinase activity in EAE and MS, concomitant with loss of MBP . Although acid proteinases were previously thought to be involved in the process, current evidence indicates neutral proteinases including calcium activated neutral proteinase (calpain), metalloprotease, multicatalytic proteinase complex (MPC), matrix metalloproteinases, and uncharacterized neutral proteinases also participate in the demyelinating mechanism8-12 . Some of these proteinases are found in myelin, suggesting myelin may be autodigestive in demyelinating diseases13 . Many of these proteinases degrade MBP while the peripheral nervous system (PNS) basic protein P2 is specifically digested by MPC and cathepsin D14,15 . Thus, in demyelinating
Role of Proteases in the Pathophysiology of Neurodegenerative Diseases, edited by Lajtha and Banik. Kluwer Academic/Plenum Publishers, New York, 2001.
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diseases, degradation of myelin proteins is critical in progression of the disease process.
PROTEINS AND PROTEOLYTIC ENZYMES OF MYELIN Since this chapter deals essentially with the role of proteolytic enzymes in myelin breakdown, it is important to briefly review the structural proteins of CNS and PNS myelin which together with lipids maintain the integrity of the myelin sheath. Several important CNS proteinases partially responsible for destabilizing the myelin sheath in diseases will also be reviewed. Myelin is largely composed of lipids (70%) and proteins (30%). The two major proteins of myelin are myelin basic protein (MBP) and proteolipid protein (PLP) which constitute 30% and 50% of all myelin proteins, respectively. PLP (24kD molecular weight) is tightly bound to lipids while MBP (18kD molecular weight) is also complexed with lipids, but with less affinity than PLP16. Minor myelin proteins with important roles include MAG (myelin-associated glycoprotein) MOG (myelin oligodendrocyte-specific glycoprotein), MOBP (myelin oligodendrocyte-specific basic protein), and DM-20 of the PLP family of proteins. Proteinases, lipases, kinases, and peptidases are also present 13,17-22 . MBP is highly susceptible to proteolysis and is digested by cathepsin B and D, calpain, metalloproteinase, trypsin, and pepsin while PLP is resistant to trypsin. The latter however, is partially digested by elastase23, trypsin and calpain, if detergent is present23-26 . The intact MBP, PLP, and MOG proteins or their proteolyzed fragments when injected into susceptible animals result in an autoimmune demyelinating disease, experimental allergic encephalomyelitis (EAE) a model for human multiple sclerosis (MS). PNS myelin also contains MBP along with P0 protein, which is similar to CNS PLP. MAG and other minor enzyme proteins, proteinases, and lipases are present as well. One of the myelin basic proteins, P2 protein, is resistant to endogenous CNS and PNS proteinases. However, P2 can be degraded by cathepsin D and MPC, in the presence of a detergent. The P2 protein, intact or fragmented, has been found to cause an autoimmune demyelinating disease of PNS, experimental allergic neuritis (EAN), which has been used as a model for Guillain Barre Syndrome, the PNS demyelinating disease of humans. Proteinase Hypothesis Acid proteinase activity was demonstrated in CNS samples in the early 1930s and 1940s27,28. Subsequently lysosomal proteinases cathepsins A, B, and D were purified from brain samples with increased activities of these proteases observed in diseases29-32. Cathepsin D was found to degrade phenylalanine-phenylalanine linkages in MBP molecules. Acid proteinase activity was also found to be localized in neurons. In contrast to acid proteinases, the demonstration of non-lysosomal neutral proteinase activity in brain was difficult since they are more unstable than lysosomal proteinases. Nonetheless, in the 1950s and 1960s Ansell and Richter33 and Marks and Lajtha34 were able to determine neutral proteinase activity in brain samples35. Since then several neutral proteinases including MPC, calpain, and metalloproteinase activity were found in the brain and later purified 14,20-22,36-43 Aminopeptidases and arylamidases are also found in myelin 29,44-47 . In contrast to CNS, the characterization ofproteinases in PNS is less extensive. The histochemical demonstration of proteinases in PNS was shown in the early 1960s24 followed by findings of calpain activity in sciatic nerve and Schwann cells48,49. Later, an
Calcium Activated Neutral Proteinase
unidentified neutral proteinase capable of degrading the P0 protein of PNS was also demonstrated50. The characterization of these various proteinases suggested myelin may be metabolically active and even autodigestive in demyelinating diseases.
MECHANISMS OF MYELIN BREAKDOWN IN VITRO AND IN VIVO Since the process of myelinolysis in demyelinating diseases was poorly defined, in vitro models were used to investigate the degradative process. Several studies evaluated the effects of snake venom, proteinases, lipases, and lysolecithin in whole brain and/or purified myelin51-55. These studies indicated that lipases or proteinases alone are not adequate for destruction of the myelin sheath.
Effects of Proteolytic Enzymes on Myelin Since the detergent-like action of lysolecithin treatment did not change the myelin ultrastructure, it was hypothesized that myelin proteins play a significant role in maintaining the structure and stability of myelin. Subsequent experiments evaluating the effects of trypsin on purified5 4 - 5 7myelin demonstrated a loss of phospholipids and MBP but no change in PLP . PLP in vitro is digestible with trypsin only in the presence of detergents such as Triton X-100 since it is embedded and protected in the membrane by lipids23,58. In order for PLP to be degraded, lipases were needed to expose it to proteinases. Thus, incubation of myelin with trypsin and phospholipase together resulted in the digestion of MBP and PLP with 58. ultrastructural dissolution of myelin into vesicles This suggested that the dual actions of proteinases and lipases were essential components in the mechanism of myelin breakdown in demyelinating disease. Studies with nerve extracts of Wallerian degeneration on purified myelin at 37°C also revealed that proteinases are important for myelin alterations59. There was greater loss of MBP and some loss of lipids when myelin was incubated with Wallerian Degeneration nerve extracts compared to controls. Acid proteinases have been implicated in these studies. From these and other studies it was suggested that MBP digestion is the initial step in myelin breakdown. Since activated inflammatory cells, including macrophages and lymphocytes have been shown to secrete proteinases into a culture medium, CNS and PNS myelin proteins were incubated in this medium with subsequent digestion10,60. Although these neutral proteases were not characterized at that time, subsequent studies from other laboratories identified calpain and metalloproteinase secreted by activated lymphocytes and macrophages capable of degrading purified myelin and MBP61,62.
Myelinolysis in Experimental Animal Models The role of proteolytic enzymes in myelinolysis associated with experimental allergic encephalomyelitis (EAE), Wallerian degeneration, diphtheritic (toxin) degeneration, and CNS injury were later studied. Of these animal models, demyelination has been studied most widely in EAE. Myelin breakdown also has been examined in several viral models, including canine distemper, scrapie, measles, and in cuprizone intoxication, i.e. non-infective and non-autoimmune perturbation. Both biochemical and histochemical studies revealed increased acid and neutral proteinase activity during the first week of degeneration. Results form these studies suggested loss of myelin basic proteins was responsible for changes in myelin structure. Subsequent in vitro studies showed losses of P0, P1, and P2 myelin proteins
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following incubation of PNS myelin with trypsin63-66. These studies suggested that the removal of proteins by various proteinases is responsible for structural changes in nerve degeneration. Increased proteinase activity was also associated with diphtheritic toxin-mediated demyelination67-69. In this model, increases in acid proteinase activity are found much earlier than that of neutral proteinase activity suggesting initial involvement of lysosomal proteinases in diphtheritic neuropathy50,67,68. Although the neutral proteinase found in this model was uncharacterized, recent studies demonstrated increased calpain activity in diphtheritic neuropathy70. The increased calpain activity has been correlated with degradation of calpain substrates (neurofilament proteins [NFP]), and elevated intracellular free calcium concentrations. CNS Wallerian degeneration was studied in optic nerve where macrophages, astrocytes, oligodendrocytes and microglia were implicated in myelin breakdown during nerve degeneration71-75. Long term intervals of optic nerve degeneration following enucleation of the eye resulted in almost complete loss of myelin proteins, while short term degeneration showed no apparent myelin lipid or protein loss76. Optic nerve degeneration caused by retinal destruction also demonstrated losses of myelin proteins MAG and MBP with structural alteration of myelin77,78, implicating involvement of proteinases. Recent in vitro and in vivo studies using experimental allergic optic neuritis (EAON) models revealed de adation of axon and myelin proteins concomitant with elevated calpain activity79 In viral animal models of demyelination, there was increased lysosomal hydrolases (e.g., ß-glucuronidase and cathepsin A) suggesting neuronal degeneration and inflammatory infiltration80 82. Increased glucuronidase and cathepsins A and D are also found in toxic cuprizone demyelination, often associated with proliferative glial cells (astrocytes and microglia). In these models increased proteinase activities concomitant with a loss of myelin proteins have been implicated in the mechanism of myelinolysis in demyelinating process. Alterations in the axon/myelin structural unit, at the morphological and biochemical levels are also common in brain and spinal cord injury83-87. In spinal cord injury lesions there is splitting of myelin lamellae with concomitant axon/myelin protein loss87,88. Various enzyme activities included calpain and cathepsins B and D are increased in and around the lesion site89-91. Use of calpain inhibitors in vivo in these injury models has been shown to prevent axon and myelin protein degradation92-95. THE CALPAIN FAMILY In autoimmune demyelinating diseases such as multiple sclerosis and EAE, the corresponding animal model, degradation of myelin proteins in CNS lesions suggested a role for calpain since all major myelin proteins are substrates of this enzyme. Calpain is a cytosolic cysteine endopeptidase (EC 3.4.22.17) that retains characteristics of the thiol proteinase, papain and calcium binding protein, calmodulin. The proteinase has been localized in every mammalian cell type studied, and calpain homologues have been identified in lower order organisms including nematodes, insects, yeast, and fungi96. The calpain family consists of at least six homologous members divided into two classes according to tissue distribution, i.e., ubiquitous or tissue specific. Recently discovered tissue specific calpains, such as p94 and nCL-2 (localized in muscle), exist as monomers or oligomers of the 80 kD catalytic subunit. Ubiquitous calpain is distributed in every cell, most often as a heterodimeric complex composed of 80 kD catalytic and 30 kD
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regulatory subunits. Both ubiquitous calpain isoforms, millicalpain (mcalpain) and microcalpain (µcalpain), share similar biochemical and catalytic properties with the exception of calcium concentrations required for activation. Approximately 1 -20µM and 250-750µM calcium levels are required for half-maximal activity of µcalpain and mcalpain, respectively, because calcium binding domains of the two isoforms differ in affinity for calcium97,98. Prior to activation, proenzyme calpain is normally associated with the endogenous inhibitor calpastatin. This association also requires calcium levels similar to the calcium activation requirement for each isoform. After calpain activation, calpastatin is also degraded by the active enzyme98,99. Calpain has been described as a biomodulator because it degrades substrates in a limited fashion, resulting in alteration rather than destruction of the ‘substrate. Calpain degrades a wide array of substrates including cytoskeletal and myofibrillar proteins, histones, enzymes, myelin proteins (myelin basic protein, MBP) and Since calpain activation often occurs along the cell receptor protein13,100-104. membrane, many membrane or membrane-associated proteins (actin-binding proteins such as fodrin, talin, filamin, a-actinin, and microtubule-associated proteins; growth factor receptors such as EGF receptors; adhesion molecules such as integrin, cadherin, N-CAM; and ion transporters such as Ca2+ -ATPase) are calpain substrates105.
CALPAIN ACTIVITY AND EXPRESSION IN ALLERGIC ENCEPHALOMYELITIS
EXPERIMENTAL
EAE is an autoimmune inflammatory disease induced in animals - most commonly rabbits, guinea pigs, monkeys, mice and Lewis rats - by injection of an emulsified suspension of whole CNS tissue, white matter, myelin, PLP or MBP, together with Freund’s complete adjuvant (FCA)106-110. The animals progressively lose weight followed by development of paralysis at 10 to 12 days after challenge. Perivascular cuffing with infiltrating lymphocytes, monocytes and plasma cells, can be observed by light microscopy 106,109,111. Electron microscopic examination has shown lamellar separation and splitting of the major dense and intraperiod lines of the myelin lamellae followed by vesicular degeneration of myelin which is ultimately phagocytozed112,113. Blood-brain barrier permeability is increased in EAE, possibly by free-radicals produced by inflammatory cells since permeability is reduced by antioxidant enzymes in EAE114. The hypothesis that MBP degradation is the initial step in myelin breakdown in demyelinating diseases59 is supported by findings of substantially increased acid and neutral proteinase activities in EAE and MS tissue6,115-123. Neutral proteinase activity is increased in EAE lymph nodes and serum124,125. Several early studies demonstrated significant increased acid proteinase activity in the lesion in rabbits and monkeys with acute EAE119. This greatly increased proteinase activity was later confirmed by Smith and colleagues66. These investigators demonstrated increased activity was due to at least two proteolytic enzymes, an uncharacterized neutral proteinase and an acid proteinase, cathepsin A. The activity of cathepsin A was found to be less than that of the neutral proteinase. Later, the increased neutral proteinase activity was shown to be elevated by 250% in the EAE lesion compared to controls. Cathepsin A activity was also found to be several fold greater in the lesion than controls80,118. Subsequently, increased activities of cathepsins B and D and neutral proteinase were observed in the lesion of monkeys with EAE66. In all these studies, MBP was preferentially degraded in the lesion.
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In contrast to monkeys with EAE, both cathepsins A and D and neutral proteinase activities are increased in rats with acute EAE. Cathepsin A activity was found to be greater than other proteinases118,127 while Boeheme et al128 reported no change in cathepsin A activity in EAE. Hirsch and colleagues reported increased cathepsin A and D in the lesion of rats with acute EAE, suggesting lymphocytic infiltration127. MBP was selectively degraded by EAE lymph node homogenate66 and later an uncharacterized neutral proteinase was partially purified from lymph nodes66 . Subsequent studies revealed an increased neutral proteinase activity in the serum of rats with acute EAE125. These studies indicated that elevated proteinase activity plays an important role in demyelination and they may derive not only from infiltrating cells, but also from endogenous cells. To examine the specific role of calpain in EAE, experiments in our laboratory were designed to evaluate and localize calpain activity and expression in the spinal cords of Lewis rats with an acute form of the disease129-132. These studies were also carried out in white matter tissue sections from human patients with various neurodegenerative diseases. Calpain activity was measured indirectly by evaluating the degradation of known calpain substrates such as fodrin, 68 kD axonal neurofilament protein (NFP) and the myelin protein MAG131. Western blot analysis revealed a 43% loss (p=0.041) of 68 kD NFP in spinal cords from animals with EAE compared to controls (Fig. 1A). This loss of NFP in EAE animals confirms earlier findings of NFP degradation in patients with MS, which suggests axonal degeneration may be present in autoimmune demyelinating diseases133,134. Although other proteases are involved in myelin degradation, recent studies suggest activated calpain plays a major role in this process since NFP and myelin protein degr adation is significantly decreased when calpain is inhibited following CNS injury 86,93,124,135142 . Similarly, spinal cords from rats with EAE demonstrated a 40% loss (p=0.014)
Figure 1. Loss of 68 kD neurofilament protein (A) and 96 kD myelin associated glycoprotein (B) in spinal cords of animals with EAE compared to adjuvant controls. Western blots (top) of samples from both groups were quantified via densitometry and analyzed by one way ANOVA (+S.E.M.). The dMAG band in (B) is marked with an (<). Shields and Banik131. With permission.
Calcium Activated Neutral Proteinase
of 96 kD MAG compared to controls. In some samples from animals with EAE, a smaller dissociated protein (dMAG) was observed as the intensity of the larger MAG band was decreased (Fig. 1B). The formation of dMAG observed in this study correlates well with dMAG production demonstrated previously in MS tissue, although the involvement of calpain in dMAG production remains poorly understood since other proteinases have been implicated in MAG degradation132,143,144 Calpain expression at the translational level was also evaluated using an antibody specific for the proenzyme form of mcalpain131. The single band observed at about 80 kD was increased by 206% (p=0.005) in the CNS of animals with EAE compared to adjuvant controls (Fig. 2).
Figure 2. Calpain translational expression in rats with EAE compared to adjuvant controls. Western blots (top) of samples using a polyclonal mcalpain antibody were quantified via densitometry and analyzed by one way ANOVA (+S.E.M.). Shields and Bank131. With permission. In contrast to calpain, calpastatin samples were characterized by multiple immunoreactive bands at 180 kD, 110 kD, 80 kD, 68 kD, 55 kD, and 43 kD (Fig. 3). Lower molecular weight bands at 55 kD and 43 kD showed no significant differences between controls and animals with EAE. Higher molecular weight bands were increased in EAE compared to controls as follows: 180 kD - 233% (p=0.0007), 110 kD - 89% (p=0.016), 80 kD - 330% (p=0.009), 68 kD - 127% (p=0.026). Additional calpastatin bands at 120 kD and 90 kD in samples from animals with EAE remain poorly understood. However, increased calpastatin translational expression suggests the expression of calpain and its inhibitor is similarly regulated in pathological conditions. Although calpastatin expression was increased in EAE, the calpain-calpastatin ratio may have been too high for complete inhibition of calpain activity, or calpastatin may have been degraded by active calpain into smaller fragments devoid of inhibitory activity. In order to evaluate calpain expression in various cell phenotypes, immunohistochemical techniques were employed. Glial cells were observed in the white matter of normal rat spinal cords by hematoxylin and eosin staining at 200x magnification (Fig. 4A). In spinal cord white matter from animals with acute EAE, perivascular cuffing and inflammatory cell infiltrates were widely distributed (Fig. 4B). Using immunoperoxidase staining, calpain-positive cells were observed throughout spinal cord sections from normal control rats (Fig. 4C). In acute EAE, calpain immunoreactivity was markedly increased in cell bodies and processes (Fig. 4D). Greater numbers of calpain-positive cells were observed in spinal cords from
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Figure 3. Calpastatin translational expression in animals with EAE compared to adjuvant controls. Western blots of samples from both groups were quantified via densitometry and analyzed by one way ANOVA. Calpastatin isoforms which showed statistically significant differences between EAE and control samples are marked by an (*). Calpastatin isoforms found only in EAE samples are marked with an (<). The molecular weights of protein standards are shown on the left. Shields and Banik131. With permission. animals with EAE - often in clusters as noted with H&E staining. To determine the cell phenotypes responsible for increased calpain expression, double immunofluorescent labeling was employed using a polyclonal calpain antibody (FITC secondary-green) and monoclonal antibodies for cell-specific markers (Texas red secondary-red) 130 . Cells positive for calpain and the cell-specific marker appeared yellow when viewed with a dual-pass fluorescence filter. The majority of calpain expression in normal CNS was observed in astrocytic cell bodies and proximal processes (identified by an antibody specific for GFAP). In EAE, calpain expression in reactive astrocytes was markedly increased in both the cell bodies and processes. To examine calpain expression in macrophages/microglia, a monoclonal antibody specific for complement receptor type 3 (localized in mononuclear phagocytes) was employed. Quiescent, ramified microglia in both normal gray and white matter expressed little calpain. In EAE, activated mononuclear phagocytes with more rounded morphology demonstrated markedly increased calpain expression. Activated mononuclear phagocytes with large increases in calpain expression were often encased by astrocytic foot processes in inflammatory foci. Activated calpain released from these cells may participate in myelin protein degradation for antigen presentation (possibly contributing to epitope spreading) since macrophages and T cells are also present in inflammatory foci.
Calcium Activated Neutral Proteinase
Figure 4. H&E and calpain immunoperoxidase staining of Lewis rat spinal cord (200x). (A ) Normal rat spinal cord white matter with H&E stain; (B) Spinal cords of Lewis rats with acute EAE demonstrated perivascular cuffing and increased numbers of glial and inflammatory cells with H&E staining; (C) Immunoperoxidase staining of control spinal cords showed some calpain expression in glial cells of white matter; (D) In white matter of spinal cords from animals with EAE, a greater number of cells demonstrated markedly increased calpain expression with immunoperoxidase staining. Shields et al131. With permission. Increased calpain expression was also observed in infiltrating inflammatory cells including activated T cells and macrophages. In normal spinal cords, the relatively few T cells observed did not express detectable levels of calpain. In contrast, activated T cells producing IFN- in EAE expressed large amounts of calpain. Interestingly, calpain immunoreactivity was not confined to the cell borders but appeared as a halo surrounding the cells130. These observations suggest activated T cells may secrete calpain in vivo - thus confirming previous in vitro studies demonstrating secretion of calpain by T cells upon activation61. Similar increases in calpain expression among activated glial/inflammatory cells were observed using experimental optic neuritis models in rats129,132.
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CALPAIN ACTIVITY AND EXPRESSION IN MULTIPLE SCLEROSIS The findings of increased calpain expression in animals with EAE suggested this enzyme may also be upregulated in MS. Calpain activity in postmortem tissue form human MS patients was measured by the production of calpain-specific degradation products. Calpain degrades the 230 kD subunit of fodrin to produce a 150 kD fragment which is recognized by the antibody used in this study. Although production of the 150 kD fodrin fragment was marginally increased in white matter from Alzheimer’s patients compared to normal controls, only increases observed in MS plaques [52.7% (p = 0.002)] and adjacent normal appearing white matter (NAWM) [40.9% (p = 0.018)] were significant (Fig. 5).
Figure 5. Calpain activity as measured by Western blot detection of 150 kD calpain-cleaved fodrin fragment production in CNS white matter from normal control, Parkinson’s, Alzheimer’s, NAWM, and MS plaque samples. Western blots (top) were quantified via densitometry and analyzed by one way ANOVA (+S.E.M.). Shields et al145. With permission.
In addition to substrate degradation, calpain activity was evaluated by measuring the relative amount of active (76 kD) enzyme. Activated µ-calpain levels were upregulated in NAWM and Parkinson’s white matter samples, but the 90.1% (p = 0.028) increase in MS plaques was alone significant compared to normal controls (Fig. 6).
Calcium Activated Neutral Proteinase
Figure 6. Levels of 76 kD activated µ-calpain in CNS white matter from normal control, Parkinson’s, Alzheimer’s, NAWM, and MS lesion samples. Western blots (top) were quantified via densitometry and analyzed by one way ANOVA (+S.E.M.). Shields et al145. With permission.
Calpain and calpastatin translational expression levels were also measured via Western blotting. Using a polyclonal antibody specific for the 80 kD catalytic subunit of proenzyme m-calpain, Western blotting revealed calpain expression was significantly increased by 462.5% (p = 0.002) in MS plaque samples compared to normal controls (Fig. 7). Alterations of calpain expression in NAWM, Parkinson’s, and Alzheimer’s white matter samples were not significantly different from normal controls. Calpastatin expression was measured with a polyclonal antibody specific for the central consensus sequence found in Domain 1 of human calpastatin (Fig. 8). 110 kD and 68 kD calpastatin isoforms were not significantly different in any sample set compared to normal controls. The 40 kD calpastatin isoform was significantly increased by 40.4% (p = 0.027) in MS plaque samples compared to normal controls, but no significant alterations were observed in Parkinson’s, Alzheimer’s or NAWM samples. Since the calpain-calpastatin ratio is critical for regulation of calpain activation, calpastatin expression must also be increased to properly control excessive calpain activation. The translational expression of both commonly observed calpastatin isoforms (110 kD and 68 kD) remained largely unchanged in MS samples compared to normal controls. An increase in the 40 kD isoform in MS
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Figure 7. Calpain translational expression measured by Western blot (top) detection of 80 kD mcalpain proenzyme in CNS white matter from nomal control, Parkinson’s, Alzheimer’s, NAWM, and MS plaque samples. Western blots were quantified via densitometry and analyzed by one way ANOVA (+S.E.M.). Shields et all145. With permission. plaque samples remains poorly understood, but may represent calpastatin fragments cleaved by calpain upon activation. Thus, calpain activity in pathological conditions such as MS may be poorly regulated by calpastatin since expression of the latter is not commensurately increased. Double immunofluorescent labeling was employed to identify calpain-positive cell phenotypes and observe differences in calpain translational expression in normal controls, NAWM, and MS plaques145. Antibodies specific for the m-calpain proenzyme were added in combination with monoclonal antibodies specific for astrocytes, T cells, and mononuclear phagocytes. While detectable levels of calpain were observed in T cells, astrocytes, and mononuclear phagocytes in normal controls, overall calpain expression was lower than that found in NAWM or MS plaques. In NAWM, calpain expression was increased in reactive astrocytes, CD4 positive T cells, and mononuclear phagocytes. Sections observed near the center of MS lesions revealed extensive cellular destruction, gliosis, and inflammatory infiltrates. Compared to normal control and NAWM samples, calpain expression was markedly upregulated in MHC class II-positive cells, GFAP-positive
Calcium Activated Neutral Proteinase
Figure 8. Expression of 110 kD, 68 kD, and 40 kD calpastatin isoforms in CNS white matter from normal control, Parkinson’s, Alzheimer’s, NAWM, and MS lesion samples. Western blots (top) were quantified via densitometry and analyzed by one way ANOVA (+S.E.M.) as depicted graphically. Shields et al145. With permission.
cells/processes, mononuclear phagocytes, and CD4 positive T cells. Thus, increased calpain expression does not appear to be confined to the lesion borders, but is associated with the number and activation state of glial and infiltrating inflammatory cells in the NAWM.
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CALPAIN ACTIVATION AND INHIBITION IN DEMYELINATING DISEASES Since increased intracellular calcium levels are required for calpain activation during demyelination, several possible mechanisms have been explored. First, cytosolic calpain may be exposed to calcium levels sufficient for activation as calcium stores (endoplasmic reticulum) are released through the inositol (1,4,5)triphosphate-mediated pathway as glial/inflammatory cells are activated146. Moreover, damaged oligodendrocyte membranes may facilitate calcium influx. In vitro studies have demonstrated vesicular disruption of the myelin sheath, as seen in demyelinating diseases, when rat sciatic nerves were exposed to calcium ionophores at physiological pH147. Previous studies have also shown myelin membranes in rats are susceptible to classical and alternative complement pathways in addition to antibody-dependent complement fixation 148-150. Complement proteins (which enter the CNS as the blood brain barrier is compromised) and perforins have been proposed as possible mechanisms allowing calcium entry into oligodendrocytes151,152. In addition, calpain released or secreted from activated cells in EAE and MS will be activated by extracellular calcium levels (approximately 2 mM)61,153. Thus, in demyelinating diseases, calpain may degrade myelin proteins intracellularly as calpain present in myelin is activated (via increased intracellular calcium levels following membrane damage) and/or extracellularly by calpain secreted from activated glial/inflammatory cells. The delineation of a putative role for calpain in demyelinating diseases has prompted several investigators to evaluate the effects of cell-penetrating calpainspecific inhibitors in various pathological conditions. The inhibition of increased calpain activity following treatment with proteinase inhibitors E-64 and leupeptin reduces muscle degeneration in muscular dystrophy154-156 with improvement in ischemic and posthypoxic recovery of cells using calpeptin and MDL-28170157. In CNS injury models, the use of calpain inhibitors (AK-295) has resulted in reduced lesion size and some prevention of protein degradation compared to controls158. Proteinase inhibitors (AMCA, pepstatin) also suppress clinical symptoms and reduce the number of CNS lesions in EAE and experimental allergic neuritis63,159-164. The results from these studies do not offer many clues to the mechanism of protection against the disease. Although proteolytic enzymes, acid proteinases and plasminogen activators (of macrophages) were inhibited, further experiments should be conducted to delineate the specific proteases responsible for each disease process63.
CONCLUSIONS Since calpain expression is upregulated in activated glial/inflammatory cells in EAE, increases in calpain activity and expression may be a result of cell activation, and thus present in all inflammatory responses. Future studies employing calpainspecific inhibitors are therefore needed to (1) elucidate the role(s) of calpain in autoimmune demyelinating diseases and (2) evaluate the potential for possible therapeutic benefits associated therewith.
Calcium Activated Neutral Proteinase
ACKNOWLEDGEMENTS This work was supported in part by grants from NIH-NINDS NS-3 1622 and NS38146, SCW-1238 from the Paralyzed Veterans of America, RG-2130B2 from the National MS Society, and MUSC Medical Scientist Training Program (D.C.S.).
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PAPAIN-LIKE CYSTEINE PROTEASES AND THEIR IMPLICATIONS IN NEURODEGENERATIVE DISEASES
Dieter Brömme1 and Suzana Petanceska2 1
Mount Sinai School of Medicine Department of Genetics New York, NY 10029 2
Department of Psychiatry New York University Nathan S. Kline Institute for Psychiatric Research Orangeburg, NY 10962
INTRODUCTION Papain-like cysteine proteases have been implicated in various pathologies of the brain such as brain tumors, Alzheimer’s disease, stroke, cerebral lesions, neurological autoimmune diseases, and certain forms of epilepsy. However, the evidence of their involvement in neuropathologies is at best circumstantial and primarily based on expression and inhibitor data. There is a clear lack of functional data verifying a critical role of papainlike cathepsins in diseases of the central nervous system. This chapter gives an overview about human papain-like cysteine proteases and summarizes the present knowledge about their potential role in neurological diseases.
CLASSIFICATION AND NOMENCLATURE The most commonly used classification of proteases is based on their catalytic mechanism to cleave peptide bonds. Presently five protease classes are known: serine, cysteine, aspartate, threonine, and metalloproteinases. The names refer to the amino acids or a metallo-ion (in generally Zn2+) which either directly or indirectly, via the polarization of a water molecule, attack the carbonyl carbon of the scissile peptide bond in substrates. It should be noted that the amino acid designations of the protease classes are not correlated to any specificity towards such residues in substrates. Each protease class is subdivided into numerous clans and subclans. The interested reader is referred to Barrett1 for a comprehensive overview of protease classification. This chapter will focus on the C1 family of cysteine proteases, which is also known as papain-like protease family 2 . Numerous other chapters of this book are focused on the C2 cysteine protease clan, which comprises the calpains. Papain-like cysteine proteases form the largest group in the cysteine protease class and are found in viruses, various mono- and multicellular parasites, and throughout the plant and animal realm. The only exceptions where C1 proteases have not been identified are bacteria. Papain-like cysteine proteases in vertebrates and invertebrates also are named cathepsins, a name introduced by Willstädter and Bamann in 1929 describing acidic intracellular protease3. The name cathepsin is derived from the Greek word “καϑεψειv” which means, “to digest.” It should be noted that the term “cathepsin” also is used for members of the Role of Proteases in the Pathophysiology of Neurodegenerative Diseases, edited by Lajtha and Banik. Kluwer Academic/Plenum Publishers, New York, 2001.
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aspartate and serine protease classes. If not explicitly noted otherwise, the term cathepsin will be used for papain-like cysteine proteases in this chapter. Due to recent advances in deciphering the human genome, the number of human cathepsins has increased from four to eleven members. In addition, three novel papain-like cysteine proteases, mouse cathepsins P4 and M5, and rat cathepsin Q6 have been described for which the human orthologues are presently unknown. It is likely that this number will increase by the conclusion of the human genome project.
CATALYTIC MECHANISM The active site of papain-like cysteine proteases is composed of the name-defining cysteine (Cys-25; papain numbering), a histidine (His-159), and an asparagine (Asn-175) residue with the cysteine and histidine forming an ion-pair. In contrast to serine proteases, the ion-pair is already present in the ground state of the enzyme and does not depend on the interaction with a substrate. Therefore, cysteine proteases can be regarded as “a priori” activated enzymes7. The negatively charged cysteine residue reacts via a nucleophilic attack with the carbonyl carbon of the scissile bond of the bound substrate and forms a tetrahedral intermediate. This unstable intermediate resolves into a covalently bound acyl enzyme (acylation) under the release of the C-terminal fragment of the substrate. This step is followed by the hydrolysis of the acyl enzyme with water, forming a second tetrahedral intermediate which finally splits into the free enzyme and the Nterminal substrate Figure 1. Mechanism of substrate hydrolysis by cysteine proteases fragment (deacylation) (Figure 1).
STRUCTURE, EVOLUTIONARY RELATEDNESS, AND CHROMOSOMAL LOCALIZATION Presently, eleven human cathepsins of the C1 clan have been cloned and characterized. Bleomycin hydrolase, which shares the cathepsins’ active site residues, forms a distinct subfamily of the C1 clan. Cathepsins are synthesized as preproenzymes. The pre or signal sequence permits the translocation of the gene product into the endoplasmic reticulum. The precursor enzymes are targeted to the lysosomes where under acidic conditions the mature and active cathepsins are proteolytically separated from their prodomains. In case of overexpression of cathepsin genes, their zymogens are targeted to the secretory pathway. For example, tumor cells are able to secrete large amounts of inactive procathepsins8. In contrast, bleomycin hydrolase does not have a signal and propeptide domain and forms a hexamer, which is active in the cytosol 9. Some of the cathepsins have been cloned simultaneously in different laboratories resulting in multiple designations. Table 1 summarizes the various names given to these proteases, the length of the protein domains in amino acids, and the chromosomal localization of the human genes. All cathepsins have in common that their genes encode proteins, which contain 15-21 long signal sequences, propeptides of varying lengths between 41-251 amino acids, and mature domains of 214-260 amino acids. The highest
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49
conservation is observed in the mature domains of the polypeptides which represent the catalytically active proteases.
Table 1. Mammalian cathepsins, their synonyms, length of their signal sequences, pro and mature domains, and the chromosomal localization of the human genes. Protease and synonyms cathepsin B (human) cathepsin L (human) cathepsin H (human) cathepsin I (rabbit) cathepsin S (human) cathepsin C (human) DPPI (human) cathepsin J (rat) cathepsin 0 (human) cathepsin K (human) cathepsin 0 (human) cathepsinO2(human) cathepsin X (human) OC2 (rabbit) JATP1 (chicken) cathepsin W (human) lymphopain (human) cathepsin V (human) cathepsin U (human) cathepsin L2 (human) cathepsin F (human) cathepsin Z (human) cathepsin X (human) cathepsin P (human) cathepsin Y (rat) cathepsin P (mouse) cathepsin J (mouse) cathepsin Q (rat) bleomycin hydrolase (human) aminopeptidase H (chicken) hydrolase H (rabbit)
length of (in amino acids) signal peptide 17 17 17
prodomain
chromosomal localization
62 96 98
mature domain 260 220 220
preproenzyme 339 333 335
8q22-23 9q21-22 15q24-25
16 21
98 209
217 233
331 463
1q21 11q14.1-3
21 15
86 99
214 215
321 329
4q31-32 1q21
19
108
249
376
11q13.1-3
17
96
22 1
334
9q21
19 20
251 41
214 242
484 303
11.q13.1-3 20q13
18
95
22 1
334
20 0
104 0
219 455
342 17q11.2
Three-dimensional structures of cathepsins show a overall conserved folding resulting into a L-domain which contains the active site cysteine residue at the N-terminal end of a conserved a-helix and an R-domain dominated by four to six beta-sheets forming a barrel10. Using a phylogenetic analysis program, cathepsins can be grouped into a cathepsin Llike group (cathepsins L, V, S, K, and H), a cathepsin F-like group (cathepsins F and W), and into a cathepsin B-like group which comprises besides cathepsin B other not well assigned cathepsins such as cathepsins Z and C. Bleomycin hydrolase forms an independent branch in the cathepsin tree (Figure 2).
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Figure 2. Phylogenetic tree of human papain-like cysteine proteases. The dendrogram was obtained using the complete amino acid sequences from all presently known 11 cathepsins and bleomycin hydrolase.
EXPRESSION OF CATHEPSINS IN BRAIN TISSUE The tissue distribution of cathepsins varies from a ubiquitous to a highly tissue-specific expression. Most of the presently known cathepsins show low level or no expression in the central nervous system. Northern blot analysis of whole brain revealed low level expression of cathepsins B, S and H, moderate levels of bleomycin hydrolase and no detectable expression for cathepsins L, K, W, V (L2), and Z9; 11; 12; l3; l4; 15; 16; 17 . Cathepsin F, a recently described papain-like cysteine protease appears the only cathepsin expressed at relatively high levels in brain13 (Fig. 3). However, to date, no detailed studies have revealed the cellular distribution of cathepsin F in brain. Cathepsin F shows a ubiquitous expression and has been demonstrated to be expressed in macrophages18. In situ hybridization and immunohistochemical analysis revealed a predominant expression of various cathepsins in microglia cells, a macrophage related brain-resident cell type. Cathepsins B, L, and H have been found glioblastomas and astrocytomas17; 19; 20. Interestingly, expression and activities of these cathepsins is significantly lower in normal brain. Cathepsin S has been demonstrated by in situ hybridization in microglia cells in rat21. Using a polyclonal antihuman cathepsin S antibody, cathepsin S polypeptide expression was shown in neocortical and hippocampal neurons and glia22. Surprisingly, northern analysis revealed only very low mRNA expression levels (see Figure 3). Bleomycin hydrolase has shown immunoreactivity in neocortical neurons and in senile plaques of Alzheimer’s disease (AD)23. AD plaques also were immuno-positive for cathepsins B, L, and S24, although no significant differences of cathepsin activities in AD and normal brain could be observed 25. To date, an exclusively brain-specific cathepsin has not been identified. Known tissue-specific cathepsin are cathepsin K which is primarily expressed in osteoclasts11; 26, cathepsin W in natural killer cells12; 27 , and cathepsin V in thymus and testis28; 29. The closely related cathepsin S is predominantly expressed in bone marrowderived antigen-presenting cells 30 .
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SYNTHETIC INHIBITORS OF CATHEPSINS Inhibitors of putative target proteases are used as fundamental tools to investigate the physiological and pathological implications of these enzymes. Measurable effects of protease inhibitors on functional parameters of brain pathology such as ischemic neuronal cell death or ataxia and biochemical observations of cleavage events on selected substrates are frequently the only indications that a certain protease class or an individual enzyme is involved. The implication of an individual protease or protease family is entirely based on the assumption that the inhibitors used are specific. Absolute specificity is achieved only in rare cases and thus caution is required for the interpretation of the results. In the past three decades, several cysteine protease-selective inhibitors have been developed which include diazo-methyl ketones, fluoromethyl ketones epoxides, and These vinyl sulfones31; 32; 33; 34 . inhibitors are irreversible inactivators which display low or no crossinhibition of serine, aspartate and metalloproteinases. However, a major problem of currently available inhibitors is their lack of specificity towards Figure 3. Northern blotanalysis of human cathepsins individual cathepsins. To date, only and bleomycin hydrolase in human tissues including brain. cathepsin B inhibitors such as CA074 and related epoxide peptidyl inhibitors35; 36 display sufficient specificity to permit a discrimination between cathepsin B and related cathepsins in complex enzyme mixtures or in cell and animal studies. Considering the time dependent mode of action of irreversible inhibitors, the apparent Ki-value of a selected compound should be at least 3 to 4 orders of magnitude lower for the target protease when compared with those of related enzymes. The second-order rate constant of inhibition for such a compound has to be sufficiently greater for the target enzyme so that no significant inhibition of other enzymes occurs during the duration of the experiment. This requirement is difficult or almost impossible to achieve for long-term treatment experiments of animals or cell cultures. Peptide aldehydes are another inhibitor type frequently used to block papain-like cysteine proteases37. However, this inhibitor class is equally reactive to serine proteases and the threonine-dependent proteasomes. Peptide aldehydes are reversible inhibitors active at nanomolar concentrations and they form unstable hemithio or hemiketo acetals with the active site cysteine, serine or threonine of the target enzyme. The differences between the Ki-values of reversible inhibitors for selected proteases should be at least 3 orders of magnitude to prevent significant cross-inhibition of other proteases. Leupeptin (Ac-Leu-LeuArg-aldehyde), a frequently used inhibitor has a low specificity among cathepsins and does not allow the identification of individual cysteine proteases. However, it has been used to demonstrate the general involvement of cathepsin activities in the accumulation of Aβ and Aβ42 in neuronal lysosomes 38 . Most of the commercially available peptide aldehydes such as leupeptin, calpeptin and calpain inhibitors I and II have Ki-values for cathepsin L and F-like cysteine proteases (cathepsins L, V, S, K, F; unpublished, DB) in the low-nanomolar concentration range and do not allow a discrimination between these enzymes. However, the more severe problem is
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that inhibitors such as calpeptin and calpain inhibitors I and II whose designation implies specificity for calpains have up to two orders of magnitude lower binding affinities for calpains than for cathepsin L- and F-like proteases (unpublished, DB). Calpains, belonging to the C2 protease clan2, have been implicated in various neurodegenerative diseases as described in other chapters of this book. Many of these implications have been concluded from studies using various types of inhibitors such as calpain inhibitors I and II or calpeptin. For example, calpain inhibitor I and calpeptin blocked the cleavage of GluR1 of AMPA receptors after N-methyl-D-aspartate stimulation or treatment with kainic acid39; 40 or calpain inhibitors 1 and 2 reduced depolarization-induced degradation of tau-protein to approximately 35% or 25% of control values in primary rat septo-hippocampal cultures41. Considering the high affinity of calpain inhibitors to cathepsins, inhibitory effects caused by these compounds must be discussed cautiously. The same specificity problem applies to other calpain inhibitors. For example, AK275, a peptidyl a-keto amide inhibitor used in stroke animal models to inhibit calpain activity has an approximately 45 to 55-fold lower Kivalue for cathepsin L when compared to those for calpains I and II 42 (unpublished DB). AK275 proved effective to protect against focal ischemic brain damage in rats 43. On the other hand, CA-074, a selective cathepsin B inhibitor and E-64c, a general cathepsin inhibitor revealed similar protective activity against ischemic hippocampal neuronal death in primates 44; 45 . AK275 caused 75% reduction in infarct volume whereas E-64c and CA074 saved 84 % and 67% of CA1 neurons from neuronal death, respectively. Table 2 compares the Ki-values of various calpain inhibitors for calpains to those obtained for cathepsins B and L.
Table 2. Inhibition of cathepsins L, B and calpains with calpain inhibitors Inhibitor
cathepsin L
K i (nM) cathepsin B calpain I
calpain II
Ac-Leu-Leu-Nle-al
0.5#
n.d.
190#
220#
Ac-Leu-Leu-Met-al (calpain inhibitor II) Z-Leu-Nle-a1 (calpeptin) Z-Leu-Abu-CONH-CH2-CH3 (AK275) Z-Leu-Abu-CONH-(CH2)2OH Z-Leu-Abu-CONH-(CH2)2O(CH2)2OH Z-Leu-Abu-CONH-CH2-2-furyl Z-Leu-Abu-CONH-CH2-2tetrahydrofuryl Z-Leu-Abu-CONH-(CH2)3- 1 -imidazolyl Z-Leu-Phe-CONH-(CH2)3-4morpholiny1 Z-Leu- Abu-CONHCH2CH(OH)C6H4(3,4-(OCH2Ph)2) Z-Leu-Phe-CONH- CH2-CH3 Z-Leu-Phe-CONH-(CH2)2-Ph ** unpublished (DB and J.C. Powers); data are from46; # data are from47
0.6#
n.d.
120#
230#
-0.4
n.d.
52
34
4.6 ± 1.2 3.9 ± 1 2.9 ± 0.1 1.5 ± 0.4 1.8 ± 0.3
2,400* 4,500* 2,000* 6,000* 4,500*
250 800* 650* 800* 330*
210 78* 160* 33* 66*
0.5 ± 0.1 0.6 ± 0.2
9,900* 13,000*
290* 120*
68* 330*
0.5 ± 0.1
320*
230*
100*
2.1 ± 0.2 0.7 ± .2
6,000* 9,300*
200* 52*
39* 24*
(calpain-inhibitor I)
Although the discussed calpain inhibitors do not allow an unambiguous identification of individual cysteine proteases or cysteine proteinase subclasses in complex animal or cellbased models, they are useful in well defined enzymatic assays. For example, the inhibition of Ca2+-dependent substrate cleavage events at neutral pH can be attributed to the action of calpains, since cathepsins are not calcium sensitive and with the exception of cathepsin S unstable at neutral pH. On the other hand, inhibitor efficacy at acidic pH can mostly be attributed to cathepsins.
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CATHEPSINS AND NEUROLOGICAL DISEASES Brain Tumors Matrix metalloproteinases, serine proteases, aspartate proteases and cysteine lysosomal proteases individually, or in concert with each other contribute to the localized destruction of extracellular matrix (ECM), which is a salient feature of malignant tumors. Overexpression, ectopic expression of cysteine lysosomal proteases, and changes in their processing have been observed in malignant tumors of different origin48; 49. Of the growing number of members of the family of cysteine cathepsins, only cathepsin B, cathepsin L and cathepsin H have been studied in regards to brain tumor invasiveness50; 51. Overexpression and ectopic expression of cathepsin B mRNA and protein have been documented in glioblastomas and astrocytomas as compared to normal brain tissue49; 52 . The ectopic expression in tumors occurs at the tissue level, i.e., in glioblastoma cathepsin B is expressed in glial cells and endothelial cells in contrast to its preponderantly neuronal expression in normal brain tissue, or at the cellular level in which case the overexpressed protease is misstargeted (membrane localization and/or secretion V.S. its normal lysosomal localization)53. Increased cathepsin B immunoreactivity in brain tumor cells and endothelial cells of the tumor can serve as a highly reliable predictor of survival rate of patients with primary brain tumors52. There is also evidence that cathepsin L and cathepsin H protein expression is increased in primary brain tumors such as glioblastomas and astrocytomas17; 19 . Studies using specific neutralizing antibodies to these proteases strongly support the role of cathepsin L and cathepsin H in tumor invasiveness. More specifically, anti-cathepsin L and anti-cathepsin H antibodies inhibit the progression of glioma cells through Matrigel17; 19. Similar experiments with neutralizing anti-cathepsin B antibodies failed to significantly decrease the ability of tumor cells to invade Matrigel, suggesting that cathepsin B might have an accessory role, i.e., activate another protease, such as cell surface urokinase plasminogen activator, which in turn can take part in ECM dissolution49. The importance of cysteine lysosomal proteases for brain tumor invasiveness was shown in an elegant experiment where a brain tumor was juxtaposed to a healthy brain tissue in an organotypic culture with or without the presence of a general inhibitor of cysteine lysosomal proteases in the media. The tumor cells quickly invaded the healthy brain tissue, a process prevented by the presence of the inhibitor in the culture medium.
Figure 4. Inhibition of glioma tumor invasion (upper spheroid) into normal brain tissue (lower spheroid) by the general cysteine protease vinylsulfone inhibitor, Mu-Leu-hPh-VS-Ph. Panel A: confrontation model in the absence of the inhibitor. Panel B: confrontation model in the presence of the inhibitor (photographs: courtesy of T. Mikkelsen, Henry Ford Hospital, Detroit, MI).
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Another level of abnormal regulation of cysteine lysosomal proteases in brain tumors can occur as a result of the defunct expression and/or targeting of the endogenous cysteine inhibitors (CPIs)50. However, except for one study showing reduced CPI activities during glioma progression compared to normal brain tissue there are no biochemical data that address these possibilities51.
Alzheimer's Disease Alzheimer' disease (AD) is the most common cause of dementia in the elderly. Although the pathogenesis of Alzheimer's disease is very complex, a large body of evidence indicates that neuritic dystrophy, neurofibrillary tangle formation, gliosis, microglial reactivity and other degenerative changes seen in the brains of AD patients, are a result of the altered metabolism of amyloid E (AE) peptides54. These highly aggregatable, 40-42 amino acid long peptides are derived by proteolysis of a ~700-amino acid, integral membrane precursor, the Alzheimer amyloid precursor protein (APP) and are the major proteinacious component of the defining pathological feature of AD the senile plaque55. The involvement of the endosomal lysosomal system in amyloidogenesis as a cardinal process in AD and the potential role(s) of cysteine and other acid proteinases in this process have been reviewed elsewhere 24. Here we will discuss some relatively recent evidence supporting the role of bleomycin hydrolase (BH) and cathepsin S in the establishing of AD pathology, as well as evidence that cysteine lysosomal proteases might participate in neurofibrillary tangle formation. The majority of AD cases are sporadic (over 90%), and identification of factors that influence the onset or progression of the disease is an important step towards understanding its mechanisms and developing successful, rational drug therapies. A recent genetic study provided evidence that the gene for BH located on chromosome 17 (17q11.1-11.2), is a novel susceptibility locus for development of AD56. This study showed that the G/G genotype (a result of an A-G substitution at position 1450) is significantly overrepresented in AD patients AD as compared to age matched controls. The biology behind this polymorphism is largely speculative. The A-G substitution results in a conservative substitution I443V in the C-terminus; deletion of the last 18 amino acids at the C-terminus, including the polymorphic site abolishes its enzymatic activity56. To date, the only known function of this enzyme is the metabolic inactivation of the chemotherapeutic glycopeptide bleomycin9;57. It is of interest that BH is a neutral protease that lacks a leader sequence, which suggests cytosolic localization9. In human brain, the mRNA for its transcript localized mostly to glial cells in the white matter58. Although BH immunoreactivity has been detected in senile plaques of AD patients, a comparative in situ hybridization histochemistry analysis of the distribution of BH mRNA in healthy v.s. AD brains failed to reveal any significant differences23; 58. In addition, the initial observation that the G/G genotype associates with increased risk of sporadic AD has been reproduced with very limited success; only in one59 out of four follow up studies this genotype was significantly overrepresented in AD patients v.s. age-matched controls59; 60; 62; 63 . AE peptides are generated by the action of E- and J-secretase activities; in an alternative, non-amyloidogenic scenario, the generation of AE is precluded by the action of a third proteolytic activity, D-secretase which cleaves at position 17 of the AE sequence55. Since the cysteine lysosomal proteases have broad substrate specificity and since the endosomal compartment is one of the cellular sites for AE generation it was hypothesized that they can subserve the role of secretases, particularly E-secretase. With the discovery of the molecular identity if E-secretase65, it seems unlikely that cysteine lysosomal proteases are the cognate Esecretases, however, it is quite plausible that they can serve as ad-hoc E secretases under conditions of cellular stress. To test this hypothesis the effect of overexpression of cathepsin B, cathepsin L, and cathepsin S, on the production of AE peptides was investigated. Only overexpression of the cDNA for cathepsin S in human embryonic kidney cells (U293) resulted in a modest but specific increase in the levels of secreted AE peptideS66. The N-terminus of the AE species generated in response to overexpression of cathepsin S is Met at position (-1) in relation to the AE sequence66. This AE species has not been detected in clinical samples. If cathepsin S indeed operates as E-secretase in vivo in scenarios of deregulated ectopic expression, an additional proteolytic activity must process the AE species beginning with Met (-1). It is
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curious that analysis of the substrate specificity of BH using methyl coumaryl substrates revealed N-terminal Met as a preferred amino acid for its exopeptidase activity9, suggesting that cathepsin S and BH might be acting in concert to produce AE peptides. In support of the role of cathepsin S in AE generation are the findings that cathepsin S immunoreactivity is increased in AD brains and brains from patients with Down’s syndrome22. Studies in rodent brain and cell culture studies offer an alternative role of cathepsin S in AD pathology. In situ hybridization histochemistry of rat brain revealed that in rodents the expression of cathepsin S mRNA is restricted to microglia and pericytes in the brain21. Purified, recombinantly expressed, human cathepsin S was shown to efficiently degrade monomers and dimers of the AE peptide as well as brain proteoglycans67; 68. Proteoglycans are known to promote AE fibril formation and contribute to the protease resistance of AE aggregates69. These observations suggest that cathepsin S is one of the microglial proteases involved in AE clearance. It has also been shown that cathepsin S can be secreted from microglia and macrophages in culture and that this process is positively regulated by both cytokines and growth factors (NGF and bFGF)67. Thus, cathepsin S may participate in AE clearance at the initial stages of aggregation both intracellularly and extracellularly. It is also attractive to postulate that secreted cathepsin S can participate in the dissolution of AE fibrils by degrading the AE associated proteoglycans and that way rendering AE fibrils vulnerable to proteolysis by other AE-degrading proteases. In further support of a role in AE clearance is the finding that cathepsin S expression is dramatically upregulated following entorhinal cortex lesion (ECL) in rats, in activated microglia/macrophages at the site of the lesion and in the deafferented dentate gyrus68. ECL is a neurodegeneration paradigm that replicates some of the deafferentation seen in AD brains70. The cellular distribution of cathepsin S mRNA in human brain is yet to be determined, so it remains questionable whether human cathepsin S is expressed and regulated like its rodent homologue. The available immunohistochemistry data from human brain suggest that this might not be the case. In AD and Down's syndrome brains, cathepsin S immunoreactivity was observed in the leptomeningies, in subpopulations of cortical and hippocampal neurons, in parenchymal astrocytes, and in astrocytes surrounding senile plaques22. Control brains did not show cathepsin S immunoreactivity in the vasculature or in glial cells. Screening of various human tissues for cathepsin S expression showed miniscule levels of cathepsin S mRNA in brain12. It is important to investigate whether AD brains contain elevated levels of cathepsin S mRNA compared to healthy brains and to determine the distribution of cathepsin S mRNA in human brain at the cellular level. Intracellular neurofibrillary tangles composed of hyper-phosphorylated tau protein are one of the cytopathogenic features of Alzheimer's disease and other neuropathies54. Along the lines of investigating how lysosomal abnormalities contribute to neuronal dysfunction in the course of normal aging and during disease, several studies have utilized brain slice preparations to assess the effects of inhibitors of lysosomal proteases on neuronal integrity. It was recently shown that treatment of entorhinohippocampal slices with a specific inhibitor for cathepsin B and cathepsin L, markedly stimulates tau phosphorylation in neuronal cell bodies and initial dendritic segments in layers II and III of the entorhinal cortex and in the CA1 field71. Neurons from these cell layers are particularly vulnerable in AD. Biochemical studies have shown that the inhibitor treatment leads to increased levels of cathepsin D in the cytoplasm, which in turn cleaves tau making it susceptible to aberrant phosphorylation and aggregation into neurofibrillary tangle-like strictures71. How does suppression of cathepsin B and cathepsin L lead to release of cathepsin D in the cytosol and what is the cause of the selective vulnerability of the neurons in layers II and II of the entorhinal cortex and the CA1 field in the hippocampus is still elusive.
Transmissible Spongiform Encephalopathies The neuropathology of transmissible spongiform encephalopathies (TSE) such as Creutzfeldt-Jakob disease, German-Straussler-Scheinker syndrome and Kuru, is characterized by the appearance in the brain of an abnormal, protease resistant, insoluble form of a host encoded protein the prion protein (PrP)72. This conformationally modified form of PrP, PrPSc, is specifically associated with TSE. Abnormal accumulation of PrPSc is often postulated to be the earliest and most central event in the infection followed by secondary changes in glial cells73: 74. Modifications of host gene expression patterns in animal models of CJD were analyzed using differential mRNA display, in order to identify genes whose altered expression might be associated or be the cause of the neurodegenerative
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changes observed in TSE. In two such studies that utilized different rodent models of CJD, cathepsin S mRNA was specifically and dramatically elevated along with other microgliaspecific genes such as the CCR5 chemokine receptor, and the gene for the microglial marker F4/8075; 76. In some rodent models of CJD cathepsin S and microglial markers were elevated prior to any PrP amyloid deposition. Previously, it was shown that the expression of cathepsin S in rats is restricted to microglia/macrophages, and its expression was shown to dramatically increase in activated microglia after brain injury68. It is also known that cathepsin S retains activity at neutral pH and has been implicated in the formation of APPderived amyloid β peptides67. In light of these observations, and based on the kinetics of cathepsin S mRNA upregulation in this animal model, cathepsin S has been implicated in the formation of amyloid deposits in CJD.
Ischemia Transient ischemia in rodents and monkeys is one of the experimental models used to study the mechanism of ischemia-induced neuronal cell death. In this paradigm, the CA1 pyramidal neurons of the hippocampus are particularly vulnerable to the ischemic insult and undergo selective and delayed death that has the morphological characteristics of apoptosis77. Since vulnerable neurons of the CA1 field contain not only enlarged lysosomes but also extralysosomal cathepsin immunoreactivity78, it has been postulated that cysteine cathepsins released from leaky lysosomes are a major cause of the ischemia-induced neuronal death in the hippocampus in this experimental paradigm. This is believed to occur in response to the ischemia-induced increase in intracellular calcium which leads to activation of µ-calpain at the lysosomal membrane36. This “calpain-cathepsin” hypothesis of neuronal death proposes that the ischemia-induced µ-calpain is targeted to the lysosomal membrane to provoke its rupture. The protein levels for both cathepsin B and cathepsin L increase in the CA1 field of the hippocampus; however, only cathepsin B activity is shown to increase after an ischemic insult79. In contrast, the activity of cathepsin L decreases inspite of an increase in cathepsin L protein content79. The relevance of this increase in cathepsin B activity is supported by the results of recent studies in which intravenous administration of CA-074, a selective cathepsin B inhibitor, or E64, a general inhibitor of cysteine cathepsins, immediately after an ischemic insult resulted in significant rescue of neurons in the CA1 field of the hippocampus36; 44; 45. The implications of these studies are two-fold: first, developing specific inhibitors to cathepsin B and other cysteine cathepsins that might participate in the post-ischemic neuronal degeneration holds great therapeutic promise for the treatment of stroke, and second, similar mechanisms of neuronal degeneration might occur during aging, since instability of the lysosomal membrane and extralysosomal cathepsin localization have been reported in aging animals. Another model of ischemia is the one occurring in response to middle cerebral artery occlusion (MCAO). MCAO leads to necrosis and apoptosis of neurons in the ischemic area80. Cathepsin B immunoreactivity increases immediately after MCAO (2 h later) in the ischemic neurons; 24 h after reperfusion, cathepsin B immunoreactivity decreases, only to increase again at 48 h after reperfusion, and remain increased for several days80. The majority of neuronal death observed after MCAO is necrotic, and while the cathepsin B content increases initially only in ischemic neurons, by 48 h cathepsin B immunoreactivity is mostly seen in macrophages/microglia in the ischemic area80. Infusion of an endogenous cathepsin B inhibitor stefin prior to MCAO can rescue a small percent of afflicted neurons80. In contrast, ischemic infractions in rat and mouse brain induced by MCAO could be dramatically reduced by cerebroventricular administration of a general caspase inhibitors81. This raises the issue of the interaction between cysteine lysosomal proteases and caspases to produce apoptotic or necrotic neuronal death.
Cysteine Proteinase Inhibitors and Neurodegeneration Loss of function mutations in the gene for human cystatin B are associated with Unvericht-Lundborg disease (EPM1)82. EPM1 is a type of progressive myoclonus epilepsy, an autosomal recessive disorder, characterized by myoclonic and tonic-clonic seizures82. To this date biochemical markers of the disease have been lacking. Cystatin B is a ubiquitously expressed reversible inhibitor of cathepsins B, H, L and S and papain; it localizes to the cytosol and most likely participates in the regulation of proteolysis in the cytosol. In the majority of cases, cystatin B-deficiency is a result of an expansion of a dodecamer sequence
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in its promoter region which results in decreased transcription of the gene83. Other EPMassociated mutations occur in the coding region, and result in a protein unable to bind the cognate proteases84. The fact that EPM1 is associated with the lack of a functional cysteineprotease inhibitor suggests that the disease might come about as a result of excessive intracellular proteolysis. The analysis of the phenotype of cystatin B knockout mice has shed some light on the establishing of disease pathology and the role of cystatin B in this process85. Cystatin B deficient mice have a phenotype that replicates some symptoms of the human disease, i.e., myoclonic seizures and ataxia. The neuropathology of these knockout mice is characterized by loss of cerebellar granule cells by what appears to be apoptotic cell death85, suggesting that cystatin B is involved in the regulation of apoptosis. There is no evidence to date that cystatin B is capable of interacting with caspases directly. Two possible scenarios of its involvement in the regulation of apoptosis in the cerebellum have been proposed. According to the first scenario, cystatin B serves as an inhibitor of cysteine cathepsins capable of activating caspases. There is some evidence that cathepsin B can act as a caspase-activating enzyme86. It is unclear however, what causes the release of cysteine cathepsins in the cytosol where they would be in a position to activate cytosolic caspases. Alternatively, cystatin B-deficiency can cause apoptosis by a general increase in cytosolic proteolysis, which results in activation of caspases. In support of a role of cystatin B in the regulation of neuronal apoptosis are the results of a recent study in which repeated seizures of rats evoked by hippocampal kindling, caused an increase in cystatin B mRNA and protein in the forebrain87. Based on these findings one can hypothesize that cystatin B counteracts apoptosis by neutralizing spurious intracellular proteolysis by cysteine cathepsins that might be released from lysosomes in response to seizures. Cystatin C is a ubiquitous, secreted, cysteine protease inhibitor that very efficiently inhibits cysteine proteases such as cathepsin B, H, L and S88; 89. It appears to have a general protective function to prevent tissue destruction by intracellular enzymes released in the extracellular environment88; 89. As a result, cystatin C is involved in several diseases associated with proteolytic anomalies, such as cancer, inflammatory lung disease, multiple sclerosis, HIV infection, and autoimmune diseases. A mutant variant of cystatin C, containing one amino-acid substitution at position 68 (L68Q) is associated with a hereditary form of cerebral hemorrhage and amyloidosis90. Decreased levels of cystatin C in the cerebrospinal fluid and accumulation of cystatin C-derived amyloid fibrils in the walls of brain arteries, leading to fatal strokes characterize this autosomal dominant disorder91. The production, processing, secretion and clearance of the wild-type and L68Q mutant cystatin C were investigated in cell lines stably expressing either variant, and revealed that the mutant variant dimerized at lower concentrations that the wild-type form, and once secreted, was more susceptible to proteolysis by serine proteases92. These findings suggest that the L68Q substitution result in a protein prone to self-aggregation and amyloid fibril formation.
SUMMARY It is apparent that most studies assessing the role of cysteine lysosomal proteases in various processes of neurodegeneration pertain to cathepsin L and cathepsin B the classical and most well characterized members of the family. With the advent of animal models for a number of neurodegenerative disorders, and the availability of high throughput analyses of gene expression it is now possible to begin assessing which members of this family of proteases participate in the establishing of various neuropathological states.
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28. Adachi, W., Kawamoto, S., Ohno, I., Nishida, K., Kinoshita, S., et al., Isolation and characterization of human cathepsin V: a major proteinase in corneal epithelium., Invest Ophthalmol Vis Sci. 39:1789 (1998) 29. Brömme, D., Li. Z., Barnes, M., Mehler, E., Human cathepsin V functional expression, tissue distribution, electrostatic surface potential, enzymatic characterization, and chromosomal localization., Biochemistry. 38:2377 (1999) 30. Villadangos, J. A., Bryant, R. A., Deussing, J., Driessen, C., Lennon-Dumenil, A. M., et al., Proteases involved in MHC class II antigen presentation., Immunol Rev. 172:109 (1999) 31. Shaw, E., ed. Peptidyl diazomethanes as inhibitors of cysteine and serine proteinases. Vol. 244. New York: Academic Press. 649 pp.(1994) 32. Rasnick, D., Synthesis of peptide fluoromethyl ketones and the inhibition of human cathepsin B, Anal Biochem. 149:461 (1985) 33. Barrett, A. J., Kembhavi, A. A., Brown, M. A., Kirschke, H., Knight, C. G., et al., L-transEpoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L., Biochem J. 201:189 (1982) 34. Palmer, J. T., Rasnick, D., Klaus, J. L., Bromme, D., Vinyl sulfones as mechanism-based cysteine protease inhibitors, J Med Chem. 38:3193 (1995) 35. Towatari, T., Nikawa, T., Murata, M., Yokoo, C., Tamai, M., et al., Novel epoxysuccinyl peptides. A selective inhibitor of cathepsin B, in vivo., FEBS Lett. 280:31 (1991) 36. Yamamoto, A., Hara, T., Tomoo, K., Ishida, T., Fujii, T., et al., Binding mode of CA074, a specific irreversible inhibitor, to bovine cathepsin B as determined by X-ray crystal analysis of the complex., J Biochem (Tokyo). 121:974 (1997) 37. Rich, D. H., Inhibitors of cysteine proteinases, In Proteinase Inhibitors, ed. A. J. Barrettt, G. Salvesen. pp. 153. Elsevier, Amsterdam, New york, Oxford: (1986) 38. Frautschy, S. A., Horn, D. L., Sigel, J. J., Harris-White, M. E., Mendoza, J. J., et al.. Protease inhibitor coinfusion with amyloid beta-protein results in enhanced deposition and toxicity in rat brain., J Neurosci. 18:8311 (1998) 39. Gellerman, D. M., Bi, X., Baudry, M., NMDA receptor-mediated regulation of AMPA receptor properties in organotypic hippocampal slice cultures., J Neurochem. 69: 131 (1997) 40. Bi, X., Chen, J., Baudry, M., Calpain-mediated proteolysis of GluR1 subunits in organotypic hippocampal cultures following kainic acid treatment., Brain Res. 781:355 (1998) 41. Kampfl, A., Whitson, J. S., Zhao, X., Posmantur, R., Clifton, G. L., Hayes, R. L., Calpain inhibitors reduce depolarization induced loss of tau protein in primary septo-hippocampal cultures., Neurosci Lett. 194:149 (1995) 42. Li, Z., Ortega-Vilain, A. C., Patil, G. S., Chu, D. L., Foreman, J. E., et al., Novel peptidyl alpha-keto amide inhibitors of calpains and other cysteine proteases., J Med Chem. 39:4089 (1996) 43. Bartus, R. T., Baker, K. L., Heiser, A. D., Sawyer, S. D., Dean, R. L., et al., Postischemic administration of AK275, a calpain inhibitor, provides substantial protection against focal ischemic brain damage., Cereb Blood Flow Metab. 14:537 (1994) 44. Yamashima, T., Kohda, Y., Tsuchiya, K., Ueno, T., Yamashita, J., et al., Inhibition of ischaemic hippocampal neuronal death in primates with cathepsin B inhibitor CA-074: a novel strategy for neuroprotection based on 'calpain-cathepsin hypothesis'., Eur J Neurosci. 10: 1723 (1998) 45. Tsuchiya, K., Kohda, Y., Yoshida, M., Zhao, L., Ueno, T., et al., Postictal blockade of ischemic hippocampal neuronal death in primates using selective cathepsin inhibitors., Exp Neurol. 155:187 (1999) 46. Li, Z., Patil, G. S., Golubski, Z. E., Hori, H., Tehrani, K., et al., Peptide alpha-keto ester, alpha-keto amide, and alpha-keto acid inhibitors of calpains and other cysteine proteases, J Med Chem. 36:3472 (1993) 47. Sasaki, T., Kishi, M., Saito, M., Tanaka, T., Higuchi, N., et al., Inhibitory effect of di- and tripeptidyl aldehydes on calpains and cathepsins, J Enzyme Inhib. 3: 195 (1990) 48. Lah, T. T., Kos, J., Cysteine proteinases in cancer progression and their clinical relevance for prognosis., Biol Chem. 379:125 (1998) 49. Keppler, D., Sameni, M., Moin, K., Mikkelsen, T., Diglio, C. A., Sloane, B. F., Tumor progression and angiogenesis: cathepsin B & Co., Biochem Cell Biol. 74:799 (1996) 50. Rooprai, H., K., McCormick, D., Proteases and their inhibitors in human brain tumours: a review., Anticancer Res. 27:4151 (1997) 51. Sivaparvathi, M., McCutcheon, I., Sawaya, R., Nicolson, G. L., Rao, J. S., Expression of cysteine protease inhibitors in human gliomas and meningiomas., Clin Exp Metastasis. 14:344 (1996)
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52. Strojnik, T., Kos, J., Zidanik, B., Golouh, R., Lah, T., Cathepsin B immunohistochemical staining in tumor and endothelial cells is a new prognostic factor for survival in patients with brain tumors., Clin Cancer Res. 5:559 (1999) 53. Mikkelsen, T., Yan, P. S., Ho, K. L., Sameni, M., Sloane, B. F., Rosenblum, M. L., Immunolocalization of cathepsin B in human glioma: implications for tumor invasion and angiogenesis., J Neurosurg. 83:285 (1995) 54. Selkoe, D., The origins of Alzheimer disease: a is for amyloid., JAMA. 283:1571 (200) 55. Gandy, S., Neurohormonal Signaling Pathways and the Regulation of Alzheimer beta-Amyloid Precursor Metabolism., Trends Endocrinol Metab. 7:273 (1999) 56. Montoya, S. E., Aston, C. E., DeKosky, S. T., Kamboh, M. I., Lazo, J. S., Ferrell, R. E., Bleomycin hydrolase is associated with risk of sporadic Alzheimer's disease., Nat Genet. 18(1998) 57. Sebti, S. M., Jani, J. P., Mistry, J. S., Gorelik, E., Lazo, J. S., Metabolic inactivation: a mechanism of human tumor resistance to bleomycin., Cancer Res. 51:227 (1991) 58. Malherbe, P., Faull, R. L., Richards, J. G., Regional and cellular distribution of bleomycin hydrolase mRNA in human brain: comparison between Alzheimer's diseased and control brains., Neurosci Lett. 281:37 (2000) 59. Papassotiropoulos, A., Bagli, M., Jessen, F., Frahnert, C., Rao, M. L., et al., Confirmation of the association between bleomycin hydrolase genotype and Alzheimer's disease., Mol Psychiatry. 5:213 (2000) 60. Farrer, L. A., Abraham, C. R., Haines, J. L., Rogaeva, E. A., Song, Y., et al., Association between bleomycin hydrolase and Alzheimer's disease in Caucasians., Ann Neurol. 44:808 (1998) 61. Nagase, H., Enghild, J. J., Suzuki, K., Salvesen, G., Stepwise activation mechanisms of the precursor of matrix metalloproteinase 3 (stromelysin) by proteinases and (4-aminophenyl)mercuic acetate., Biochemistry. 29:5783 (1990) 62. Namba, Y., Ouchi, Y., Asada, T., Hattori, H., Ueki, A., Ikeda, K., Lack of association between bleomycin hydrolase gene polymorphism and Alzheimer's disease in Japanese people., Ann Neurol. 46:136 (1999) 63. Thome, J., Gewirtz, J., Sakai, N., Zachariou, V., Retz-Junginger, P., et al., Polymorphisms of the human apolipoprotein E promoter and bleomycin hydrolase gene: risk factors for Alzheimer's dementia?, Neurosci Lett. 274:37 (1999) 64. Page, A. E., Warburton, M. J., Chambers, T. J., Pringle, J. A., Hayman, A. R., Human osteoclastomas contain multiple forms of cathepsin B, Biochim Biophys Acta. 1116:57 (1992) 65. Vassar, R., Bennett, B. D., Babu-Khan, S., Kahn, S., Mendiaz, E. A,, et al., Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE., Science. 286:735 (1999) 66. Munger, J. S., Haass, C., Lemere, C. A., Shi, G. P., Wong, W. S., et al., Lysosomal processing of amyloid precursor protein to A beta peptides: a distinct role for cathepsin S, Biochem J. 3 11 :299 (1995) 67. Liuzzo, J. P., Petanceska, S. S., Devi, L. A., Neurotrophic factors regulate cathepsin S in macrophages and microglia: A role in the degradation of myelin basic protein and amyloid beta peptide., Mol Med. 5:334 (1999) 68. Petanceska, S., Canoll, P., Devi, L. A., Expression of rat cathepsin S in phagocytic cells., J Biol Chem. 271 :4403 (1 996) 69. Kisilevsky, R., Fraser, P., Proteoglycans and amyloid fibrillogenesis., Ciba Found Symp. 199:58 (1996) 70. Shukla, C., Bridges, L. R., Regional distribution of tau, beta-amyloid and beta-amyloid precursor protein in the Alzheimer's brain: a quantitative immunolabelling study., Neuroreport. 10:3785 (1999) 71. Bi X, Z. J., Lynch G, Lysosomal protease inhibitors induce meganeurites and tangle-like structures in entorhinohippocampal regions vulnerable to Alzheimer's disease., Exp Neurol. 158:312 (1999) 72. Prusiner, S., Prion diseases and the BSE crisis., Science. 278:245 (1997) 73. Brown, D. R., Schmidt, B., Kretzschmar, H. A., Role of microglia and host prion protein in neurotoxicity of a prion protein fragment., Nature. 380:345 (1996) 74. Williams, A., Lucassen, P. J., Ritchie, D., Bruce, M., PrP deposition, microglial activation, and neuronal apoptosis in murine scrapie., Exp Neurol. 144:433 (1997) 75. Baker CA, L. Z., Zaitsev I, Manuelidis L, Microglial activation varies in different models of CreutzfeldtJakob disease., J Virol. 73:5089 (1999)
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76. Dandoy-Dron, F., Guillo, F., Benboudjema, L., Deslys, J. P., Lasmezas, C., et al., Gene expression in scrapie. Cloning of a new scrapie-responsive gene and the identification of increased levels of seven other mRNA transcripts., J Biol Chem. 273:7691 (1998) 77. Nitatori, T., Sato, N., Waguri, S., Karasawa, Y., Araki, H., et al., Delayed neuronal death in the CA1 pyramidal cell layer of the gerbil hippocampus following transient ischemia is apoptosis., J Neurosci. 15:1001 (1995) 78. Nitatori, T., Sato, N., Kominami, E., Uchiyama, Y., Participation of cathepsins B, H, and L in perikaryal condensation of CA1 pyramidal neurons undergoing apoptosis after brief ischemia., Adv Exp Med Biol. 389:177 (1996) 79. Kohda, Y., Yamashima, T., Sakuda, K., Yamashita, J., Ueno, T., et al., Dynamic changes of cathepsins B and L expression in the monkey hippocampus after transient ischemia., Biochem Biophys Res Commun. 228:616 (1996) 80. Seyfried, D., Han, Y., Zheng, Z., Day, N., K., M., et al., Cathepsin B and middle cerebral artery occlusion in the rat., J Neurosurg. 87:716 (1997) 81. Hara, H., Friedlander, R. M., Gagliardini, V., Ayata, C., Fink, K., Huang, Z., et al., Inhibition of interleukin 1 beta converting enzyme family proteases reduces ischemic and excitotoxic neuronal damage., Proc Natl Acad Sci USA. 94:2007 (1997) 82. Serratosa, J. M., Gardiner, R. M., Lehesjoki, A. E., Pennacchio, L. A., Myers, R. M., The molecular genetic bases of the progressive myoclonus epilepsies., Adv Neurol. 79:383 (1999) 83. Lalioti, M. D., Scott, H. S., Buresi, C., Rossier, C., Bottani, A., et al., Dodecamer repeat expansion in cystatin B gene in progressive myoclonus epilepsy., Nature. 386:847 (1997) 84. Pennacchio, L. A., Lehesjoki, A. E., Stone, N. E., Willour, V. L., Virtaneva, K., et al., Mutations in the gene encoding cystatin B in progressive myoclonus epilepsy, Science. 271:1731 (1996) 85. Pennacchio, L. A., Bouley, D. M., Higgins, K. M., Scott, M. P., Noebels, J. L., Myers, R. M., Progressive ataxia, myoclonic epilepsy and cerebellar apoptosis in cystatin B-deficient mice., Nat Genet. 20:251 (1998) 86. Vancompernolle, K., Van Herreweghe, F., Pynaert, G., Van de Craen, M., De Vos, K., et al., Atractyloside-induced release of cathepsin B, a protease with caspase-processing activity., FEBS Lett. 438:150 (1998) 87. D’Amato, E., Kokaia, Z., Nanobashvili, A., Reeben, M., Lehesjoki, A. E., et al., Seizures induce widespread upregulation of cystatin B, the gene mutated in progressive myoclonus epilepsy, in rat forebrain neurons., Eur J Neurosci. 12:1687 (2000) 88. Barrett AJ, D. M., Grubb A, The place of human gamma-trace (cystatin C) amongst the cysteine proteinase inhibitors., Biochem Biophys Res Commun. 120:631 (1984) 89. Abrahamson, M., Human cysteine protease inhibitors, Scand J Clin Lab Invest. 48:21 (1988) 90. Levy, E., Lopez-Otin, C., Ghiso, J., Geltner, D., Frangione, B., Stroke in Icelandic patients with hereditary amyloid angiopathy is related to a mutation in the cystatin C gene, an inhibitor of cysteine proteases., J Exp Med. 169:1771 (1989) 91. Abrahamson, M., Molecular basis for amyloidosis related to hereditary brain hemorrhage., Scand J Clin Lab Invest Suppl. 226:47 (1996) 92. Wei, L., Berman, Y., Castano, E. M., Cadene, M., Beavis, R. C., et al., Instability of the amyloidogenic cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type., J Biol Chem. 273: 11806 (1998)
THE ROLE OF THE CALPAIN SYSTEM IN NEUROMUSCULAR DISEASE
Darrel E. Goll, Valery F. Thompson, Hongqi Li, and Jinyang Cong Muscle Biology Group University of Arizona Tucson, Arizona 85721
INTRODUCTION The first report of a neutral calcium-activated proteinase appeared in 1964 in a paper describing a Ca2+-stimulated proteolytic activity having a pH optimum between 7.7 and 8.0 in 0.25 M sucrose supernatants of rat brain extracts1. The proteolytic activity was partly purified by using ammonium sulfate precipitation at 40% saturation and chromatography on a DEAE-anion exchange column, where the activity eluted between 0.27 and 0.30 mM NaC1. Activity of the partly purified enzyme was dependent on the presence of Ca2+, was completely inhibited by 1 mM EDTA, stimulated by 2-mercaptoethanol, optimal between pH 7.1-7.3 (partial purification seemed to lower the pH optimum), and inhibited by Zn2+, Co2+, Cu2+, Hg2+, and iodoacetate, but affected very little by Mn2+, Mg2+, Ba2+,or diisopropyl fluorophosphate. These properties are identical to those described for m-calpain when it was first purified2,3. and it seems very likely that the proteolytic activity described by Guroff1 was m-calpain4. Interestingly, although Guroff found Ca2+-dependent proteolytic activity in extracts of brains from a number of species, including bovine, porcine, mouse, guinea pig, and rabbit, he did not detect it in kidney or spleen extracts, although these tissues are now known to contain substantial quantities of both µ-calpain and m-calpain5,6. It seems likely that Guroff's failure to detect Ca2+-dependent proteolytic activity in kidney or spleen extracts was due to the presence of calpastatin in these extracts. Although brain also contains calpastatin, the calpastatin levels in rat brain are slightly lower than the calpain levels, whereas calpastatin is present in excess over calpain in the kidney and spleen5,6. If Guroff had assayed the fractions of his DEAE-cellulose column for inhibitory activity, he would have also identified calpastatin, which was not identified until 12 years later7. The calpain system has been implicated in a number of neuromuscular diseases and other tissue pathologies. In general, inappropriate activity of the calpains is suspected whenever an alteration in Ca2+ homeostasis occurs, and this alteration is accompanied by limited degradation of cytoskeletal proteins such the neurofilament proteins, spectrin/fodrin, talin, vinculin, myelin basic protein, MAP2, and tau8,9. Administration of synthetic inhibitors that have some selectivity for inhibiting the calpains has often been used to support the conclusion that the calpains are responsible for the observed degradation. It has been impossible, however, to obtain a molecule that inhibits only the calpains9,10 (other than its natural inhibitor, calpastatin), and it is necessary to use a battery of different inhibitors with differing selectivities for different proteolytic enzymes such as the proteasome and cathepsins B, L, and S before concluding that the calpains are involved. Even then, the conclusion should be tempered by the possibility that other proteases may also be involved. Similarly, the cytoskeletal substrates of the calpains are degraded by a number of proteolytic enzymes, and with the exception of spectrin, where the bond cleaved by the calpains has been Role of Proteases in the Pathophysiology of Neurodegenerative Diseases, edited by Lajtha and Banik. Kluwer Academic/Plenum Publishers, New York, 2001.
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identified11,12 and an antibody that specifically recognizes the calpain-produced spectrin fragment has been developed 13,14, the pattern of proteolytic fragments of cytoskeletal proteins does not prove that these fragments originate from the calpains. The role of the calpain system in cerebral ischemia, neuronal death, Alzheimer’s disease, demyelinating diseases and other neuronal pathologies has been recently reviewed15-18 or is discussed elsewhere in this book. Therefore, this article will begin with a brief review of some properties of the calpain system and then focus on its role neuromuscular diseases, emphasizing the muscular dystrophies.
THE CALPAIN SYSTEM The general properties of the three major, well-characterized proteins in the calpain system, µ-calpain, m-calpain, and calpastatin, have been reviewed a number of times19-21; some of these properties are summarized in Table 1. Screening cDNA libraries from different organisms or tissues during the last 10 years has identified at least 12 mRNAs that encode polypeptides having sequence homology to µ- or m-calpain22. With only three exceptions, however, the proteins encoded by these mRNAs have not been isolated, so very little is known about their properties. One of these calpain-like mRNAs is expressed at a high level (10-fold greater than the µ- or m-calpain messages) specifically in skeletal muscle23 (Table 1). Disruption of the gene for this muscle-specific calpain, skm-calpain or p94 or calpain 3, has been shown to be the cause of limb girdle muscular dystrophy type 2A24, and a discussion of skm-calpain will be included in this review. Table 1. Some general properties of the calpain system.
Occurrence Found in all vertebrate cells that have been examined; calpain-like proteins have been isolated from Drosophila and from a blood fluke, Schistosoma; mRNAs encoding for molecules having sequence homology to the calpains have been identified in C. elegans, Drosophila, and Schistosoma.
Ubiquitous Calpains Polypeptides
µ-calpain
80,28 kDa
[Ca2+]required for half maximal activity 3-50 µM
m-calpain
80,28 kDa
400-800 µM
Name
Tissue-specific calpains Polypeptides Name Tissue skm-calpain, p94, calpain 3 94,82 kDa skeletal muscle, rat lens Eight other tissue specific calpains identified only as mRNAs All calpains that have been isolated in protein form are cysteine proteases with pH optima of 7.2-8.2.
Calpastatin Multiheaded protein inhibitor that inhibits only the calpains; expressed in several different isoforms that have one, three, or four inhibitory domains and different N-terminal sequences.
Cellular Distribution The calpains and calpastatins studied thus far are located exclusively intracellularly; various proportions of the calpains are associated with subcellular organelles, which are primarily myofibrils in skeletal muscle, but may include the plasma membrane, mitochondria, and nuclei.
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Most tissues contain both µ- and m-calpain in varying proportions, but some tissues contain only one; human platelets and erythrocytes have only µ-calpain and some smooth muscle cells have only m-calpain. As will be discussed later, whole brain tissue contains largely m-calpain with very little µ-calpain. The subsite specificities of the two calpains are very similar if not identical, and the current evidence suggests that µ- and m-calpain cleave the same polypeptides in cells but respond to different signals. Ever since the first calpain, m-calpain, was purified 2 and characterized 3, it has been clear that the Ca2+ concentrations required for proteolytic activity of the calpains was much higher than the 30-300nM free Ca2+ that exits in normal cells25,26 (see Table 1 for the Ca2+ requirements of µ- and m-calpain in in vitro assays). Consequently, cells contain some mechanism to reduce the Ca2+ requirement of the calpains to the physiological range. Because inappropriate calpain activity in cells usually occurs in response to an increase in intracellular Ca2+ concentration 15-18,27,28 , it may be reasonably suggested that the mechanism that regulates Ca2+ requirement of the calpains also involves fluctuations in [Ca2+], but in the physiological range (from 30-300nMto 400-800nM). The nature of this mechanism remains unknown despite the efforts of a number of investigators. A popular suggestion for lowering the Ca2+ requirement of the calpains proposed that the calpains were proenzymes similar to prothrombin or trypsinogen and that they required autolysis for activation. Both µ- and m-calpain autolyze rapidly when incubated with sufficient calcium2933. Although it is not uncommon for proteolytic enzymes to autolyze, autolysis of the calpains is unusual. Brief autolysis, up to 2 min at 25°C, reduces the [Ca2+] required for half-maximal proteolytic activity of µ-calpain from 3-50 µM to 0.5-2.0 µM and that of mcalpain from 440-800 µM to 50-150 µM without affecting the specific activity of either enzyme33. Autolysis removes the N-terminal 19 or 27 (m- or µ-calpain) amino acids from the 80kDa subunit, reducing its mass to 78- or 76-kDa, respectively, and removes the Nterminal 91 amino acids from the 28kDa subunit, reducing its mass to 18kDa33. The [Ca2+] required to initiate autolysis are just slightly greater than those required for proteolytic activity34, but phospholipids, with phosphatidylinositol 4,5-bisphosphate (PIP2) being the most effective, lowers the [Ca2+] required for autolysis by 5- to 10-fold in in vitro assays30,31. Consequently, in the presence of PIP2, only 0.8-50µM Ca2+ is required to initiate autolysis of µ-calpain32, 34. It was proposed that the unautolyzed, “proenzyme” calpains were translocated to the plasma membrane where they bound to a PIP2 molecule to initiate autolysis at intracellular [Ca2+]. The autolyzed molecules, having a reduced Ca2+ requirement (0.5-2.0 µM for µ-calpain) were released from the membrane as enzymes active at physiological Ca2+ concentrations. It was clear from the beginning that membrane activation could not apply to mcalpain, which requires 90-140µM Ca2+ to initiate autolysis even in the presence of phospholipids, and which requires 50- 150µM Ca2+ for a half-maximal rate of proteolytic activity after autolysis. The Ca2+ requirements for autolysis of µ-calpain in the presence of PIP2 are just above the physiological range, and it is not clear that locally high transients of Ca2+ would be sufficiently reliable to serve as a mechanism to activate µ-calpain. Finally, several recent studies have shown that both unautolyzed µ-calpain20,32,35 and m-calpain36-38 are active proteases. It is clear, however, from observations in a variety of systems, especially platelets, that the calpains are autolyzed under conditions that would be expected to elicit their activity. Hence, it seems likely that autolysis has some physiological significance, but the nature of this role is unknown. It also is not known whether under some conditions the calpains can be proteolytically active without experiencing autolysis. If so, it may be misleading to use the absence of autolysis as indicating the absence of calpain activity.
The Calpain System in the Brain The brain contains a wide variety of different cells, so purification of the calpains from brain tissue results in a “composite” calpain from the different cell types. Initial attempts to purify the calpains from brain produced confusing results. Two forms of calpain were obtained from calf brain, both having a single subunit of 78kDa, with one requiring 2µM Ca2+ for maximal activity and the other requiring 700µM Ca2+ for maximal activity39,40. Because the enzymes had been exposed to Ca2+ during purification, it seems
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likely in retrospect that they had undergone autolysis, which would account for the molecular weight of 78kDa, but absence of the small, 28kDa subunit was puzzling. It may have been degraded by autolysis during purification. Approximately three times more m-calpain was obtained than µ-calpain (the 2µM-requiring form). Subsequent studies by the same investigators isolated a third protease from calf brain having a single 78kDa polypeptide but requiring an intermediate Ca2+ concentration (260µM)41. A single Ca2+ activated neutral protease was obtained from monkey brain42; this enzyme, which had been exposed to 5mM Ca2+ during purification, had two subunits of 74- and 20-kDa and required ~500µM Ca2+ for half-maximal proteolytic activity. In a third study, three peaks of Ca2+-activated proteolytic activity were obtained from bovine brain43. Peak I had a major polypeptide at 6668kDa; the peak II polypeptide was 48-50kDa; and the peak III polypeptide was 30-32kDa. The Ca2+ concentrations required for proteolytic activity were ~1 mM for peak I and ~250µM for peaks II and III. Because these three peaks were all obtained from a hydroxylapatite column of a fraction that had eluted at ~220-250mM KC1 from an anion-exchange column, they must all have been derivatives of m-calpain. Degradation of the calpains on hydroxylapatite columns has been observed (Szpacenko and Goll, unpublished). Three different Ca2+-dependent proteases having molecular weights of 154kDa, 96kDa, and 76kDa were obtained from rat brain44. Purification of these proteases also used a casein affinity column that required exposure to Ca2+ for the proteolytic activities to bind. The 154kDa polypeptide was converted to the smaller forms in the presence of Ca2+, and it was suggested that the larger polypeptide was the native, undegraded enzyme. The Ca2+ concentrations required for proteolytic activity ranged from 10µM to 1000µM. No small 28kDa subunit was detected. None of the attempts at purifying the brain calpains reported the presence of calpastatin in the brain extracts. Because of this and because of the widely varying polypeptides that were obtained in the different studies, we used two preparations of bovine brain to learn what kind of results would be obtained by using the procedures that we use to purify calpains from skeletal muscle and platelets and that carefully avoids any exposure to Ca2+ during the purification45. The elution profile off an anion-exchange column showed that brain calpastatin, like calpastatin in skeletal muscle and other tissues, elutes early, between 30 and 120mM KCl, from an anion-exchange column at pH 7.5 (Fig. 1). In contrast to the earlier results reported by Murachi and coworkers5,6 on rat brain, our studies found that bovine brain contains significant amounts of calpastatin activity, enough to inhibit all the m-calpain present (Fig. 1). It is unclear whether this is due to a difference between the two species, or to the different procedures used to separate and assay the calpains and calpastatin. Thus, it seems that Guroff 1 may have been fortuitously fortunate that he used rat brain instead of bovine brain tissue in his studies of Ca2+-dependent proteolytic activity because he likely would not have detected Ca2+-dependent proteolytic activity in crude extracts of bovine brain. We did not detect any µ-calpain in our extracts of bovine brain (Fig. 1). Depending on
Figure 1. Elution profile of a bovine brain extract that had been salted out P25-65, dialyzed, and loaded onto a DEAE-cellulose column at pH 7.5. The extract was from 2,247 g bovine brain tissue.
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the tissue, µ-calpain elutes at the same KCl concentration as calpastatin or at a KC1 concentration just slightly greater than that required to elute calpastatin45. Thus, our results do not eliminate the possibility that bovine brain contains a small amount of µ-calpain that coelutes with calpastatin off an anion-exchange column. Previous studies also have found very little µ-calpain in either rat5,6 or bovine brain.46, and a small amount in rat peripheral nerves47,48. Subcellular fractionation of rat glioma C649 or transformed Schwann cells50 found that these cell lines contained -8-1049 or ~0.550 times as much m-calpain activity as µ-calpain activity and that the m-calpain activity was largely (~ 70-75%) associated with the membrane fraction, whereas the µ-calpain fraction was largely (~80%) cytosolic. Hence, unless precautions are taken to ensure that all calpain is extracted from the membranes when homogenizing brain tissue, estimates of m-calpain activity in brain may actually underestimate the total amount of m-calpain present in brain. We obtained an average of 6 mg of partly purified m-calpain having 80- and 28-kDa subunits from 2000 g of bovine brain tissue in the two preparations with a 1,320-fold purification. Activity of bovine brain m-calpain was completed inhibited by leupeptin, iodoacetamide, and antipain, was unaffected by soybean trypsin inhibitor, and required ~900µM Ca2+ for half-maximal activity. We did not purify the bovine brain calpastatin, but it was as effective in inhibiting either bovine brain or bovine kidney m-calpain activity (100% inhibition) as turkey gizzard smooth muscle inhibitor was. In sum, although the calpain system in brain has not been as well characterized as in some other tissues, the available evidence indicates that brain tissue contains ~10-15% as much µ-calpain as m-calpain, and that the calpains in the brain do not differ substantially from the calpains in other tissues of the same organism ( i.e., they contain 28- and 80-kDa subunits, require the same [Ca2+] for proteolytic activity, etc.). It seems likely that a few cells in brain contain significant amounts of µ-calpain, but that the majority of the cells in the brain contain largely m-calpain and very little if any µ-calpain. Much of the m-calpain in the brain is associated with the cell membranes. On the other hand, there is very little definitive information available on brain calpastatin. Because calpastatin can exist in a number of different isoforms generated by different start sites of transcription /translation or alternative splicing events8 and because these different isoforms may have different calpain inhibitory properties51, it would be important to learn more about the nature of brain calpastatin and the types of calpastatin isoforms that are expressed in the brain.
THE CALPAIN SYSTEM IN NEUROMUSCULAR DISEASE-THE MUSCULAR DYSTROPHIES The Muscular Dystrophy Association(MDA) supports research in over 40 different kinds of muscular dystrophies ranging from diseases caused by defects in mitochondrial function and deficiencies in particular metabolic enzymes such as phosphorylase or maltase to endocrine abnormalities and diseases of the peripheral nerves (Charcot-Marie-Tooth disease, Friedreich’s ataxia) to the more widely recognized Duchenne and Becker’s muscular dystrophy. Many but not all of these muscular dystrophies are associated with abnormalities in Ca2+ homeostasis. A convincing amount of circumstantial evidence indicates that the loss of muscle mass or cytoskeletal structures in those diseases that are accompanied by changes in Ca2+ homeostasis is due to inappropriate or unregulated calpain activity52-56. Research spearheaded with support from the MDA has identified the genes responsible for or associated with many of the muscular dystrophies, but with the exception of limb girdle muscular dystrophy type 2A24(LGMD type IIA), none of the genes identified so far encode for the calpains directly. Hence, the role of the calpains in most neuromuscular diseases is an indirect one; loss of Ca2+ homeostasis due to a gene defect results in unregulated, elevated calpain activity and increased degradation of the myofibrillar proteins.
Duchenne and Becker’s Muscular Dystrophy Association of the calpains with loss of muscle mass in the muscular dystrophies has been studied most thoroughly in the Duchenne and Becker muscular dystrophies. Disruption of the gene encoding a large protein named dystrophin is responsible for Duchenne and Becker’s muscular dystrophy57-59. The 427kDa dystrophin molecule is associated with the
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intracellular face of the sarcolemma of skeletal muscle cells where it is part of a complex of 8 different cytoskeletal and transmembrane proteins that link the actin cytoskeleton to laminin in the extracellular matrix59. Mutations in the dystrophin gene lead to loss of part (Becker’s) or most (Duchenne) of the dystrophin molecule. Loss of dystrophin is thought to result in weakening of the sarcolemma60 and in loss of Ca2+ channel regulation61, both of which would result in an increased influx of extracellular Ca2+ into the muscle fiber62. Several studies have shown that the intracellular [Ca2+] is elevated approximately two-fold, from ~ 40nM to ~90nM27,28,62, in skeletal muscle from the mdx mouse, an animal model of Duchenne muscular dystrophy whose skeletal muscle lacks dystrophin. Even these elevated Ca2+ concentrations in dystrophin-deficient muscle, however, are much lower than the [Ca2+] required for activity of the calpains in in vitro assays (Table 1). It seems likely that the elevated Ca2+ concentrations in dystrophic muscle are sufficient to activate the mechanism that regulates calpain activity in vivo (see the previous discussion in The Calpain System), causing an increase in calpain proteolysis. Skeletal muscle fibers contain sufficient calpain to completely degrade the Z-disks and other key cytoskeletal proteins in these fibers in 10 min or less, so most of the calpain in skeletal muscle fibers is in an “off” state most of the time. Several studies have shown that the structural changes observed in dystrophic63 or denervated64 muscle are similar to the changes observed when myofibrils are incubated with calpain55, 65. The muscle degradation that occurs in mdx mice66,67 or in dystrophic68,69 or denervated70 chicken pectoralis muscle is prevented by treatment of these muscles or cultures from these muscles with leupeptin, which inhibits the calpains and several other Ca2+independent, cysteine proteases. In one of the studies, extracts from muscles that had been injected in vivo with leupeptin were assayed and shown to have decreased levels of calpain activity66. Combaret and coworkers53 reported that expression of m-calpain but not of components of the proteasome complex were increased in mdx mouse muscle. Spencer et al. 71, on the other hand, found that expression of both µ-calpain and m-calpain was the same in mdx and normal mouse muscle. The amount of total calpain protein, however, was greater in mdx mouse muscle than in the muscle from normal mice, and it was suggested that the increased calpain concentration in mdx muscle resulted from changes in posttranslational regulation71. Quantitative immunohistochemical studies showed that the amount of calpain was greater in muscle from patients afflicted with Duchenne or Becker’s muscular dystrophy72 and in muscle from patients with inflammatory myopathy73 than in muscle from normal patients. The increase in amount of immunologically reactive calpain was especially noticeable in the early stages of Duchenne dystrophy, suggesting that the calpains are involved in the early stages of muscle degradation in the muscular dystrophies72. Assays of calpain and calpastatin activity in muscles of normal and dystrophic (UM-X7.1) Syrian hamsters showed that dystrophic muscle in this animal model had higher m-calpain and calpastatin activities than normal muscle; activity of µ-calpain was slightly higher at 4 weeks of age but was the same at 10 weeks in muscle from the dystrophic and normal hamsters74. Assays of calpain activity in extracts of mdx and normal mouse muscle, however, found no difference in net calpain activity in the muscle from these two lines of animals, even though the mdx muscle had more calpain protein as measured by Western analysis than muscle from normal mice54. The assays were done on whole muscle extracts, so the difference between extractable calpain activity and extractable calpastatin activity in the muscle was measured. Consequently, it is not clear whether increased calpastatin negated any increase in calpain activity, or whether the calpain protein in the mdx mouse was not fully active proteolytically. In sum, four lines of evidence obtained both from animal models of muscle dystrophy and from human dystrophic patients indicate that the calpain system has an important role in the muscle degeneration that is the hallmark of the Duchenne and Becker’s muscular dystrophies. 1. Intracellular free Ca2+ concentration is elevated approximately 2-3fold in muscle lacking dystrophin, and this increase in free Ca2+ is associated with an increase in proteolytic degradation. 2. The structural changes that accompany muscle degeneration in the muscular dystrophies are mimicked by the effects of the calpains on myofibrillar proteins. 3. Treatment of degenerating muscle with compounds that inhibit the calpains prevents muscle degeneration in dystrophic or denervated muscle. 4. The
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expression and in some instances the activities of the calpains are elevated in dystrophic muscle.
Limb Girdle Muscular Dystrophy Type 2A and skm-Calpain The finding that disruption of the gene for skeletal muscle-specific calpain, skmcalpain, resulted in limb girdle muscular dystrophy type 2A(LGMD2A)24 has been confirmed by several laboratories75 and many of the mutations in the skm-calpain gene that result in LGMD2A have been characterized76.It has, however, been unclear how disruption of a gene encoding for a putative proteolytic enzyme could lead to a muscle wasting disease. This uncertainty has been exacerbated by failure to isolate the protein form of skm-calpain, despite many attempts by a number of laboratories. Hence, all information on the properties of skm-calpain has been obtained indirectly by study of its mRNA or expression of the cDNA for skm-calpain in cell lines. These studies have lead to the conclusion that the skmcalpain protein rapidly autolyzes and is turned over in muscle with a half-life of 27 min77, and that this rapid turnover accounts for failure to isolate the protein from muscle cells. Assays of human muscle biopsy samples, however, detected skm-calpain immunologically even though the biopsy samples had been at room temperature for over an hour75. Also, although the predicted amino acid sequence for skm-calpain indicates that it is a Ca2+dependent protease, it has been impossible to prevent its putative rapid autolysis by addition of large concentrations of EDTA, a Ca2+ chelator, or by including a variety of protease inhibitors in muscle extracts78. Finally, because the protein form of skm-calpain has not been isolated, antibodies to skm-calpain have all been elicited against peptides synthesized to duplicate areas of amino sequence that are unique to skm-calpain. Although these antibodies label the skm-calpain polypeptide in systems where the skm-calpain has been expressed from its cDNA, it is uncertain what they label in skeletal muscle. In the absence of skm-calpain protein, the only way to determine what antibodies to synthetic skm-calpain peptides label in skeletal muscle is to isolate the polypeptide labeled and sequence it to learn whether its sequence matches the predicted sequence for skm-calpain. Studies in our laboratory have found that different antibodies label a)phosphorylase, b)gelsolin, c)muscle-type 6phosphofructokinase, d) fructose 1,6-bisphosphatase, and e) skeletal-muscle-specific carbonic anhydrase. We have not yet obtained an antibody that, based on this criteria, labels skm-calpain in muscle tissue. Consequently, the nature of the skm-calpain polypeptide and its catalytic properties remain a mystery. It has been recently reported that skm-calpain deficiency may initiate apoptosis in muscle nuclei, and that apoptosis is the cause of the muscle wasting in LGMD2A79. Coexpression of skm-calpain and INBD in insect cells resulted in rapid degradation of INBD whereas INBD was stable and was not degraded when it was expressed alone. INBD is an inhibitor of the NF-NB-rel transcription family. Interaction of INB with NF-NB masks the nuclear localization signal on the p65 subunit of NF-kB, and prevents translocation of NFNB to the nucleus, where it initiates expression of genes involved in cell survival. Hence, it was suggested that, in the absence of skm-calpain, the IκB complexed to NF-NB is not degraded, the NF-kB is not translocated to the nucleus, and the nucleus initiates an apoptotic sequence. TUNEL-positive nuclei were observed in LGMD2A muscle that was not expressing skm-calpain, but not in muscle from patients expressing skm-calpain, including some Duchenne muscular dystrophy patients who were experiencing muscle atrophy but who were expressing skm-calpain79. Although the number of TUNEL-positive nuclei in the LGMD2A patients was small (0.27 to 0.65% of all nuclei), such apoptosis may be sufficient to induce the relatively mild (compared with Duchenne) muscle wasting characteristic of LGMD2A. The proposal offers the first mechanism to explain how disruption of a gene encoding a putative proteolytic enzyme could initiate a muscular dystrophy.
Other Neuromuscular Diseases There have been significant advances during the past 10 years in identifying genes and their protein products that are associated with different muscular dystrophies. In many instances, the genes encode for cytoskeletal proteins that are part of the dystrophin complex80-82. Disruption of the G-sarcoglycan gene, for example, is responsible for one of the limb-girdle muscular dystrophies, and loss of a number of other cytoskeletal membrane
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proteins 82 results in muscular dystrophy. Although these dystrophies have not as yet been studied as extensively as the Duchenne and Becker’s dystrophies have, it seems likely that loss of these cytoskeletal proteins also may lead to loss of Ca2+ homostasis and a calpain involvement in a manner similar to that observed for Duchenne and Beckers. Charcot-MarieTooth disease may involve five different gene defects, but one of the results of these defects is loss of myelin; the myelin basic protein is rapidly degraded by the calpains in in vitro assays.
ACKNOWLEDGEMENTS The research results described in this review were supported in part by grants from the Department of Agriculture(USDA)National Research Initiative Competitive grants program(98-03619; 98-01191); the Muscular Dystrophy Association; and by the Arizona Agriculture Experiment Station, Project 28, a contributing project to USDA Regional Research Project No. NC-131. We thank Janet Christner for her assistance in preparing a manuscript in camera-ready form under severe time constraints.
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41. M.N. Malik, A.M. Sheikh, M.D. Fenko, and H. Wisniewski, Purification and degradation of purified neurofilament proteins by the brain calcium-activated neutral proteases, Life Sci. 39: 1335-1343 (1996). 42. T. Hirao and K. Takahashi, Purification and characterization of a calcium-activated neutral protease from monkey brain and its action on neuropeptides, J. Biochem. 96:775-784 (1984). 43. N.L. Banik, E.L. Hogan, M.G. Jenkins, J.K. McDonald, W.W. McAlhaney, and M.B. Sostek, Purification of a calcium-activated neutral proteinase from bovine brain, Neurochem. Res. 8: 13891405 (1983). 44. U.J.P. Zimmerman and W.W. Schlaepfer, Multiple forms of Ca2+-activated protease from rat brain and muscle, J. Biol. Chem. 259:3210-3218 (1993). 45. V.F. Thompson and D.E. Goll, Purification of µ-calpain, m-calpain, and calpastatin from animal tissues. In: “Methods in Molecular Biology: Calpain Methods and Protocols”, J.S. Elce, ed., Humana Press, Inc., Totowa, NJ. pp.3-16 (2000). 46. S. Kubota, T. Onaka, H. Murofishi, N. Ohsawa, and F. Takaku, Purification and characterization of high Ca2+-requiring neutral proteases from porcine and bovine brains, Biochemistry 25:8396-8402 (1986). 47. K. Kamakura, S. Ishiura, H. Sugita, and Y. Toyokura, Identification of Ca2+-activated neutral protease (CANP) in the rat peripheral nerve, Biomed. Res. 3:91-94 (1982). 48. K. Kamakura, S. Ishiura, and H. Sugita, µ-Type calcium-activated neutral protease in the rat peripheral nerve, J. Neurosci. Res. 15167-173 (1986). 49. N.L. Banik, A.K. Chakrabarti, G.W. Konat, G. Gantt-Wilford, and E.L. Hogan, Calcium activated neutral proteinase (calpain) activity in C6 cell line: compartmentation of µ and m calpain, J. Neurosci. Res. 31:708-714 (1992). 50. N.L. Banik, G.H. DeVries, T. Neuberger, T. Russell, A.K. Chakrabarti, and E.L. Hogan, Calciumactivated neutral proteinase (CANP; calpain) activity in Schwann cells: immunofluorescence localization and compartmentation of µ and mCANP, J. Neurosci. Res. 29: 346-354 (1991). 51. G.H. Geesink, D. Nonneman, and M. Koohmaraie, An improved purification protocol for heart and skeletal muscle calpastatin reveals two isoforms resulting from alternative splicing, Arch. Biochem. Biophys. 356:19-24 (1998). 52. N.C. Kar and C.M. Pearson, A calcium-activated neutral protease in normal and dystrophic human muscle, Clin. Chim. Acta 73: 293-297 (1976). 53. L. Combaret, D. Taillandier, L. Voisin, S.E. Samuels, 0.Boespflug-Tanguy, and D. Attaix, No alteration in gene expression of the components of the ubiquittin-proteasome proteolytic pathway in dystrophin-deficient muscles, FEBS Lett. 393:292-296 (1996). 54. M.J. Spencer and J.G. Tidball, Calpain concentration is elevated although net calcium dependent proteolysis is suppressed in dystrophin-deficient muscle, Exp. Cell Res. 203: 107-114 (1992). 55. S. Ishiura, I. Nonaka, and H. Sugita, Ca2+-activated neutral protease: its degradative role in muscle cells, in: “Proceedings of the International Symp. on Muscular Dystrophy”, S. Ebashi, ed., Japan Medical Research Foundation Publication No. 16, University of Tokyo Press, Tokyo, Japan. pp.265-282 (1982). 56. J.P. Leonard and M.M. Salpeter, Agonist-induced myopathy at the neuromuscular junction is mediated by calcium, J. Cell Biol. 82:811-819 (1979). 57. E.P. Hoffman, R.H. Brown, Jr., and L.M. Kunkel, Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 51:9190928 (1987). 58. M. Koenig, A.P. Monaco, and L.M. Kunkel, The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein, Cell 53:219-228 (1988). 59. J.M. Ervasti and K.P. Campbell, Dystrophin and the membrane skeleton, Curr. Opin. Cell Biol. 582-87 (1993). 60. A. Menke and H. Jockusch, Extent of shock-induced membrane leakage in human and mouse myotubes depends on dystrophin, J. Cell Sci. 108,727-733 (1995). 61. P.R. Turner, R. Schultz, B. Ganguly, and R.A. Steinhardt, Proteolysis results in altered leak channel kinetics and elevated free calcium in mdx muscle, J. Membr. Biol. 133:243-251 (1993). 62. F.W. Hopf, P.R. Turner, W.F. Denetclaw, Jr., P. Reddy, and R.A. Steinhardt, A critical evaluation of resting intracellular free calcium regulation in dystrophic mdx muscle, Am. J. Physio1.271:C1325C1339 (1996). 63. M.J. Cullen and J . J Fulthorpe, Phagocytosis of the A band following Z line and I band loss. Its significance in skeletal muscle breakdown, J. Path. 138: 129-143 (1982). 64. M.J. Cullen and M.G. Pluskal, Early changes in the ultrastructure of denervated rat skeletal muscle, Exptl. Neurol. 56:115-131 (1977). 65. H. Sugita, S. Ishiura, K, Suzuki, and K. Imahori, Ca-activated neutral protease and its inhibitors: in vitro effect on intact myofibrils, Muscle & Nerve 3: 335-339 (1980).
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66. M.A. Badalamente and A. Stracher, Delay of muscle degeneration and necrosis in mdx mice by calpain inhibition, Muscle & Nerve 23:106-111 (2000). 67. J.H. Sher, A. Stracher, S.A. Shafiq, and J. Hardy-Stashin, Successful treatment of murine muscular dystrophy with the protease inhibitor leupeptin, Proc. Natl. Acad. Sci. 78:7742-7744 (1981). 68. A. Stracher, E.B. McGowan, and S.A. Shafiq, Muscular dystrophy: inhibition of degeneration in vivo with protease inhibitors, Science 200:50-51 (1978). 69. E.B. McGowan, S.A. Shafiq, and A. Stracher, Delayed degeneration of dystrophic and normal muscle cell cultures treated with pepstatin, leupeptin, and antipain, Exptl. Neurol. 50:649-657 (1976). 70. A. Stracher, E.B. McGowan, A. Hedrych, and S.A. Shafiq, In vivo effect of protease inhibitors in denervation atrophy, Exptl. Neuro1.66:611-618 (1979). 71, M.J. Spencer, D.E. Croall, and J.G. Tidball, Calpains are activated in necrotic fibers from mdx dystrophic mice, J. Biol. Chem. 270: 10909-10914 (1995). 72. T. Kumamoto, H. Ueyama, S. Watanabe, K. Yoshioka, T. Miike, D.E. Goll, M. Ando, and T. Tsuda, Immunohistochemical study of calpain and its endogenous inhibitor in the skeletal muscle of muscular dystrophy, Acta Neuropath. 89:399-403 (1995). 73. T. Kumamoto, H. Ueyama, R. Sugihara, E. Kominami, D.E. Goll, and T. Tsuda, Calpain and cathepsins in the skeletal muscle of inflammatory myopathies, Eur. Neurol. 37:176-181 (1997). 74. S. Kawashima, M. Nakamura, and M. Hayashi, Activities of calcium-activated proteases and its endogenous inhibitor in skeletal muscle of dystrophic hamster, Biol. Chem. Hoppe-Seyler 371:205210 (1990). 75. M.J. Spencer, J.G. Tidball, L.V.B. Anderson, K.M.D. Bushby, J.B. Harris, M.R. Passo-Bueno, H. Somer, M. Vainzof, and M. Zatz, Absence of calpain 3 in a form of limb-girdle muscular dystrophy(LGMD2A), J. Neurol. Sci. 146:173-178 (1997). 76. Y. Ono, H. Shimada, H. Sorimachi, I. Richard, T.C. Saido, J.S. Beckmann, S. Ishiura, and K. Suzuki, Functional defects of a muscle-specific calpain, p94, caused by mutations associated with limb-girdle muscular dystrophy type 2A, J. Biol. Chem. 273:17073-17078 (1998). 77. H. Sorimachi, N. Toyama-Sorimachi, T.C. Saido, H. Kawasaki, H. Sugita, M. Miyasaka, K-i. Arahata, S. Ishiura, and K. Suzuki , Muscle-specific calpain, p94, is degraded by autolysis immediately after translation, resulting in disappearance from muscle, J. Biol. Chem. 268: 10593-10605 (1993). 78. K. Kinbara, S. Ishuira, S. Tomioka, H. Sorimachi, S-Y. Jeong, S. Amano, H. Kawasaki, B. Kolmerer, S. Kimura, S. Labeit, and K. Suzuki, Purification of native p94, a muscle-specific calpain, and characterization of its autolysis, Biochem. J. 335:589-596 (1998). 79. S. Baghdiguian, M. Martin, I. Richard, F. Pons, C. Astier, N. Bourg, R.T. Hay, R. Chemaly, G. Halaby, J. Loiselet, L.V.B. Anderson, A. Lopez de Munain, M. Fardeau, P. Mangeat, J.S. Beckmann, and G. Lefranc, Calpain 3 deficiency is associated with myonuclear apoptosis and profound perturbation of the IkBa/NF-kB pathway in limb-girdle muscular dystrophy type 2A, Nature Medicine 5:503-511 (1999). 80. K. Ohlendieck and K.P. Campbell, Dystrophin-associated proteins are greatly reduced in skeletal muscle from mdx mice, J. Cell Biol. 115:1685-1694 (1991). 81. A.A. Hack, M.E. Groh, and E.M. McNally, Sarcoglycans in muscular dystrophy, Microsc. Res. Tech. 48:167-180 (2000). 82. R.M. Grady, R.M. Grange, K.S. Lau, M.M. Maimone, M.C. Nichol, J.T. Stull, and J.R. Scanes, Role for a-dystrobrevin in the pathogenesis of dystrophin-dependent muscular dystrophies, Nature Cell Biol. 1:215-220 (1999).
THE ROLE OF CALPAIN PROTEOLYSIS IN CEREBRAL ISCHEMIA
Dwaine F. Emerich and Raymond T. Bartus Alkermes, Inc. Cambridge, MA 02139
INTRODUCTION Strokes occur when blood flow is acutely restricted within a circumscribed region of the brain. The immediate alteration in blood flow produces an ischemic reaction and unleashes a devastating series of cellular events capable of producing significant neuronal damage and/or cell death. Ischemic brain injury is a major public health concern that ranks third behind cancer and cardiovascular disease in medically-related deaths in the United States.1 Moreover, stroke is second only to Alzheimer's disease as a cause of neurological impairment. Although preventative care and continued refinements in medical practice have decreased both the rate of stroke and the extent of mortality, nearly 500,000 stokes will occur in the United States alone this year alone.2 Tragically, approximately 150,000 of these people will die. Effective treatments for stroke are currently limited, though there has been an explosion in our understanding of the biochemical events that occur following ischemia. The ever growing list of intracellular changes that are temporally and perhaps causally related to the ensuing neural damage provides optimism that pharmacological strategies may be developed to prevent, minimize or reverse that pathological cascade. The target of current pharmaco-therapies is the ischemic penumbra; a highly vulnerable area of tissue that exists between the ischemic core and the normally perfused brain. While the central core of ischemic tissue is virtually devoid of blood flow and will certainly die unless reperfusion is immediately achieved, the pen-infarcted penumbral region consists of a zone of variably reduced blood flow and compromised, but salvageable tissue.3,4 Animal and human imaging studied demonstrate that a pathological cascade of intracellular events occurs within this tissue that will eventually compromise its Function and eventually lead to cellular death. These events do, however, require time to manifest themselves providing a temporal window of opportunity for treating stroke, after the schemic events but prior to the development of irreversible neuronal death. The opportunity to devise innovative therapies for stroke is tightly linked to an accurate understanding of the cascade of cellular, molecular and biochemical events that occur immediately following and secondary to the ischemic event. While our understanding of the sequence of events between the ischemic episode and the delayed neuronal death is not completely understood, it has been clear for some time now that n elevation in intracellular calcium triggers a variety of intracellular events that, when left
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uncontrolled, are capable of producing marked pathological changes in vulnerable neurons.6,7 A growing body of evidence suggests that calcium-dependent proteolyis is a likely mediator of cell death following ischemia. By its nature, proteolysis produces irreversible degradation or modification of cellular proteins. When it occurs in the absence of its normal regulatory controls (as can occur in stroke), the irreversible nature of its effects can have significant pathological consequences. Although a variety of different lysosomal and cytosolic proteases could be considered pathogenic candidates, the roperties and localization of the calciumactivated calpains have drawn the most attention.8,9 Interest in the role of calpain as a mediator of ischemic damage has been intensified by two sets of observations. First, brain levels of numerous calpain substrates are reduced after an ischemic event. Secondly, the exogenous administration of calpain inhibitors attenuates neuronal damage in animal models of ischemia. Together, these data suggest that calpains might provide a useful pharmacological target for treating ischemic cell death. This chapter reviews the current data implicating calpain proteolysis in the neuronal pathology that results from cerebral ischemia. First, the general properties of calpain are discussed with particular attention to its cellular location, regulation, and proteolytic targets. Next, the evidence for calpain proteolysis secondary to ischemia is discussed. Finally, attempts to attenuate ischemia-induced degeneration by pharmacologically inhibiting calpain are reviewed. Collectively, these data point to an emerging theme that calpain inhibition might be a viable therapeutic strategy for minimizing the neuronal damage caused by ischemia.
GENERAL PROPERTIES OF CALPAINS Calpain refers to a class of homologous cytosolic thiol proteases that are distributed in all animal cells examined to date. 10 Calpains are non-lysosomal proteases, that normally exist in an inactive or quiescent state, but are activated by increased intracellular calcium. While at least six isozymes have been identified thus far, two major forms, calpain I and II, (also referred to as u- and m-calpain) are ubiquitously and constituitively expressed in the mammalian brain. Since the vast majority of data regarding calpain involves these two forms, this chapter will focus exclusively on them. Within the brain, relatively modest differences exist in the subcellular distribution of calpain, with calpain I found primarily in dendrites and cell bodies, and calpain II typically associated with axons and glial cells.11-13 Both calpain I and II are expressed as heterodimers, with an 80 kDa catalytic subunit and a smaller 30 kDa subunit that helps regulate its activity. The primary difference between these two isozymes resides within the calcium binding domains of their catalytic subunits which, in turn, affect their relative affinity for calcium.11 In vitro, calpain I binds calcium relatively easily, requiring only micromolar concentrations for activation, while calpain II exhibits poorer calcium binding properties and requires millimolar concentrations for activation. Given the relatively high levels of calcium needed for activation, the normal physiological role of calpain I was initially perceived to be limited, while the role of calpain II was believed to be nonphysiological altogether. However, the realization that the threshold for activation via calcium binding is considerably less in the presence of phospholipids (which are abundant in vivo) makes it likely that the requirements initially determined, in vitro, are much higher than what is truly necessary, in vivo.14 This realization not only made it more reasonable to assume that calpain played a role in normal physiological processes but also made it much more likely that calpain could play a prominent role in pathological conditions including ischemia. Calcium exerts potent regulatory control on calpain activity by binding to specific domains on both the catalytic and regulatory subunits. Calcium binding to cal ain, in turn, is modulated by calmodulin, providing additional control over calpain's activation.12 In its normally inactive
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state, calpain exists with its catalytic and regulatory subunits arranged in a conformational state which prohibits substrate binding and proteolytic activity. Calcium binding stimulates a conformational change in calpain leading to a rapid sequence of events including autolysis and the migration of calpain from the cytosol to the phospholipid membrane.11,15 The majority of the calpain is then released from the membrane into the cytosol, allowing the catalytic and regulatory subunits to dissociate.15 Thus, a soluble, highly active form of calpain is derived which requires much lower (physiologic) levels of calcium for continued activity and is much less responsive to most of its normal modulators (e.g., calcium, calmodulin, phospholipids). In addition to calcium concentrations, the proteolytic activity of calpain is regulated by the endogenous inhibitor protein, calpastatin. Calpastatin is highly specific for both forms of calpain and exists as both 70 kDa and 110 kDa forms, with internal repeat sequences that reversibly bind to calpain's proteolytic domain. 11,15 Because most tissues contain high levels of both calpastatin and calpain, calpastatin is in a unique position to play a critical role in the inhibition of calpain activity. Because the formation of the calpain/calpastatin complex is calcium-dependent, it is likely that it occurs only after calpain activation. It has been suggested that calpastatin is involved in the regulation of calpain under conditions where calcium concentrations increase rapidly and exceed physiologic levels.11 This implies that calpastatin plays a crucial role in limiting the proteolytic activity of calpain under conditions where all the other control mechanisms have failed. Not withstanding this possibility, there is clear evidence that under extreme pathologic conditions, calpastatin's regulatory controls are insufficient. In these instances, widespread calpain proteolysis persists unchecked, inducing irreversible and potentially lethal changes to multiple cellular proteins. The substrates that calpain affects are both numerous and diverse, including many cytoskeletal proteins (such as spectrin, MAP2, tau, and neurofilament proteins), membrane proteins (including epidermal growth factor receptor, ryanodine receptor), and various regulatory cell signaling proteins (such as protein kinase C, calmodulin-binding proteins, and G-proteins). Because uncontrolled, simultaneous proteolysis of these multiple substrates would seriously compromise the vitality of any cell, calpain proteolysis has become a focal point in the effort to identify potential pathogenic variables following ischemia. Although much is known about the structural and enzymatic properties of calpain, the normal functions of calpain have yet to be clearly defined (9 for a discussion). Calpain is present in all vertebrates, is highly conserved across species and has been characterized in a wide variety of cell types and tissues. The ubiquitous distribution of calpain has led to it being associated with a wide variety of different calcium-related proteolytic changes. This ubiquitous expression together with the diversity of calpain substrates imply an important, but poorly elucidated physiologic role. As a general rule, calpains cleave a limited number of specific sites in endogenous proteins. The nature of calpain proteolysis is therefore limited and tends to produce large polypeptide fragments rather than digesting proteins to small peptides and amino acids.16 Still, partial proteolysis can have profound cellular effects. Even limited proteolysis by calpain can cause destabilization of the structural integrity of proteins, making them more susceptible to attack by other cellular proteases. Moreover, the large polypeptide fragments produced by calpains may retain their enzymatic activity but not remain subject to the controls that regulate the intact protein, For example, calpain cleavage of protein kinase C produces a kinase that is constituitively active but less dependent upon normal regulatory cofactors, including calcium and phospholipids.17 Calpain also cleaves a limited number of sites in cytoskeletal proteins such as MAP2, spectrin, and neurofilament proteins. Even so, the large remaining polypeptide fiagments typically lose their ability to cross-link or bind to their normally associated proteins. These changes can have dramatic effects, resulting in the remodeling of disassembly of the cytoskeleton, Finally, calpain can make specific cleavages in hormone receptors and
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membrane-bound proteins such as calcium channel proteins. These cleavages, however, tend to have little or no effects on the function or activity of the receptor. Together, these data indicate that calpain participates in events linked to calcium-mediated changes in cellular structure and function. Consistent with this concept is evidence for an important role of calpain during neuronal differentiation of PC1216 and SH-SY-5Y cells.18 Similarly consistent are the data supporting the intriguing hypothesis that calpain participates in mediating long term memory by physically altering synaptic strength within the brain.19 Finally, it is interesting that levels of both calpain isozymes fall sharply during early post-natal development (when relatively speaking- most neuronal differentiation has occure).20 One might expect, therefore, that calpain plays an important role in the adult nervous system during axonal sprouting, regeneration, and neurite extension and retraction. Considerably more work will be required, however, before calpain's role in these phenomena might be more clearly established.
THE ROLE OF CALPAIN IN CERBRAL ISCHEMIA A significant amount of the tissue loss that occurs following an ischemic episode occurs after the initial alteration in blood flow. Over time, a substantial amount of cellular necrosis develops because of the sequence of biochemical events triggered by the initial perturbation. In recent years, much of the biochemical sequelae have been elucidated, consistently implicating excessive intracellular calcium as a key variable. Since it is commonly believed that calcium itself is not directly responsible for the majority of the secondary necrosis associated with cerebral ischemia, efforts have focused on the specific calcium-dependent events that might themselves be cytotoxic. Several lines of evidence suggest that calcium-dependent activation of calpain plays a key role in the etiology of cerebral ischemia. Studies in both global and focal ischemia have implicated calpain proteolysis as an important pathogenic event with two general types of studies providing the bulk of the evidence. One type of study tracks the initiation and progression of calpain proteolysis relative to cell loss. The second type attempts to reduce the ischemia-induced pathology by pharmacologically inhibiting calpain activity. The evidence supporting a contributory role for calpain activation in the pathology of cerebral ischemia is discussed below.
Foal ischemia Focal ischemia, involves a significant reduction of blood flow to a circumscribed or focal region of the brain. It is most often associated with the pathogenesis which accompanies stroke. The rate at which neurons and glia die following focal ischemia varies with the severity and type of vascular ischemia employed. Cell death typically requires several hours to complete its course. Tracking the initiation and degree of calpain proteolysis can be accomplished by quantifying the breakdown of spectrin, a calpain substrate. Spectrin is a cytoskeletal protein that contributes to the shape and structural integrity of the cell, as well as to the regulation and motility and function of various transmembrane proteins.21 Using either polyclonal antibodies in conjunction with Western blots, or monoclonals specific to the spectrin breakdown products, it is possible to accurately quantify when calpain becomes proteolytically active. This time point can then be compared to when neurons first begin to show measurable damage, thus providing insight into the temporal sequence of intracellular, ischemia-induced pathogenic events. Recent studies have characterized changes in spectrin breakdown products and the evolution of ischemia-induced infarction following a middle cerebral artery (MCA) occlusion in rats.22-24 All provide evidence that calpain-induced proteolysis of spectrin begins soon after the
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ischemia (within an hour) and precedes both an ensuing metabolic crisis24 as well as the development of a necrotic infarct.22,23 Importantly, one study quantified cell loss in both the striatum and cortex and observed substantial elevations in calpain-induced spectrin proteolysis well in advance of significant cell loss.23 Significant cell hypertrophy and changes in cell shape occurred together with increased spectrin breakdown during the first hour of MCA occlusion, with continuing and more dramatic changes in these markers occurring over the next several hours. While some evidence of cell loss was seen at 1 hour, the vast majority of cells, even those in the center of the infarcted region, remained intact and viable through 6 hours. Therefore, the increase in spectrin breakdown products was temporally correlated with structural changes in size and shape of the neurons prior to their necrotic demise. These data clearly indicate that calpain proteolysis occurs rapidly following occlusion and preceeds significant cell loss. Together, these data satisfy an important prerequisite toward establishing a cause-effect relationship between calpain proteolysis and the neural damage and loss associated with stroke. Studies which histologically track the time course of expression of calpain within ischemic tissue have provided confirmation of the relationship between calpain activation and cell death. A recent study used immunocytochemistry to study the presence of calpain within neurons of rat cortex following occlusion of the MCA for 3 hours.25 In control animals, only 2% of the neurons examined were calpain-positive, indicating a normally low level of endogenous calpain activation. In contrast, an orderly, time-related increase in the number of calpain-positive cells was observed within the cortex of animals subjected to focal ischemia. At three hours following the ischemic event, 31% of the cells were calpain-positive; a number that increased to 41% at 6 hours and 88% at 24 hours. Double-labeling experiments determined that approximately 80% of the stained cells were neurons, as opposed to glia. While these studies did not confirm the temporal relationship between cell loss and calpain expression, they do demonstrate a pattern of calpain activation within infarcted neurons that is generally in accordance with the detection of the breakdown products (spectrin) in cerebral ischemia. This line of research was substantially bolstered by the development of inhibitors that are both selective for calpain and able to cross neuronal membranes. Calpain inhibitors including CX216, AK275 and AK295 have been developed that are competitive inhibitors of both type I and type II calpain, have Ki values within the nanomolar range, and poorly inhibit other classes of proteases.23,26 The administration of calpain inhibitors to rats subjected to focal ischemia has provided more direct evidence for an important pathologic role of calpain in stroke. In Vivo studies have demonstrated significant infarct reductions following both pre-ischemia intravenous treatment27 of a calpain inhibitor and post-ischemia intracarotid treatment.28 Substantial reduction in infarct volume (i.e., 75%) was also observed when a calpain inhibitor was infused directly onto the ischemic tissue, beginning three hours after the occlusion, thus eliminating many interpretive possibilities often associated with systemic delivery of drugs 29 which are incompletely characterized. These pharmacologic studies collectively demonstrate that inhibition of calpain is sufficient to substantially reduce the volume of infarct. Moreover, the fact that substantial protection can be achieved by direct application of a specific calpain inhibitor 3 hours post-ischemia supports the hypothesis that calpain proteolysis contributes to much of the secondary necrosis that follows focal ischemia. Interestingly, calpain inhibition was recently shown to effectively inhibit the spread of tissue damage that occurred secondary to cortical aspiration in rats.30 These findings implicate calpain activation in the underlying secondary pathology that occurs following a wide-range of neural insults and support the potential use of calpain inhibition as a general means of minimizing neural degeneration secondary to trauma. While the results of these experiments clearly indicate the potential of calpain inhibition for reducing the pathological consequences of ischemia, many of the existing inhibitors are unsuitable for clinical evaluation due to poor solubility in vehicles suitable for injection,
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inadequate pharmacokinetic profile, poor penetration across the BBB and an inabiity to permeate neuronal membranes. Markgraf and colleagues31 recently reported that systemic (intravenous) administration of the CNS-penetrating calpain inhibitor MDL 28,170 significantly inhibited brain protease activity and reduced infarct volume in rats. Maximal inhibition of protease activity was observed 30 minutes after injection of MDL 28,170. Time-course studies further revealed that infarct volume was maximally reduced (80%) when the drug was given 3 hours following ischemia. Impressively, even when the drug was administered 6 hours following the initial perturbation, infarct volume was still decreased 46%. Further improvements in the pharmaco-kinetic and biodistribution profiles of selective calpain inhibitors could conceivably result in clinically effective calpain inhibition.
Global Ischemia In contrast to focal ischemia, global ischemia refers to conditions when the normal oxygen and glucose supply to the forebrain is transiently reduced to near zero levels, such as during reversible cardiac arrest or during episodes of near drowning or asphyxiation. Animal models of global ischemia typically involve stopping blood flow to the entire forebrain by transiently occluding the major arteries of the neck (i.e., carotid and basilar). The brain damage associated with global ischemia involves two unique characteristics: a "delayed cell death" phenomenon (typically taking several days to manifest itself) and selective vulnerability (whereby pyramidal neurons in the CA1 area of the hippocampus exhibit the greatest pathology). As with focal ischemia, evidence supporting an important cytotoxic role for calpain has been generated by comparing the timing of calpain proteolysis relative to that of neuronal death, as well as by pharmacologically inhibiting calpain. Several studies using a gerbil model of global ischemia demonstrated that significant calpain-induced spectrin proteolysis occurs days prior to the characteristic, selective degeneration of CA1 neurons, suggesting a possible cause-effect relationship between calpain proteolysis and eventual cell death. 32-34 In a more recent study (using rats), changes in spectrin breakdown were quantified over time, in parallel with the functional status of the CA1 neurons (using ex vivo synaptic transmission) and the histologically-defined condition of the pyramidal cells.35 This study confirmed that calpain proteolysis precedes evidence of cell death following global ischemia in the rat, and more importantly demonstrated that the timing of calpain proteolysis was closely associated both with the loss of pyramidal cell function and the earliest evidence of subcellular damage to the neurons. These data have recently been confirmed and extended in a monkey model of global ischemia which used antibodies specifically directed at activated calpain.36 Increased PIP2 immunoreactivity and increased intracellular free calcium content in CA1 neurons was observed to closely parallel calpain activation and occur prior to the loss of CA1 neurons. Electron microscopy revealed that calpain activation was particularly striking at the level of the lysosomal membrane. Together, these data raise the interesting possibility that calcium-induced calpain activation, with the aid of PIP2, results in the release of lysosomal constituents that contribute to the ensuing neuronal degeneration that occurs in ischemia. Calpain inhibitors also have protected CA1 neurons from the effects of global ischemia, further corroborating an important pathogenic role for calpain proteolysis. Pre-ischemia infusions of general calpain inhibitors into the ventricular system of rats salvaged CA1 neurons,35,37 reduced spectrin breakdown products35 and protected hippocampal synaptic transmission.35 Significant protection of CA1 neurons was also achieved when small amounts of a selective calpain inhibitor were infused directly into the most vulnerable area of the CA1 following global ischemia.38 Protection near the infusion site ranged from 40 to 100% whereas the contralateral hippocampus exhibited complete cell loss. Two recent studies also demonstrated that systemic (intravenous)39 administration of a selective calpain inhibitor (MD
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28,170) and a calpain inhibitor entrapped within liposomes40 produced significant neuroprotection of CA1 neurons. Together, these data offer compelling evidence that, despite the numerous pathologic events that occur following global ischemia, inhibition of calpain is sufficient to product significant neuroprotection.
CONCLUSIONS During the past 10 years, the number of biochemical events that follow ischemia has grown providing a lengthy list of intracellular changes that are temporally and perhaps causally related to the ensuing neural damage. As our knowledge of the sequence of cellular events underlying ischemia has grown, so has the list of possible pharmacological strategies aimed at preventing, minimizing or reversing that pathological cascade. A disregulation in calcium function has been consistently implicated in cerebral ischemia. While numerous factors might be attributed to the calcium disregulation, the end result is essentially the same. Because calcium levels are normally orders of magnitude greater outside cells than inside, calcium disregulation at the cellular level typically leads to excessive and persistent elevations of calcium within the cellular cytoplasm. This necessarily leads to unregulated calpain proteolysis which compromises the integrity of the cell's phospholipid membrane, interferes with exclusion of calcium through receptor/ion channel complexes, reduces transport of essential cell products to and from the cell body and terminal areas, impairs numerous signaling events within the cell, modifies certain transcription factors and possibly interacts with numerous other pathogenic events, including activation of an immune response or inflammatory reaction within the brain (see 8 for a review). When allowed to proceed unchecked for any extended period, any one of these events could be expected to impair the viability of the cell. When several of these events are induced simultaneously, their combined effects must necessarily be pathologic. At the same time, however, other intracellular events are also implicated as pathogenic in acute neurodegeneration (e.g., nitric oxide, lipid peroxidation and other forms of free radical damage) and might not fit as neatly into a strict calpain hypothesis.7 Thus, these data must eventually be integrated into a more complete and unifying, calpain-oriented hypothesis. Similarly, it must be recognized that several non-calpain pharmaceutical approaches have achieved a level of neuroprotection which approximates that achieved with calpain inhibitors.41,42 In fact, when one considers all the work published over the past several years with animal models of neurodegeneration, it seems remarkable that a number of very different therapeutic approaches have reported neuroprotective effects in the range of 40 to 70%, while none have yet consistently or substantially outperformed any other. It seems likely that one of two very different possibilities may explain this circumstance. First, the data may reflect the fact that a relatively high threshold of cellular destruction might be required before neurons are committed to die, and therefore, blocking any one of several parallel pathways is sufficient to shift the balance toward neuropreservation. Alternatively, it may be that the animal models commonly employed are far too forgiving. That is: a) they may not be able to differentiate between drugs that work effectively from those that do not, and b) therefore, certain drugs may appear to be effective in animal models, but ultimately will do very poorly when tested in the clinic. Only after many more clinical trials are completed, which employ sufficiently short perturbation-to-treatment intervals (i.e., < 4 hours), will it be possible to determine which of those two possibilities is more accurate. A second issue is whether necessary improvements in current, peptide-based calpain inhibitors can be made to fulfill the therapeutic implications of the calpain hypothesis. Even if one accepts all of the tenets of the calpain hypothesis, it remains uncertain whether effective calpain inhibitors might ever be developed into usable drugs for human diseases. One or more limitations in bioavailability with existing inhibitors, involving their relative insolubility in
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vehicles suitable for injection, pharmacokinetic profile, penetration across the BBB and ability to permeate neuronal membranes, continue to challenge drug development programs. To resolve this issue, it will be necessary to either: a) determine whether the presumed changes required in the physico-chemical properties of current peptide-based inhibitors are consistent with enhancing or even maintaining their potency and selectivity toward calpain, b) discover and develop smaller, completely different, non-peptide drugs that have sufficient potency and specificity against calpain, but a superior bioavailability profile, compared to current inhibitors, or c) develop innovative drug delivery techniques that circumvent the bioavailability limitations of existing drugs.
REFERENCES 1. R Bonita, Epidemiology of stoke, Lancet 339:3420350 (1992). 2. RD. Brown, J.P. Whisnant, J.D. Sicks, W.M. O’Fallon and D.O. Wiebers, Stroke incidence, prevalence, and survival. Secular trends in Rochester, Minnesota, through 1989. Stroke 27:373-380 (19%). 3. J. astrup, B.K. Siesjo, and L. Symon, Thresholds in cerebral ischemia - The ischemic penumbra, Stroke 12:723-725 (1981). 4. W.I. Rosenblum. Histopathological clues to the pathways of neuronal death following iscehmia/hypoxia, J. Neurotr 14:313-326 (1997). 5. W.-D. Heiss, M. Huber, G.R Fink. K. Herholz, U. Pietrzyk, R Wagner, and K. Wienhard, Progressive derangement of periinfarct viable tissue in ischemic stroke, J. Cereb. Blood Flow Metab., 12:193-200 (1992). 6. B.K. Siesjo and F. Bengtsson, Calcium fluxes, calcium antagonists, and calcium-related pathology in brain ischemia, hypoglycemia, and spreading depression: A unifying hypothesis, J. Cereb. Blood Flow Metab. 9: 127 (1989). 7. D.F. Emerich, and RT. Bartus, Intracellular events associated with cerebral ischemia, in: Stroke Therapy Basic, Preclinical and Clinical Directions, L.P. Miller, ed., Wiley-Liss, Inc., New York, NY (1999). 8. RT. Bartus, The calpain hypothesis of neurodegeneration: Evidence for a common cytotoxic pathway, Neuroscientist 3:3 14-327 (1997). 9. RT. Bartus, Calpain Inhibition: A common therapeutic rationale for treating multiple neurodegenerative conditions? Bar PR, Beal MF, eds. Neuroprotection: Fundamental and Clinical Aspects, Marcel Dekker, Inc., New York, NY pp71-86 (1997). 10. H. Sorimachi, T.C. Saido, and K. Suzuki, New era of calpain research. Discovery of tissue-specifi c calpains, FEBS Lctters 1994;343:1-5. 11. E. Melloni and S. Pontremoli, The calpains, TINS 12:438 (1989). 12. P. Seubert and G Lynch, Plasticity to pathology: Brain calpains as modifiers of synaptic structure. In Intracellular Calcium Dependent proteolysis, RL. Mellgren and T. Murachi, eds., CRC Press, Boca Raton (1990). 13. K. Onizuka, M. Kunimatsu, Y. Ozaki, K. Muramatsu, M. Sasaki, and H. Nishino, Distribution of u-calpain proenzyme in the brain and other neural tissues in the rat, Brain Res. 697:179-186 (1995). 14. K. Suzuki and S.. Ohno, Calcium activated neutral protease structure-function relationship and functional implications, Cell Struct. Funct 156:1-6 (1990). 15. K. Suzuki, H. Sorimachi, T. Yoshizawa, K. Kinbara, and S. Ishiura. Calpain: novel family members, avtivation and physiological function, Biol. Chem Hoppe-Seyler 376 (1995). 16. T. Saido, H. Sorimachi. and K. Suzuki, Calpain: New perspectives in molecular diversity and phusiologicalpathological involvement, FASEB J 8:814 (1994). 17. A. Kishimoto, K. mikawa, K. Hashimoto, I. Yasuda, S. Tanaka, M. Tominaga, T. Kuroda, and Y. Nishizuka, Limited proteolysis of protein kinase C subspecies by calcium-dependent neutral protease (calpain), J. Biol. Chem. 264:4088-4092 (1989).
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18. T.B. Shea, C.M. Cressman, M.J. Spencer, M.L. Beermann, and RA. Nixon, Enhancement of neurite outgrowth following calpain inhibition is mediated by protein kinase C, J. Neurochem. 517-527 (1995). 19. G. Lynch, and M. Baudry, The biochemistry of memory: A new and specific hypothesis, Science 224: 10571063 (1984). 20. RA. Nixon, Calcium-activated neutral proteinases as regulators of cellular function: Implications for Alzheimer's disease pathogenesis, Ann. N. Y. Acad. Sci. 568: 198-206 (1989). 21. S.R Goodman, and I.S. Zagon, The neural cell spectrin skeleton: a review, Invited Review, Ameri. Physiol. Soc. C347 (1986). 22. S.C. Hong, G. Lanzino, Y. Goto, S.K. Kang, F. Schottler, N.F. Kassell, and K.S. Lee, Calcium-activated proteolysis in rat neocortex induced by transient focal ischemia, Brain Res. 661:43 (1994). 23. RT. Bartus, RL. Dean, K. Cavanaugh, D. Eveleth, D. Carriero and G. Lynch, Time-related neuronal changes following middle cerebral artery occlusion: Implications for therapeutic intervention and the role of calpain, J. Cereb. Blood Flow Metab. 15:969 (1995). 24. H. Yao, M.D. Ginsberg, D.D. Eveleth, J.C. LaManna, B.D. Watson, O.F. Alonso, J.Y. Loor, J.H. Foreman, and R Busto, Local cerebral glucose Utilization and cytoskeletal proteolysis as indices of evolving focal ischemic injury in core and penumbra, J. Cereb. Blood Flow Metab. 15:398 (1995). 25. M. Liebetrau, B. Staufer, E.A. Auerswald, D. Geiger, H. Fritz, C. Zimmerman, T. Pfefferkorn, and G.F. Hamann, Increased intracellular calpain detection in experimental focal cerebral ischemia, NeuroReport 10:529-534 (1999). 26. Z. Li, G.S. Patil, and Z.E. Golubski, Peptide alpha-ketoester, a-ketoamid, and alpha-ketoacid inhibotirs of calpain and other cysteine proteases. J. Med. Chem. 36:3472-3480 (1993). 27. S.-C. Hong, Y. Goto, G. Lanzino, B.A. Soleau, N.F. Kassell, and K.S. Lee, Neuroprotection with a calpain inhibitor in a model of focal cerebral ischemia, Stroke 25:3:663 (1994). 28. RT. Bartus, N.J. Hayward, P.J. Elliott, S.D. Sawyer, K.L. Baker, RL. Dean, A. Akiyama, J.A. Straub, S.L. Harbeson, Z. Li, and J. Powers, Calpain inhibitor AK295 protects neurons from focal brain ischemia: Effects of postocclusion intra-arterial administration, Stroke 25:11:2265-2270 (1994). 29. RT. Bartus, K.L. Baker, A.D. Heiser, S.D. Sawyer, RL. Dean, P.J. Elliott, and J.A. Straub, Postischemic administration of AK275, a calpain inhibitor, provides substantial protection against focal ischemic brain damage, J. Cereb. Blood Flow Metab. 14:537-544 (1994). 30. RT. Bartus, E.-Y. Chen, G. Lynch, and J.H. Kordower, Cortical ablation induces a secondary calciumdependant pathogensis which can be reduced by inhibiitng calpain, Exper. Neurol. 155:315-326 (1999). 31. C. Markgraf, N.L. Velayo, M.P. Johnson, D.R McCarty, S. Medhi, J.R. Koehl, P.A. Chmielewski, and M.D. Linnik, Six-hour window of opportunity for calpain inhibition in focal cerebral ischemia in rats, Stroke 29:152-158 (1998). 32. P. Seubert, K. Lee, and G. Lynch, Ishemia triggers NMDA receptor-linked cytoskeletal proteolysis in hippocampus, Brain Res. 429:366-370 (1989). 33. T.C. Saido, M. Yokota, S. Nagao, I. Yamaura, E. Tani, T. Tsuchiya, K. Suzuki, and S. Kawashima, Spatial resolution of fodrin proteolysis in postischemic brain, J. Biol. Chem. 268:33:25239 (1993). 34. J.M. Roberts-Lewis, M.J. Savage, V.R Marcy, L.R Pinsker, and R. Siman, Immunolocalization of Calpain I-mediated spectrin degradation to vulnerable neurons in the ischemic gerbil brain, J. Neurosci. 14:6:3934 (1994). 35. RT. Bartus, R Dean, S. Mennerick, D. Eveleth, and G. Lynch, Temporal ordering of pathogenic events following transient global ischemia, Brain Res. 790:1-13 (1998). 36. T. Yamashima, T.C. Saido, M. Takita, J. Miyazawa, A. Miyakawa, H. Nishijyo, J. Yamashita, S. Kawashima, T. Ono and T. Yoshioka, Transient brain ischemia provokes Calcium, PIP2 and calpain responses prior to delayed neuronal death in monkeys, Eur. J. Neurosci. 8:1932-1944 (19%). 37. A. Rami and J. Krieglstein, Protective effects of calpain inhibitors against neuronal damage caused by cytotoxic hypoxia in vitro and ischemia in vivo, Brain Res. 609:67-70 (1993). 38. RT. Bartus, P.J. Elliott, N.J. Hayward, R.L. Dean, S. Harbeson, J.A. Straub, Z. Li, and J.C. Powers, Calpain as a novel target for treating acute neurodegenerative disorders, Neurological Res. 17:249-258 (1995).
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39. P. Li, W. Howlett, Q.P. He, H. Miyashita, M. Siddiqui, and A. Shuaib, Postischemic treatment with calpain inhibitor MDL 28170 ameliorates brain damage in a gerbil model of global ischemia, Neurosci. Lett. 247: 17-20 (1998). 40. M. Yokata, E. Tani, S. Tsubuki, I Yamura, I. Nakagaki, S. Hori, and T.C. Saido, Calpain inhibitor entrapped in liposome rescues ischemic neuronal damage, Brain Res. 819:8-14 (1999). 41. M.D. Ginsberg, Neuroprotection in brain ischemia: An update (Part 1), Neuroscientist 1:95-103 (1995). 42. M.D. Ginsberg, Neuroprotection in brain ischemia: An update (Part II), Neuroscientist 1:l64-175 (1995).
CALPAIN ISOFORMS IN THE EYE
T.R. Shearer1, H. Ma1, M Shih1, K.J. Lampi1, C. Fukiage2 and M. Azuma2 1
Department of Oral Molecular Biology, School of Dentistry Oregon Health Sciences University, Portland, OR 97201 2 Research Laboratories, Senju Pharmaceutical Co. Ltd. Kobe 651-2241 Japan
RETINA-SPECIFIC CALPAINS An explosion in the discovery of tissues-specific calpains has recently occurred. For example, twelve isoforms of p94 have been described in muscle1. At least five eye-specific calpains are present in the rodents, in addition to the ubiquitous µ- and m-calpains2,3. These isoforms are termed Rt88, Rt88' and Rt90 in retina and Lp82 and Lp85 in lens (Fig. 1). Interestingly, all are splice variants of muscle-preferred p94. In place of the N-terminal NS region of p94, the eye isoforms use an alternative exon 1, termed AX1 (Fig. 2). This is a portion of the 3' end of intron 1 from the p94 gene1. Such data suggest that a common promoter may be present in eye tissues. Other splice deletions or insertions are also present in the calpain isoforms found in eye. For example, Rt88 shows deletion of exons 15 and 16 to delete the IS2 region in the predicted protein (Fig. 3). Rt90 has the same IS2 deletion, but intron 18 from p94 was retained as in Lp851. This would insert a 28 amino acid sequence into domain IV of the calcium-binding region. The consequences of such an insertion are unknown, but we speculate that it may influence calcium sensitivity. The other retina isoform, Rt88', contains a stop codon in exon 12 resulting in a truncation starting in domain III if any protein were expressed. Detection of protein and enzymatic activity for Rt88, Rt88' and Rt90 in rat retina has been hampered because of the instability caused by the presence of the IS1 region (Fig. 3). Likewise, this region is responsible for the autolytic cleavage sites and instability of p94 in muscle4. To help circumvent the problems in purifying Rt88 from retina, we recently expressed recombinant Rt88 in the baculovirus system. rRt88 was then partially purified by Ni affinity chromatography and assayed by immunoblotting and casein zymography. A large band of rRt88 caseinolytic activity was detected after incubating the zymogram gel in 2 mM calcium (Fig. 4A, lane 1, arrow). In contrast, incubation of the gel in EGTA prevented caseinolysis (lane 2). rRt88 was inhibited by cysteine protease inhibitor E64 (lane 3). Immunoblots from SDS-PAGE gels also showed that all of the Rt88 band at 88 kDa was lost when incubated with calcium (Fig. 4B, lane 1), confirming calcium sensitivity. Rt88 seems very unstable since much of the intact 88 kDa band was already broken down to 51 and 52 kDa fragments even when no calcium was added (-Ca lane) or after incubation with EGTA (lane 2) or E64 (lane 3). This in vitro lability helps explain the current difficulty in detecting Rt88 protein or enzyme activity in retina. The precise function of Rt88 is unknown. However, constant levels of mRNA for Rt88 were found in retina of mature rats up to at least one year of age. This suggests an important function for Rt88 throughout life, rather that just during development of retina.
Role of Proteases in the Pathophysiology of Neurodegenerative Diseases, edited by Lajtha and Banik. Kluwer Academic/Plenum Publishers, New York, 2001.
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Figure 1. Known tissue-specific calpains from rodent eyes compared to muscle p94. NS= novel sequence, IS1 and IS2=insert sequences in p94, AX1=alternative exon IS3=insert region derived from intron 18 of p94, and dashed lines are deleted sequences.
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Figure 2A. Comparison of cDNA sequences for retina-specific calpains.
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Figure 2B. Comparison of cDNA sequences for retina-specific calpains (continued).
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Figure 2C. Comparison of cDNA sequences for retina-specific calpains (continued).
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Figure 3A Comparison of the predicted protein sequences for retina- and lens-specific calpains to the sequence for muscle p94.
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Figure 3B Comparison of the predicted protein sequences for retina- and lens-specific calpains to the sequence for muscle p94.
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Fig. 4 (A) Zymogram showing enzymatic activity of recombinant Rt88 run on native-PAGE gels containing 0.05 % casein. Lane 1 = partially purified rRt88 with gel incubated in 2 mM calcium, 2 = gel incubated with 2 mM calcium and 5 mM EDTA, 3 = gel incubated in 2 mM calcium and 0.25 mM E64. Arrow indicates caseinolysis produced by presumptive rRt88. (B) Immunoblot for rRt88 from SDS-PAGE gels with same lane designations as above, except that samples were incubated with calcium, EGTA or E64.
ROLE OF OVER-ACTIVATION OF CALPAIN IN RODENT CATARACTS Compared to retina, the role of calpains in the lens of rodents is well-defined (See review)5. The lens data may also help our understanding of the function of calpain isoforms in retina since retina-specific calpains share some structural similarities to lens-specific calpains. Calpain-induced proteolysis of lens crystallin proteins and subsequent cataract formation in rodents provides one of the most clear cut examples of the consequence of overactivation of calpains (Fig. 5). Most cataracts show massive increases in lens calcium, which sometimes exceed 1000 µM6. This is high enough to activate the abundant levels of m-calpain in young rat lens. Calpains then cause specific truncations on the N-termini (and possibly C-termini) of the β-crystallin structural proteins7 and truncations on the C-terminus of the molecular chaperone α-crystallin8. These partially proteolyzed crystallins become insoluble and scatter light in cataract. Calpain-induced proteolysis and formation of excess insoluble protein occurs in most rodents models of cataracts including those induced by buthionine sulfoximine (BSO), selenite, calcium ionophore A23 187, hydrogen peroxide, diamide, xylose, galactose, streptozotocin, Nakano mouse genetics, UPL hereditary rat cataract, and transgenic mice expressing HIV protease9. Evidence supporting the scheme in Figure 5 has been provided by observing calpainlike cleavage sites on insoluble proteins from rodent cataracts7 and by mimicking light scattering after in vitro proteolysis of crystallins by calpain9. For example, addition of calcium alone to soluble lens proteins from normal rats causes activation of m-calpain and Lp82, proteolysis, and light scattering in vitro (Figure 6, bold arrows). Recent evidence also indicates that oxidation plays a major role in producing light scattering after rodent crystallins are truncated by calpains. Note the five-day lag period after calpain proteolysis and before light scattering (Fig. 6). This lag period is probably needed for oxidation of calpain-truncated crystallins. The lag period is shortened and the extent of
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light scattering is increased by the addition of the xenobiotic oxidizing reagent diamide (Fig. 6A)10 and by the natural oxidant UV-B (Fig. 6B). In contrast, when the anti-oxidants DTE or GSH are added to the incubations, light scattering is suppressed. These data clearly indicate that oxidation is an essential component of calpain-induced light scattering in rodent crystallins. Since no light scattering occurred unless calpain proteolysis was first present, truncation may increase the susceptibility of rodent crystallins to oxidation. Proteolysis of young human and bovine proteins with m-calpain enhances heat-induced light scattering (Fig. 7). Thus, increased light scattering after truncation of crystallins may be a general phenomenon and not limited to rodents. Recent evidence also indicates that calpain-truncated crystallins may not interact normally and that this would promote formation of insoluble proteins. Intact β-crystallins polypeptides do not normally exist as monomers, but as dimers and other soluble, higher order soluble aggregates. Evidence supporting the idea that truncation is deleterious to formation of these normal oligomers was provided in an experiment where recombinant human βBl crystallin was truncated with purified m-calpain (Fig. 8). Truncation caused loss of βΒ1 homodimers. We postulate that the monomers may then form abnormal associations with other crystallins, which are insoluble and scatter light. Significant amounts of active m-calpain are present in human lenses11. We do not yet know if m-calpain is activated during aging of human lenses. However, truncation of crystallins12 and UV exposure13 both occur in aging human lenses. Whatever protease or truncation mechanism is active in human lens, the above data from rats suggest that truncation and loss of normal oligomerizations could be synergistically linked to formation of light scattering elements in human cataracts.
Figure 5. The proteolysis-insolubilization hypothesis describing the role of calpains in rodent models of cataract. Note the central role of oxidation in precipitation of truncated and intact crystallins. Modified from Shearer et al., 19995.
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Figure 6. Enhancement of light scattering by diamide (A) and UV-B (B) following truncation of lens crystallins by calpains. Bold arrows indicate light scattering with no added oxidative challenge. From Nakamura et al., 1999 and Nakamura et al., in press10,14.
Minutes at 55 0C Figure 7. Enhancement of heat-induced light scattering by pre-treatment of lens soluble proteins from 3 year old human donor with recombinant m-calpain for 3 hours.
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Figure 8. Size exclusion chromatography (TSK-Gel 3000 SW-XL) of recombinan EB1-crystallin before (A) and after (B) incubation with m-calpain. The elution positions of major classes of crystallins on this calibrated column are indicted on the top. Before calpain, EB l (monomer = 27,891 mass units) eluted near the position of EL dimers, suggesting homodimer formation (vertical dashed line). After calpain, proteolyzed EBl (monomer = 23,040 mass units) eluted with the buffer peak, indicating an interaction with the column. Light scattering analysis confirmed that the truncated bB1 was a monomer15.
PARTICIPATION OF LENS-SPECIFIC CALPAINS MATURATION AND CATARACT FORMATION
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LENS
Until recently, cataract formation in rodent models as discussed above was thought to be solely due to m-calpain. This was because abundant levels of m-calpain were found in rodent lenses16. However, recent use of RT-PCR with degenerate primers for the active site of calpain17 and the increased sensitivity of casein zymography2 demonstrated the presence of the lens-specific calpains termed Lp82 and Lp85. In young mice, Lp82 is actually the dominant calpain over the ubiquitous m- and µ-calpains18. As with retina-specific calpains, Lp82 and Lp85 are splice variants of muscle calpain p94 (Fig. 1). They both contain the alternative AX1 for exon 1, but they contain deletions of both the IS1 and IS2 regions yielding in vivo stability. Lp82 and Lp85 have very similar biochemical and physiological characteristics. For example, they share 96% sequence identity, have similar isoelectric pH’s at 5.86 versus 5.67, migrate to the same position on native acrylamide gels used in casein zymography, show similar elution off DEAE columns, have a distribution in lens of nucleus>ortex>epithelium, show highest concentrations in the insoluble fraction, are both activated during lens maturation and cataract formation, and both disappear with lens aging. Due to these similarities, even partially purified Lp82 preparations contain 20% Lp85. However, independent, transient expression of each in COS-7 cells shows that Lp82 and Lp85 are proteolytically active independent of each other19. The three most urgent questions regarding Lp82/Lp85 mixtures are their calcium requirements, substrate specificity, and biologic functions.
Calcium Requirement The concentration of calcium needed for activation of Lp82 is important because the calcium concentration in normal lens is approximately 0.1- 0.2 µM. Yet calpains are somehow activated during normal rodent lens maturation. The same calpain cleavage sites found in cataract are also found in normal maturing rodent lenses. Our preliminary data suggested that 25 µM caused 50% activation of Lp82 (Fig. 9), and this is approximately 5 to 10 times lower than the published value for m-calpain16.
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In contrast to the autolysis involved in the activation of µ-calpain, our immunoblotting results indicate that Lp82 can be active without the appreciable autolysis20. This makes sense because the IS1 region, missing in Lp82, was shown to be the cleavage site for p94 autolysis4. However, 25 µM is still approximately 50 times higher than the physiologic levels of calcium in normal lens. We speculate that in vivo activators lower calcium activation requirements of both Lp82 and m-calpain in normal lens. Lp82 was also found to be less sensitive to calpastatin than m-calpain21, and this would also help promote Lp82 activity under lower calcium conditions found in maturing lenses.
Substrate Specificity Discovery of the in vivo substrates for Lp82 is important because such information might suggest physiological functions. Ail calpain isoforms discovered so far in the eye contain the papain-like active site found in m-calpain. Thus, we expected cleavage sites produced by Lp82 to be similar to those produced by m-calpain. Initial experiments showed this to be only partially true. In vitro proteolysis of EB1 and EA3-crystallin by Lp82 or mcalpain appeared to produce the same truncated polypeptide depending on the substrate (Fig. 10A). However, against DA-crystallin, Lp82 produced a unique cleavage site five amino acids in from the C-terminus, which was not produced by m-calpain (Fig. 10B). Production of this unique cleavage site may serve a specific function and may also be useful in identifying Lp82 activity within specific regions of the lens.
Figure 9. Calcium activation curve for partially purified Lp82 from 12 day old rat lens. fluorescence units released from hydrolyzed substrate.
FU = relative
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A. EB1 crystallin
B. DA crystallin AcMDVTIQHPWFKRALGPFYPSRLFDQFFGEGLFEYDLLPFLSSTISPYYRQSLFRT VLDSGISEVRSDRDKFVIFLDVKHFSPEDLTVKVLEDFVEIHGKHNRQDDHGYIS REFHRRYRLPSNVDQSALSCSLSADGMLTFSGPKVQSGLDA
Figure 10. (A) SDS-PAGE showing in vitro proteolysis of recombinant βBl by Lp82 or m-calpain. E64 = control inhibited by cysteine protease inhibitor. Molecular weight markers on left in kDa. (B) Cleavage sites on isolated bovine DA crystallin produced by L82 and m-calpain as revealed by mass spectrography. (From Nakamura et al., submitted)14.
Functions of Lens-Specific Calpains Because Lp82 is found in high concentrations in young lenses and then it disappears with lens maturation, we believe that the major physiological function of Lp82 is for lens development. Other roles are also possible, but they are speculative. For example, redundancy of calpain activities may be another function of Lp82. This was suggested in a recent experiment using a transgenic mouse harboring a mutant gene for inactive m-calpain. The strategy was that an over abundance of inactive m-calpain would suppress wild type mcalpain activity by binding to calcium and substrates. As yet we do not know if this dominant negative was effective in knocking out wild-type m-calpain because m-calpain activity was decreased (Fig. 11B, lane marked "3") and BSO cataracts formed equally well in normal and transgenic mice (Fig. 11A). Rather than an autolytic decrease in m-calpain, this decrease could have been due to breakdown of m-calpain by the large amounts of Lp82. Note that Lp82 was also decreased (Fig. 11B). Thus, lenses have apparent redundancy in the case of calpain isoforms. Even if m-calpain activity were to be totally knocked out, cataracts may still form due to Lp82-induced proteolysis. Another consequence of over-activation of Lp82 may be in light scattering. Calpaininduced in vitro light scattering was abolished when mature rat lenses were used as a source of soluble proteins22. We previously reasoned that this was due to the fact that maturation of lenses is accompanied by normal proteolysis of E-crystallins and slow insolubilization. Essentially no more precipitation-susceptible crystallins remained in the older lenses. However, this age-related loss in ability to undergo in vitro precipitation (Fig. 12) is also well correlated with age-related loss in Lp82 (Fig. 12 insert). Partially purified Lp82 is able to cause in vitro light scattering23. Thus, loss in ability to undergo in vitro precipitation may be partially related to the specific cleavages caused by Lp82.
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Figure 11. (A) Lenses in transgenic mice (inactive m-calpain) and in normal mice (FBV/NJ). Both groups received BSO injections. Dark areas indicate cataract. (B) Casein zymograms of the soluble proteins from transgenic and normal mice receiving BSO injections, showing activities (white areas) for Lp82, mcalpain, µ-calpain, and their activated forms.
Figure 12. Attenuation of light scattering with maturation of mouse lens. The casein zymogram (above) shows normal maturational loss of Lp82 (upper band)
CONCLUSIONS Retina and lens from rodents contain at least five newly discovered isoforms of calpain (Lp82, Lp85, Rt88, Rt88’ and Rt90), in addition to the ubiquitous calpains. Unexpectedly, these isoforms were related to muscle-preferred p94. Although some of their biochemical characteristics are known, the current challenge is to discover the functions of calpain isoforms under normal and pathological conditions. Further, because of stop codons, orthologues of the isoforms discussed above do not exist in man. Another challenge is to relate the rodent data to the human situation. For example, do undiscovered
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calpain isoforms performing the same functions as Lp82, Lp85, Rt88 and Rt90 exist in human eye?
Acknowledgements Partially supported by NIH grants EY03600, EY05786 to TRS and EY12239 to KJL. REFERENCES 1. M. Herasse, Y. Ono, F. Fougerousse, E. Kimura, D. Stockholm, et al., Expression and functional characteristics of calpain 3 isoforms generated through tissue-specific transcriptional and posttranscriptional events, Mol. Cell. Biol. 19(6):4047 (1999). 2. H. Ma, M. Shih, I. Hata, C. Fukiage, M. Azuma, and T.R. Shearer, Protein for Lp82 calpain is expressed and enzymatically active in young rat lens, Exp. Eye Res. 67(2):221 (1998). 3. M. Azuma, C. Fukiage, M. Higashine, T. Nakajima, Y. Kawamoto, et al., Identification of a retinaspecific calpain (Rt88) from rat, Curr. Eye Res. (submitted). 4. K. Kinbara, S. Ishiura, S. Tomioka, H. Sorimachi, S.Y. Jeong, et al., Purification of native p94, a muscle-specific calpain, and characterization of its autolysis. Biochem. J. 335:589 (1998). 5. T.R. Shearer, H. Ma, M. Shih, C. Fukiage, and M. Azuma, Calpains in lens of the eye, In: CALPAIN: Pharmacology and Toxicology of Calcium-Dependent Protease, K.K.W. Wang and P.-W. Yuen, EDS., Philadelphia, Taylor & Francis (1999). 6. T.R. Shearer and L.L. David, Role of calcium in selenium cataract, Curr. Eye Res. 2(11): 777 (1982). 7. L.L. David, M. Azuma, and T.R. Shearer, Cataract and the acceleration of calpain-induced ß-crystallin insolubilization occurring during normal maturation of rat lens, Invest. Ophthalmol. Vis. Science 35(3):785 (1994). 8. M.J. Kelley, L.L. David, N. Iwasaki, J. Wright, and T.R. Shearer, alpha-Crystallin chaperone activity is reduced by calpain II in vitro and in selenite cataract, J. Biol. Chem. 268(25): 18844 (1993). 9. T.R. Shearer, M. Shih, T. Mizuno, and L.L. David, Crystallins from rat lens are especially susceptible to calpain-induced light scattering compared to other species, Curr. Eye Res. 15(8):860 (1996). 10. Y. Nakamura, C. Fukiage, M. Azuma, and T.R. Shearer, Oxidation enhances calpain-induced turbidity in young rat lenses, Curr. Eye Res. (19): 33 (1999). 11. L.L. David, M.D. Varnum, K.J. Lampi, and T.R. Shearer, Calpain II in human lens, Invest. Ophthalmol. Vis. Sci. 30(2):269 (1989). 12. M.S. Ajaz, Z. Ma, D.L. Smith, and J.B. Smith, Size of human lens beta-crystallin aggregates are distinguished by N-terminal truncation of betaB1, J. Biol. Chem. 272:11250 (1997). 13. J. Dillon, UV-B as a pro-aging and pro-cataract factor, Doc. Ophthalmol. 88(3-4):339 (1994). 14. K.J. Lampi, J. Oxford, T.R. Shearer, L.L. David, H.P. Bachinger, and D.M. Kapfer, Human bB1 crystallin structure and altered structure by truncation and deamidiation, (Submitted). 15. Y. Nakamura, M. Azuma, and T.R. Shearer, Calpain-induced light scattering in young rat lenses is enhanced by UV-B, Exp. Eye Res. (2000) In press. 16. L.L. David, and T.R. Shearer, Purification of calpain II from rat lens and determination of endogenous substrates, Exp. Eye Res. 42(3):227 (1986). 17. H. Ma, C. Fukiage, M. Azuma, and T.R. Shearer, Cloning and expression of mRNA for calpain Lp82 from rat lens: splice variant of p94, Invest. Ophthalmol. Vis. Sci. 39(2):454 (1998). 18. H. Ma, M. Shih, C. Fukiage, Y. Nakamura, M. Azuma, and T.R. Shearer, Lp82 is the dominant form of calpain in young mouse lens, Exp. Eye Res. 68:447 (1999). 19. H. Ma, M. Shih, I. Hata, C. Fukiage, M. Azuma, and T. Shearer, Lp85 is an enzymatically active rodent-specific isozyme of Lp82, Curr. Eye Res. 20(3):183 (2000). 20. I. Shih, H. Ma, and T.R. Shearer, unpublished. 21. Y. Nakamura, C. Fukiage, H. Ma, M. Shih, M. Azuma, and T. Shearer, Decreased sensitivity of lensspecific calpain Lp82 to calpastatin inhibitor, Exp. Eye Res. 69:155 (1999). 22. C. Fukiage, M. Azuma, Y. Nakamura, Y. Tamada, and T.R. Shearer, Calpain-induced light scattering by crystallins from three rodent species, Exp. Eye Res. 65(6):757 (1997). 23. Y. Nakamura, C. Fukiage, M. Shih, H. Ma, L.L. David, et al., Contribution of Lp82-induced proteolysis to experimental cataractogenesis in mice, Invest. Ophthalmol. Vis. Sci. 41: 1460 (2000).
METALLOENDOPEPTIDASE EC 3.4.24.15 IN NEURODEGENERATION
Carmela R. Abraham and Franchot Slot Boston University School of Medicine Boston, Massachusetts 02 118
INTRODUCTION The metalloendopeptidases represent a fascinating class of enzymes involved in neurodegenerative diseases. Many metalloendopeptidases are integrally involved in brain processes. This family boasts enkephalinase (24.11), neurolysin (24.16), and others. One of the most important members of this family is 24.15, also known as Thimet oligopeptidase or ThopI. 24.15 has been strongly implicated in Alzheimer’s disease and a multitude of analgesic pathways and is a regulator of major reproductive hormones. In addition, it has been recently suggested to serve important roles in antigen presentation and immunity. Here, 24.15 will be the focus, but in discussing 24.15 there will be reference to 24.11 and 24.16, each of which shows some overlap in function and activity. While direct links have been made between 24.15 activity and Alzheimer’s disease, numerous suggestive links have been considered as well. These may be contributors to Alzheimer’s disease pathology and may play roles in other neurodegenerative phenomena as well. Both the direct and indirect links will be explored here. One of the prevailing aspects of this enzyme that will become apparent is the large number of very different activities. Coupled to this is the fact that these activities don’t necessarily quickly form an integrated picture of the physiological “role” of the enzyme. Finding that role has been and continues to be elusive. Even the direct links to disease leave many questions open. Its importance in many pathways is clear and there are many hints that suggest the beginnings of a coherent function for the enzyme. Nevertheless, as will be seen, much more work is necessary to come to a clear understanding of the full and precise influence of this enzyme. The common practice of naming an enzyme by its cleavage assay, (i.e. Pz-peptidase is the enzyme cleaving the Pz peptide) has led to multiple names for the metalloendopeptidases. In some cases more than half a dozen names refer to the same molecule. The Enzyme Commission (EC) classification system was developed, in part, to unite these many names under a common description. We will use this system where possible for the sake of clarity and refer, therefore, to the metalloendopeptidase as 24.15, rather than the ThopI or the full EC 3.4.24.15.
Role of Proteases in the Pathophysiology of Neurodegenerative Diseases, edited by Lajtha and Banik. Kluwer Academic/Plenum Publishers, New York, 2001.
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HISTORY OF 24.15 When collagen was first sequenced, the repetitive nature of the amino acids led to the design of a synthetic peptide with a similar sequence. Synthesized in 1963, the peptide was called the Pz-peptide for the N-terminal, yellow, exopeptidase-blocking group, “Pz” (phenylazobenzyloxycarbonyl)1. The degradation and subsequent extraction of the Pzpeptide fragments were used as a marker for collagen breakdown. The assumption here was that the enzyme responsible for the Pz-peptide degradation would be the same enzyme responsible for degrading collagen itself. As a result of this assumption Pz-peptidase was considered nearly synonymous with collagenase. Research into collagen mechanics, maintenance, and modeling used this assay extensively2. Indeed, it seemed to be a good marker system since it was shown to correlate well with tissue breakdown in a variety of systems. In 1972, Aswanikumar and Radhakrishnan purified the Pz-peptidase from rat granuloma tissue (known to be high in Pz-peptidase activity)3. It was given the number EC 3.4.99.31. The same authors later showed Pz-peptidase to be a 56 KDa protein inhibited by EDTA, with an optimum pH of 7.04. Further characterization, however, showed that Pz-peptidase was a poor candidate as a collagenase. The enzyme was not able to cleave native mammalian or avian collagen5, nor was it even effective in breaking down denatured collagen (gelatin), or gelatin constituents4,6,7. In fact, it was determined that the purified Pz-peptidase had a very narrow substrate specificity, almost exclusively restricted to small peptides less than 18 amino acids in length8. In addition, further work confirmed that “Pz-peptidase” was probably not a single enzyme. This has resulted in some confusion in the literature. The ability to cleave the Pz-peptide is not a specific effect of a single enzyme. Later purifications of the Pz-peptide cleaving activity resulted in preparations with different molecular weights, inhibitor profiles, and cofactor responses4,9,10. Data used here under the aegis of Pzpeptidase, therefore, will be specified as such. Obviously we have excluded data from consideration that derive from irrelevant “Pz-peptidases” (judged by physico-chemical attributes). Camargo and colleagues in 1972, isolated Endo-oligopeptidase A (originally called neutral endopeptidase) from rabbit brain11. Endo-oligopeptidase A (EOPA) was deemed to be a cysteine protease and in keeping with that was given the number EC 3.4.22.19 (3.4.22.19 refers to the 19th enzyme purified in the cysteine endopeptidase family). Subsequent to the purification of EOPA, Orlowski and colleagues identified “Soluble Metalloendopeptidase” in 198312. Accordingly their enzyme was labeled EC 3.4.24.15 (metalloendopeptidase family). The International Union of Biochemistry (IUB) recommended name for 24.15 became Thimet oligopeptidase, for its thiol and metal dependence as well as its restriction to small peptides less than 18 amino acids8,13. At the outset Pz-peptidase, Endo-oligopeptidase A, and Soluble Metalloendopeptidase appeared to be very different enzymes. In 1989, however, Tisljaf et al. determined by “substrate swapping” that Pz-peptidase and EOPA were identical.7 Shortly thereafter it was also shown that 24.15 and Pz-peptidase were identical14 . By extension, it was naturally argued that 24.15 and EOPA were the same as well. This proved to be problematic though. The enzyme had been identified alternatively as a cysteine, metallo-, or simply unclassified peptidase based on inhibitor profile. As a result of the inhibitor differences in addition to substrate specificity differences, it was convincingly argued that these were in fact different molecules15. Significant differences in assay design and animal models confounded results and comparisons. Publications in 1989 and 1991, though, argued that EOPA and 24.15 were indeed the same. They attributed the apparent differences in inhibitor profile and substrate specificity, to a cysteine near the metallopeptidase active site, and to species differences, respectively7,13,14. These enzymes were all believed to be the same until EOPA was very recently cloned. Sequence analysis 76 of EOPA has indicated finally that the two enzymes are separate . With the resolution of the nomenclature, there was continuing evidence for 24.15 as an important neuropeptidase. Other evidence also came to light linking it with Alzheimer’s disease (AD). The first of these links indicated that 24.15 was responsible for the cleavage of a synthetic peptide mimicking the E-secretase cleavage16,17,18 of the amyloid precursor protein (APP). APP gives rise to the Amyloid beta (AE) peptide, which accumulates and is thought to be causative for AD pathology. Subsequently, we
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determined that 24.15 activated a serine protease, which in turn, degraded AE19. Our work has also indicated that D -antichymotrypsin (ACT) inhibits this serine protease. ACT has long been identified as an important factor in AD20 . The role of 24.15 in AD may be a critical one and will be discussed later. In addition to these important and direct roles in AD for the metalloendopeptidase 24.15, the last decade has shown multiple indirect associations with other disease pathology. In at least five different pain and analgesia pathways 24.15 has been noted as a key regulator. The array of neuropeptides processed by this one enzyme is intriguing. Many of these transmitters have proven to be directly involved with a host of neuropathologies, Since 24.15 is critical for the regulation of these transmitters and peptides, one may speculate that these pathways are interrelated through the enzyme. It invites a search for a unifying and coherent role of the enzyme in the organism. Neuropeptides may be neurotransmitters or hormones or both. As a primary regulator of both, 24.15 hints at integrating these and other physiologic systems. Early and continuing work has identified the enzyme in the clearance of Gonadotropin releasing hormone (GnRH)21,22. This master reproductive hormone in turn controls the levels of a number of other hormones in the body. The potential interplay between these and the wide variety of other activities of 24.15 is fascinating and will also receive more attention later. The most recently discovered activity of 24.15 is in its coordination with the function of the proteasome23,24. Peptides processed from this complex seem to associate tightly with the 24.15 peptidase, (even without being cleaved), and serve an as yet unclear role in delivering the “cargo” to the MHC antigen presenting molecules at the cell surface. Other work has strongly implicated 24.15 in related immune functions and as a potential player in the inflammatory response. The history, therefore, of this single metallo-endopeptidase is very colorful. The many seemingly disparate effects and potential roles are both extraordinary and perplexing. Each of them, however, may be readily associated with aspects of neurodegeneration. It is the goal of this chapter to continue our growth toward a comprehensive and coherent understanding of the overarching role of this important peptidase.
BIOCHEMICAL CHARACTERIZATION Molecular Weight Molecular weights reported for Thimet oligopeptidase (24.15) vary from species to species and tissue to tissue. In fact, even within a single cell there seems to bemore than one form. The enzyme is found in the nucleus, cytosol, on the cell surface, and even secreted. Selective trafficking may be the result of differential splicing, proteolytic processing, or alternative starts of transcription. Such processes would be expected to result in size changes. In addition, a particular tissue or species may selectively use the numerous potential glycosylation and phosphorylation sites. Lastly, there are some reports of the enzyme forming multimers under certain conditions25,26. This may explain some of the larger sizes reported, especially since some of these analyses were done in SDS-free systems9,10,27 The molecular weight determined from amino acid composition of a rat clone is approximately 73 kDa2 and the human is 78.5 kDa28. Most preparations fall within a 70-85 kDa range. This 32 includes rabbit skeletal muscle (74 kDa),29 rat testis (70 kDa)31, rat epidermis (80 kDa) , bovine brain (75 kDa)33, chicken liver (80 kDa)31, human brain (85 kDa)17, monkey brain (80 kDa)34,35, and others. The high molecular weight form was seen in bovine dental follicle and adrenal gland (220 kDa)9,27 and rabbit serum10. Human erythrocytes showed a molecular weight of 75 kDa36. In addition, numerous lower molecular weight forms have been seen. Most of these center around 50 kDa. Indeed, the first size determination of Pz-peptidase by Aswanikumar and Radhakrishnan from monkey tissue sizes were human brain (55 kDa),34,37 monkey brain (55 kidne was 56 kDa4. Other kDa)35 human testis (55)38, rat brain (43)39, etc. There have also been many lower molecular weight bands noted in the 20-30 kDa range, however, it is unclear whether these are degradation products or isolation artifacts. Multiple forms of the enzyme have been seen in the same tissues and are often seen in the same preparation. We consistently find two molecular weight forms in AD brain that migrate at 85 kDa and 43-55 kDa17,34,35.
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Only a few of the isozymes noted have been tested for activity. When the primary form found has been in the 55 kDa range, it has been active37.
Isoelectric Point Most reports have found isoelectric points ranging between 4.8 and 5.2. One report using a preparation from rabbit uterus notes a 4.4 pI40, and another in human pituitary is as high as 5.538. In rat liver mitochondria, two isoelectric forms were separated, one at 4.9 and another at 5.241
pH optimum Reports of pH optima have ranged between 6.0 and 8.5 nearly exclusively. Two papers showed dual pH optima with 6.5 and 8 reported by one group9 and 7.1 and 7.9 by another10. Both of these were looking at Pz-peptidase activity, however, and it is likely that these activities reflect enzymes other than 24.15. (As mentioned previously, several different enzymes cleaving the Pz-peptide have been found – only one of them being 24.15). The few studies discussing pH inactivation show significant loss of 24.15 activity both below and above these optima. A 50% loss of activity was seen below 6 and above 8.5 in one study32. We showed a pH optimum closer to 6 with inactivation below pH 5 and above 7.617. A further study determined that irreversible inactivation was achieved at a pH less than 3.542. These results are not consistent with data indicating a role for 24.15 in the low pH endosomal vesicles43. It is possible that immunohistochemical cross-reactivity is responsible for this. It may be also that this is one route to degradation of 24.15, and its presence in endosomes may not reflect activity.
Thiol Activity It is a consistent finding that 24.15 requires a low level of thiol content for full activation. This is found to be sufficient at concentrations less than 0.5mM dithiothreitol. On the other hand concentrations above 5mM reversibly block activity25. The reason for this inactivation is not entirely clear. One group showed that the low thiol content was necessary to convert the enzyme from a multimeric to a monomeric form25 . This study has been neither confirmed nor refuted in the literature but seems that it might fit well with some other indications of complex formation discussed. Thiol activation is thought to be the result of reduction of cysteine residues that lead to intermolecular bridges and complex formation, which block substrate access to the active site25,44 The inhibitory effect of high thiol concentrations may, on the other hand, be a result of the proximity of a cysteine residue near the active site. This cysteine has also been implicated as the reason for inhibition by N-ethylmaleimide, iodoacetate, and iodoacetamide; known cysteine protease inhibitors25. This effect of thiol-sensitive inhibition is pronounced and explains the original classification as a cysteine protease (EC 3.4.22.19), as well as the confusion with Endo-oligopeptidase A.
Inhibitor Profile Table 1 indicates the inhibitor profile of Thimet oligopeptidase (24.15). By virtue of its inhibition by EDTA, EGTA, and 1,10-phenanthroline, and the ability to restore activity with a variety of metals, 24.15 has been classified as a metallo-protease. In keeping with this, the enzyme displays the characteristic HEXXH motif for coordinating a zinc ion and zinc has been seen in pure preparations to be a constituent of the protein41. As mentioned, the inhibition by certain cysteine protease inhibitors has been suggested to be due to a cysteine residue 5 amino acids from the active site (Cys-483 in rat)25. The failure of inhibition by E-64, a universal cysteine peptidase blocker, indicates also that the thiol inhibition is an effect of Cys-483 rather than 24.15 being a cysteine peptidase, In keeping with 24.15 as a metallopeptidase, a variety of metals have been shown to restore enzyme activity subsequent to EDTA treatment. Zn fully restores activity at the lowest concentrations (50uM). Ca, Sr, Mg, Ba, Mn, and Cd are also effective in varying degrees of reactivation. 2mM Mg seems to be important for full enzymatic activity17. Ca and Mn in concentrations approaching 20mM can actually induce an activity above and
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beyond what is normally seen in an unaltered preparation35,45. This depends in part on the preparation and species used but has been seen for the recombinant enzyme as well35. Importantly, both aluminum and iron salts can completely inhibit recombinant 24.15 at low millimolar and micromolar concentrations, respectively35 . Aluminum and iron have both been shown to accumulate in AD brain46,47. Specific inhibitors to 24.15 have also been developed. Orlowski and colleagues produced the first set of these. They were peptide analogues capable of coordinating the active site zinc with a carbonyl on the C-terminus. The best of this group displayed a Km of 16.6nM48. Later, Jiracek et al. reported the creation of phosphinic peptide moieties (which were also peptide analogues). These studies showed that alanine was preferred in the Pl’, an aromatic residue in the P1, arginine or lysine in the P2’, and methionine in the P3’ positions. The phosphinic peptides are the most specific and effective inhibitors known to date with a Km in the picomolar range (the best being 70 picomolar). One of these inhibitors displayed a 1,000 fold selectivity for 24.15 over the very closely related metallo-endopeptidase, neurolysin (24.16.)49
Substrate Specificity The synthetic substrates and inhibitors for 24.15 have provided considerable insight into the specificity of the enzyme. Nevertheless, there are no good rules for cleavage yet, and it is impossible to predict where the peptidase will cleave. Table 1 lists both the endogenous and synthetic substrates that are known for the enzyme along with the cleavage sites. It should be noted here, however, these cleavage sites are specific and well defined even though the rules guiding the cleavage are neither specific nor well defined. The cleavage sites noted are the only sites utilized, and only in a small number of peptides is there more than one. Processing of the peptides by 24.15 is clearly not a promiscuous activity. Most of these cleavages occur with Km values in the low micromolar range and a random peptide population is not cleaved23.
Table 1. Substrates for EC 3.4.24.15 Peptide
Peptide Structure
Reference
Synthetic Substrates Pz-peptide (for continuous rate) analysis (flourescent) “E-secretase peptide” “E-secretase peptide” (recomb.)
Pz-PL+GP-D-R Suc-G-PL+GP-MCA Bz-G+AAF-pAB DNP-PL+GPW-D-K Mcc-PL+GP-D-K(DNP) HSEVKM+DAEF ISEVK+M+D+AE+FRHDS
2 2 2 2 2 17 68
NATURAL SUBSTRATES Bradykinin Neurotensin GnRH Angiotensin I Angiotensin II Somatostatin Substance P Dynorphin A 1-8 Dynorphin B D- Neoendorphin E- Neoendorphin Orphanin FQ Cholecystokinin-8
RPPGF+SPFR pELYGNKPR+RPYIL pEWSY+GLRPG DRVY+IHP+FHL DRVY+IHPF AGCK+NFFW+KTFTSC RPKPQQF+F+GLM YGGFL+RRI YGGFL+RRQF+KWT YGGF+M+TSELSE+TPLVT YGGFL+RKYP FGGFTGA*RKSA*R*KLANQ DYMGW+MDF- NH2
12,26,96 12,26,85,96,33 26,12,85,100 97 85,97 85,97 12,85 26,84,99 96,33 84,99 26,84,99 98 101
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Of the few guidelines for substrate specificity, Orlowski reported that there was selectivity for hydrophobic residues in the P1’ and P3’ sites48. 24.15 seems to be able to cleave peptides with a proline in the P1 position as well50. 24.15 is strictly an endo-peptidase. The enzyme does not cleave N or C-terminal residues from a peptide unless the peptide is reduced to a minimal size. The peptide-binding pocket accepts approximately 5 amino acid residues48 and the enzyme is incapable of cleaving peptides smaller than 4 amino acids12. Conversely, no peptide chain longer than 18 amino acids has been shown to be reproducibly cleaved (APP serving as a possible exception). These data, then, ensure 24.15’s definition as an endo-oligopeptidase.
Active Site Mechanism Thimet oligopeptidase is a zinc-metalloendopeptidase. The coordination of zinc and the reaction mechanism of the active site are performed by the HEXXH motif. In 24.15, this is seen as HEFGH. In this mechanism, zinc is coordinated by Histidines-473 and 477 and by the distant Glutamate-502. Glu-474 then forms a hydrogen bond with water and coordinates the zinc with the water’s oxygen. This creates a nucleophilic center, which makes an attack on the carbonyl carbon of the peptide bond. The carbonyl carbon is released, and the enzyme is freed for another reaction51. Gene Structure, RNA, Protein Motifs Our lab determined that Thimet oligopeptidase was located on Chromosome 19q13.3 in 199652. This was very promising because of the proximity of the risk factor for AD, Apo E. Since then we went on to identify its localization on the p-arm at position 13.353 . Because Apo E is on the q-arm it is unlikely that there is any connection between these two genes from a chromosomal regulation standpoint. Recently, the Lawrence Livermore National Laboratory has read through this region of human chromosome 19 and the entire genomic sequence is now known. Previous to this sequencing, however, the cDNA sequence and intron/exon sites were identified in the pig. It was found that 24.15 bears a strong homology and very close relationship to 24.16. The two enzymes were over 80% identical and had the great majority of intron splice sites in common. Exons 5-1 6 of 24.16 matched exactly to exons 2- 13 of 24.15 despite varying lengths of introns54. The chromosomal localization of human 24.16 is currently unknown. Substrate specificities differ and the two enzymes cleave at distinct locations (even when they work on the same peptide). We routinely see a single mRNA transcript of 2.5Kbp for Thimet oligopeptidase (aside from the heterogeneous nuclear transcript) in human and monkey brain35. Kato et al. report the porcine 24.15 RNA to exist as a single transcript, in contrast to 24.16 with six transcripts which span 45 kbp in 13 exons54. The fact of a single transcript is interesting considering that 24.15 seems to be located in both the cytosol and nucleus as well as secreted to the extracellular milieu. One might expect different transcripts to cover this function. Pierotti et al. found three potential 5’ starts by primer extension. The size differences of these are small and may explain the single band seen by the relatively poor resolution of Northern blot55. Porcine 24.16 has been shown to make use of alternative splicing and multiple starts of transcription to direct its trafficking to the mitochondria, cytosol, or cell surface54. The different 5’ ends may be alternative start sites and therefore may be responsible for the alternative localization of the enzyme. 24.15 does contain a consensus nuclear localization sequence but its functionality and relationship to our observations and previous studies that have seen 24.15 in the cell nucleus is not known. 18,34,35,56 This may in part be explained by some confusion in the promoter sequence of 24.15. Original Genbank sequences for human 24.15 and other data showed neither a TATA box nor an initiation region for the enzyme (Accession # U29367)55. Porcine 24.15 is also reported to contain no TATA box54. The more recent sequencing by the Lawrence Livermore National Laboratory, however, indicates the presence of a TATA box (Accession # AC006538). There are multiple potential transcription factor consensus sequences but until the actual start of transcription is determined by primer extension analyses, or any discussion of the nuclear regulation of the enzyme is speculation. The protein sequence of 24.15 reveals a host of potential phosphorylation and glycosylation sites. It is currently unknown which, if any, of these are utilized. Other potential protein motifs have not been tested or published.
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CONSERVATION AND TISSUE DISTRIBUTION Assayed by the ability to cleave a test peptide, 24.15 has been found in varying concentrations in nearly every tissue tested. It has been seen in humans, monkeys, rabbits, rats, cows, pigs, plants, and a close homologue exists in yeast57. The greatest amount of study has been in human, rabbit, and rat species. In all organisms, 24.15 seems to be highly associated with rapidly dividing cells, or in tissues undergoing high turnover. Although its role as a mammalian collagenase has been disproven, (Pz-peptide is not a substrate)5, clearly the association of the enzyme to these high-turnover areas and its utility as a marker for tissue re-modeling is strong58,59. The only organ where this does not seem to fit is in the brain, where ironically it is high in all species tested. In fact, the brain contains such high levels of 24.15 that in most animals it is second only to the levels in the testis. These two tissues, the brain and testis have often been seen to display concentrations an order of magnitude higher than all others. The reason for this is unknown. Certainly, the enzyme is not playing a critical role in the brain for cell division, though tissue remodeling leading to memory formation or synapse plasticity is a possibility. Generally, however, the role in the brain is assumed to be as a neuropeptidase. Why 24.15 is found in such high concentrations in the testis is also less than clear. Here its role in cell division/tissue modeling is easier to justify. Equally plausible, though, is the strong connection the enzyme has with hormonal regulation. This will be discussed later. At any rate, the enzyme has been highly conserved over evolution and is likely to serve an important function beyond those already identified. It has been suggested that 24.15 and 24.16 diverged approximately 500 million years ago60. The enzymes have changed remarkably little, it would seem, even since the early eukaryotes. The yeast yscD gene shares nearly 35% open reading frame homology and very similar substrate and inhibitor profiles with 24.1557. Our recent studies have shown that 24.15 is very important to cells in culture. Growth rate and morphology are altered when 24.15 expression is disturbed23,35. Clearly, in culture, cleavage of neuropeptides is not of preeminent importance and so the question remains open as to what else 24.15 may be doing for the cell. Recent data in our laboratory and in Silva et. al.61 provides clues to an important role in cell division itself.
ALZHEIMER’S DISEASE Alzheimer’s disease is a neurodegenerative disorder that takes a tremendous toll on the aging population around the world. In nations with longer life expectancies, and therefore, where a larger percentage of the population is aged, it is seen to be a major health care crisis. Beyond the tragic effect it has on families and individuals, as well as the loss of productivity, costs of health care alone have been estimated to surmount 100 billion dollars in the United States alone. Alzheimer’s disease accounts for 60-70% of dementia cases in Europe as well62. First diagnosed by Alois Alzheimer in 190763, the pathology of the disease is characterized by amyloid plaques, neurofibrillary tangles, dystrophic neurites, microgliosis, and astrocytosis. The amyloid plaque is an extracellular protein deposit composed of the Amyloid E peptide (AE) and surrounded by dystrophic neurites, activated microglia and astrocytes. The neurofibrillary tangle is an intracellular deposition of hyperphosphorylated tau protein that occurs in selected neurons. A review of these deposits is available64 . The Amyloid precursor protein has long been seen as a major factor in AD. The single-pass transmembrane protein has been shown to give rise to the neurotoxic AE, a 3942 amino acid peptide that aggregates to form an SDS insoluble amyloid plaque65. Amyloid is defined as an insoluble protein precipitate of E-pleated sheet structure, which stains with Congo Red to give a classic apple-green birefringence under polarizing light. A large number of proteins have been shown to form amyloid, however, and these lead to the diseases termed amyloidoses. The mechanism for this conversion is not completely understood.
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In the case of the APP, two cleavages must be made in the 100-135 KDa protein to liberate the AE peptide. The enzymes responsible for these cuts have been termed secretases. The E- and J-secretases generate the N- and C-termini of the AE-peptide, respectively. The sequencing of AE in 1984 by Glenner66 paved the way for the cloning and sequencing of the APP molecule. With this, the E-secretase and J-secretase cleavage sites were made apparent. Armed with this sequence, our lab constructed a 10 amino acid synthetic test peptide which flanked the N-terminus of AE . This peptide, known as P1, recapitulated the E-secretase cleavage site. The P1 peptide was used to assay for Esecretase activity in AD brain. The P1 peptide cleavage activity occurring between the methionine and aspartic acid (leaving the aspartic acid as the common N-terminus of Aβ) was purified from AD brain and partially characterized in 1991 67 . In 1992, by means of an inhibitor study, McDermott et al. showed that the cleavage of a shorter test peptide indicated 24.15 to be the enzyme generating the N-terminus of Aβ16. Our purification of the enzyme confirmed these results. We showed that a homogeneous preparation of 24.15 was indeed active against the test peptide and also reported that the protease cleaved recombinant APP17 in vitro. It was shown, in addition, that 24.15 cleaved APP in multiple sites including the β-secretase site68, which may suggest 24.15 is a decision point between an Aβ and a non-Aβ generating event. The data that 24.15 cleaves APP, unfortunately, has not been reliably reproduced by other groups. Koike et al., nevertheless, recently reported that 24.15 was capable of cleaving APP, citing insensitive fluorescent assay schemes for the previous failure of reproducibility18. While this data is compelling, the reports that claim 24.15 is not functional in cleaving native APP make strong arguments69. These issues travel beyond the simple failure to detect 24.15-induced APP cleavage. The largest of these is the repeated observation that 24.15 is severely restricted by substrate size8,70 . Thompson et al. showed no increase in AE formation when 24.15 was transfected with APP into HEK-293 cells68. Specific 24.15 inhibitors did not block AE formation in the same cell line71. When normally cleaved peptides have been increased to beyond 15 or 18 amino acids in length, 24.15 loses all ability to process them. Indeed, no peptide larger than this has ever been shown to be cleaved by the peptidase other than APP. (Studies indicating the cleavage of collagen and gelatin by Pz-peptidase have been disproven). Because of this substrate size restriction it seems unlikely that the APP protein being greater than 100kDa would be cut. Still, as has been discussed previously, the cleavage specificity of 24.15 is far from understood. It is known that the enzyme is active in the presence of a proline-induced chain bend in the substrate50. Therefore, it may be possible that under certain conditions there is a looping out of the β-secretase site of APP either freely or induced by another protein that makes this area uniquely accessible to the action of 24.15. The cloning of a major β-secretase (β-site APP-cleaving enzyme) in 1999, makes 24.15’s role in this process more suspect. Nevertheless, this β-secretase seems to be responsible for only approximately 70% of Aβ-generating activity72,73,74. So it may still be that the remaining E-secretase fraction (or β-secretase activity in other cell types) is a result of 24.15. We sought to further explore the role that 24.15 may have in AD. Our lab generated unique and specific monoclonal antibodies to the enzyme. Strikingly, two antibodies used in immunohistochemistry were shown to strongly label neurofibrillary tangles (NFT), a major pathologic feature of AD. In addition to labeling neurofibrillary tangles, the antibodies were seen to stain neurites of senile plaques as well as neuropil threads34. Given the suggestions for a role of 24.15 in tissue remodeling (as Pz-peptidase), it is interesting that it is found associated with several disturbances of cell shape and microtubule structure seen in AD. Additionally, the metallopeptidase co-localizes with APP in the dystrophic neurites surrounding senile plaques as well34. Therefore the enzyme is importantly, and intimately, involved with both of the two cardinal features of AD. In order to identify the role of 24.15 in cell culture, we used a human neuroblastoma cell line, SKNMC, shown to have certain features in common with neurons. Three stable transfectants were created with this cell line. The transfectants were established with a plasmid alternatively expressing 24.15 cDNA (giving an overexpressing cell line), antisense cDNA to 24.15 (giving an underexpressing cell line), or simply the plasmid vector used for each of the other two (giving the mock cell line)19. Since 24.15 had been implicated as being a possible β-secretase, we expected to see that the overexpressing cell line would produce a higher level of Aβ. Similarly we
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believed the antisense transfectant would show little to no Aβ. The actual results were exactly contrary to our expectations. Conditioned medium from overexpressing cells in fact showed significantly lower levels of Aβ and that of the antisense cells showed higher levels19. It was unclear why this would be. Conditioned medium (CM) of the three cell lines was then incubated in vitro with iodinated synthetic Aβ peptide. The results were consistent. Aβ-degradation was increased after incubation with sense CM and decreased after incubation with the antisense CM. Beyond this, we found that the degradation of Aβ1-42 proceeded more slowly than did the degradation of Aβ 1-40. Furthermore, aggregated Aβ 1-42 showed almost no degradation whatsoever19. Aβ 1-42 has been previously shown to aggregate more quickly than 1-40, therefore it is more amyloidogenic64 . These results were intriguing. We attempted to show absolute specificity by adding a 24.15-specific inhibitor to the incubations. However, we only saw a slight reduction with this inhibitor (approximately 30%). As a result a panel of other protease inhibitors was tested. Again we were surprised, for pretreatment of the CM with serine protease This was done initially with 4-(2inhibitors entirely suppressed Aβ-degradation. aminoethyl)benzenesulfonyl flouride (AEBSF) and diisopropylfluorophosphate (DFP) 19. With a serine protease invoked in a mechanism of Aβ clearance we wondered about the possible effect of α1-antichymotrypsin (ACT). ACT is a serine protease inhibitor of the serpin family. Abraham et al. 1988, showed that ACT mRNA was highly elevated in AD brain75, serum, and cerebrospinal fluid77. ACT was shown to not only co-localize with both diffuse and neuritic amyloid plaques but also to be tightly associated with them. It is also known that ACT increases the rate of Aβ 1-42 fibril formation78,79. Consistently, APP/ACT doubly transgenic mice display a plaque load that is increased over APP singly transgenic animals further implicating ACT in Aβ clearance80. ACT is an acute phase reactant, as well, and rises significantly in an inflammatory response. Inflammation is currently an intensively studied area in Alzheimer’s disease research and seems to play a very significant role in the downstream pathology of AD. Until now, however, the role that ACT might be playing has often been hinted at but remained unclear. This is despite its involvement in Alzheimer’s being depicted as early as 1988. It is nevertheless interesting to speculate that this molecule seems to play a role in several aspects of AD. We therefore sought to determine whether it might also have a role in Aβ-clearance. Our results showed that ACT blocked up to 60% of Aβ-degradation in the assay system. In addition, incubation of ACT with the conditioned medium of overexpressing cells led to the formation of an SDS-resistant complex with the inhibitor. Recombinant 24.15 alone, on the other hand, was not capable of degrading Aβ. Yet if CM of overexpressing cells was treated with radiolabeled diisopropylfluorophosphate there were more resultant serine proteases labeled. In all, the conclusion is that 24.15 activates possibly several serine proteases that are functional in degrading Aβ19. The mechanism of activation of the serine proteases is not currently understood. It is possible that 24.15 cleaves a proenzyme to activate it. Of course for the same reasons that 24.15 is unlikely to be the β-secretase, namely the substrate size restriction of the enzyme, it does not seem reasonable that this is the explanation. A better possibility is that 24.15 cleaves a peptide inhibitor of the one or more serine proteases and therefore activates them. Perhaps also, in a converse manner, 24.15 cleaves a peptide precursor to generate an activator of the serine protease. Currently the mechanism of serine protease activation remains unclear. We are seeking now to isolate the serine protease(s), activated by 24.15, likely to be a final step in Aβ clearance. The possibility exists, of course, that if the serine protease can be induced (perhaps by the induction of 24.15), Aβ levels can be reduced. This would lead to a lower plaque load and reduce the severity and progression of the disease. Similarly, if ACT indeed plays as important a role as it seems to, the increased activation of the serine protease may draw ACT away from its role in inflammatory mediation and Aβ deposition by mass action. With less Aβ, there will be an additional decrease in the resultant microgliosis and astrocytosis. With fewer reactive astrocytes, ACT release is likely to be slowed thus ending the vicious cycle of Aβ deposition and pathology as described81. It has been shown that 24.15 declines significantly in AD82. If so, it would be reasonable to expect that the serine protease declines as well. This may or may not be a causative event but it would seem likely to at least make a contribution to the pathology.
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OTHER POTENTIAL ROLES AS A NEUROPEPTIDASE The fact that 24.15 cleaves Somatostatin, Substance P, Neurotensin, Neoendorphins, Dynorphin, Metorphinamide, GnRH, and other neuropeptides is striking. Even more striking are the roles of these peptides in many degenerative diseases including AD. Somatostatin, for example, has been shown to be significantly deficient in AD83. It is important for memory and experimental lowering triggers a severe impairment of memory71. Therefore, if connections can be drawn between these many disparate peptides acted upon by 24.15, they can lead to functional clues. These insights may indicate the specific overarching role that 24.15 is playing. We expect this function to be critical to both normal and pathologic physiology. We will take a brief look here at some of the more interesting of these connections.
Pain and Analgesia It has been noted already that 24.15 acts upon α - and β-Neoendorphin, Metorphinamide, and Dynorphin A. It has also been shown that by selectively inhibiting the metallopeptidase, rats display an increased pain threshold and tail-flick latency84a as would be expected. These effects can be blocked by administration of the opiate antagonist, naloxone. So it may be assumed, by this alone, that 24.15 plays an important role in analgesia. In the degradation of these peptides by 24.15, however, the enzyme in turn generates both Met-and Leu-enkephalins. These latter two peptides display analgesic properties of their own including antinociception84b. These different opioids are suspected of mediating different types of analgesic effects in the brain. Because 24.15 is capable of converting one type of opioid peptide into another, it may be in part responsible for tailoring the analgesic response to match the organism’s need. There is greater significance to 24.15 than its action on opioid peptides alone however, for the enzyme acts on at least four other distinct analgesia-mediating pathways. The Neurotensin/Neuromedin system and the Orphanin system both point to 24.15 as a major regulator. Similarly, Bradykinin and Substance P mediate pain sensation and processes and are effectively degraded by the same enzyme32,85. What might 24.15 be doing that it is so intimately linked to at least 5 different pain and analgesia systems? Clearly, it begs the question whether some or even all of these pathways are linked. It is possible that regulation of this single peptidase could be in part responsible for a coordinated analgesic response. It is also not unreasonable to suggest that these peptides may be intimately linked with learning and memory processes. Therefore it may be a foregone conclusion that 24.15 plays a unique role in mediating these learning and memory functions. Looking at its role in Aβ-clearance may be enough in itself to make this case. Soluble Aβ has been seen to be associated with synaptic loss86. Additionally, it takes only a small leap to argue that a misregulation or dysfunction of this enzyme could be a serious contributing factor to memory loss, learning difficulties, and neurodegeneration. Estrogen, which is under the influence of 24.15 as discussed below, has a clear role in learning as well87. Or, on the other side of the coin, that 24.15 could be a valuable target for the remedy of these ailing systems. Remarkably, little work has explored these possibilities.
Hormonal Implications Prolyl Endopeptidase and 24.15 have been long known to be the two principal posttranslational regulators of Gonadotropin Releasing Hormone (GnRH.). This critical peptide is responsible for determining the secretion of Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH). LH and FSH, in turn, determine the pattern of secretion of all the major sex hormones of the body including estrogen, progesterone, testosterone and their derivatives. For this reason GnFW has been called the “Master Hormone”. The involvement of 24.15 here is compelling for a variety of other indications surrounding the enzyme in both a reproductive as well as neural sense. 24.15 is routinely seen at highest concentrations in the testis. Why? It is unclear, but it is known that both enkephalins and endorphins (generated and degraded by 24.15, respectively) are potent activators of smooth muscle tone in the male reproductive tract.
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Similarly, Substance P (also degraded by 24.15) has been shown to have a role in increasing contractility of vas deferens smooth muscle88 . In addition, 24.15 (studied then as Pz-peptidase), was shown to double in activity in uterine tissue during the course of gestation and then double again during labor. It has been long known that estradiol plays a very large role in neural remodeling and cognition. Studies of post-menopausal women who are given estradiol exogenously routinely fare better on tests for cognitive function and decline. Pertinent to our own research in AD, women who received estradiol post-menopausally demonstrated an increased age of disease onset and a slower rate of progression towards dementia89. The role of estradiol is still unclear but the effect is pronounced. One very interesting side note to the possible involvement of 24.15 in a hormonally regulatory role is found with female athletes. High performance female athletes frequently experience amenorrhea90. This is believed to result from a lack of GnRH pulsatility.91 GnRH pulsatility has been suggested to be mediated by an autofeedback loop whereby the GnRH breakdown products (after Prolyl Endopeptidase and 24.15 processing) may act to modulate GnRH release92. Endorphin and enkephalin release are pronounced in these types of activities. Therefore 24.15 is in a key position on each side of the issue. Does the athleticism and toll on the body result in a change in 24.15 levels such that greater enkephalins are produced and consequently GnRH is shut down? Or is it that the enkephalin/endorphin-release trigger a 24.15 response that reacts consequently on GnRH This is also unknown. But here we see as well that the enzyme is uniquely poised to be a key player in this physiology.
Inflammation role Inflammation has been repeatedly proven to be a critical player in a number of neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, Amyotrophic Lateral Sclerosis, Multiple Sclerosis, and many others. Strangely, this diverse metallopeptidase 24.15 has a number of potential connections to the role of inflammation as well. Substance P, Neurotensin, endorphins93 and Bradykinin32 (all degraded by 24.15 as discussed) are primary mediators of the inflammatory response. Substance P induces cytokine release and activates mast cells93a. Neurotensin has been seen to bind to peritioneal macrophages and modulate phagocytic activation93. Enkephalins and endorphins stimulate interferon release, chemotaxis, superoxide production, antibodydependent cytotoxicity and a variety of other functions93 . Bradykinin is markedly increased in human skin during severe inflammation. It induces vasodilation, prostacyclin synthesis, and leads to intracellular calcium influx32. Also at least one paper reported that PGE2 upregulates 24.1594. It is known that chronic users of non-steroidal antiinflammatory drugs (NSAIDS) are consistently seen to have a reduced incidence of AD, and a consequent diminished rate of progression to dementia. Therefore, the effect of PGE2 on 24.15 (which is in turn inhibited by NSAIDS) is another possible connection that travels hand in hand with the degradation of the other neuropeptides that are also strong inflammatory mediators. Most intriguing of all, however, is the recent 1999 data by Silva et al. In their studies of the potential role of 24.15 in antigen presentation, this group performed an experiment theoretically similar to our own with Alzheimer’s disease. In one series of experiments they used a liposome transfection method to transfer 24.15 directly into T-cells. T-cells which were transfected with the enzyme had a diminished doubling time (ie divided more rapidly), whereas introduction of 24.15 specific inhibitors had the opposite effect. They also showed that the 24.15-containing immune cells were more efficient at killing a mycobacterium-containing macrophage. Again, the 24.15 inhibitor blocked this effect61. This correlates very well to our own work where we see neuroblastoma cells transfected with 24.15 antisense RNA grow at a significantly reduced rate compared to overexpressing cells35. In another set of experiments published side by side with this one they showed that 24.15 bound tightly to a set of randomly generated peptides23 . However, the enzyme only actually cleaved a very small fraction of them. This led the group to hypothesize that 24.15 may be instrumental in trafficking these peptides. Instead of cleaving them, it acts to protect the majority of them from cleavage. The antigens are transported intact to the antigen presenting machinery of the cell. Rather than modifying
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the post-proteasomally processed peptides to make them suitable for presentation, 24.15 may somehow select the appropriate antigens and then protect them on route to the cell surface. Other work in our own laboratory with the protease Bleomycin Hydrolase (BH) has suggested that both BH and 24.15 may be considered as “chaperases”. BH and 24.15 both serve on the one hand as a protease or peptidase, and on the other hand with the ability to act as a chaperone to a protein or peptide, respectively95. The specifically coordinated role of 24.15 in inflammation and antigen presentation is being actively pursued. Obviously much work here remains and speculation still dominates the field.
FUTURE DIRECTIONS Much work needs to be done to clarify all of these issues and fully define the role 24.15 is playing. The connections it makes to a wide variety of disease processes are exciting. Currently, its greatest potential role is in halting or reversing the causes and effects of neurodegeneration. But beyond this, great discoveries are forthcoming in the fields of immunology and endocrinology. The possibilities for useful interventions and therapies through this enzyme are significant. The very nature of the ties it makes between physiological systems suggest that it may serve to draw together a more unified understanding of the interplay and integration of the mind and health and the mediation by the messengers that travel between.
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16. J.R. McDermott, J.A. Biggins, and A.M. Gibson, Human brain peptidase activity with the specificity to generate the N-terminus of the Alzheimer beta-amyloid protein from its precursor, Biochem. Biophys. Res. Comm. 185:746 (1992). 17. G. Papastoitsis, R. Siman, R. Scott, and C.R. Abraham, Identification of a metalloprotease from Alzheimer's disease brain able to degrade the beta-amyloid precursor protein and generate amyloidogenic fragments, Biochemistry 33: 192 (1994). 18. H. Koike, H. Seki, Z. Kouchi, M. Ito, T. Kinouchi, S. Tomioka, H. Sorimachi, T.C. Saido, K. Maruyama, K. Suzuki, and S. Ishiura, Thimet oligopeptidase cleaves the full-length Alzheimer amyloid precursor protein at a beta-secretase cleavage site in COS cells, J. Biochem. 126:235 (1999). 19. R. Yamin, E.G. Malgeri, J.A. Sloane, W.T. McGraw, C.R. Abraham, Metalloendopeptidase EC 3.4.24.15 is necessary for Alzheimer's amyloid-beta peptide degradation., J. Biol. Chem. 274: 18777 (1999). 20. C.R. Abraham, D.J. Selkoe, and H. Potter, Immunochemical identification of the serine protease inhibitor alphal-antichymotrypsin in the brain amyloid deposits of Alzheimer's disease, Cell 52:487 (1988). 21. A.I. Smith, T. Tetaz, J.L. Roberts, M. Glucksman, I.J. Clarke, R.A. Lew, The role of EC 3.4.24.15 in the post-secretory regulation of peptide signals, Biochimie 76:288 (1994). 22. B. Horsthemke and K. Bauer, Characterization of a nonchymotrypsin-like endopeptidase from anterior that hydrolyzes luteining hormone-releasing hormone at the tyrosyl-glycine and histidyl-tryptophan bonds, Biochemistry 19:2867 (1980). 23. F.C. V. Portaro, M.D. Gomes, A. Cabrera, B.L. Fernandes, C.L. Silva, E.S. Ferro, L. Juliano, and A.C.M. Camargo, Thimet oligopeptidase and the stability of MHC class I epitopes in macrophage cytosol, Biochem. Biophys. Res. Comm . 255:596 (1999). 24. I.A.York, A.L. Goldberg, X.Y. Mo, K.L. Rock, Proteolysis and class I major histocompatibility complex antigen presentation, Immunol. Rev. 172:49 (1999). 25. C.N. Shrimpton, M.J. Glucksman, R.A. Lew, J.W. Tullai, E.H. Margulies, J.L. Roberts, and A.I. Smith, Thiol activation of endopeptidase EC 3.4.24.15. A novel mechanism for the regulation of catalytic activity, J. Biol. Chem. 272:I7395 (1997). 26. M. Orlowski, S. Reznik, J. Ayala, and A.R. Pierotti, Endopeptidase 24.15 from rat testes. Isolation of the enzyme and its toward synthetic and natural peptides, including enkephalin-containing peptides, Biochem. J. 261:951 (1989). 27. K. Mizuno, A. Miyata, K. Kangawa, H. Matsuo, A unique proenkephalin-converting enzyme purified from bovine adrenal chromaffin granules, Biochem. Biophys. Res. Comm. 108:1235 (1982). 28. A. Thompson, G. Huber, and P. Malherbe, Cloning and functional expression of a metalloendopeptidase from human brain the ability to cleave a beta-APP substrate peptide, Biochem. Biophys. Res. Comm. 213:66 (1995). 29. U. Tisljar and A.J. Barrett, Purification and characterization of Pz-peptidase from rabbit muscle, Arch. Bioch. Biophys. 274:138 (1989). 30. A.C.M. de Camargo, H.Caldo, and P.C. Emson, Degradation of neurotensin by rabbit brain endooligopeptidase A and endo-oligopeptidase B (proline-endopeptidase), Biochem. Biophys. Res. Comm. 116:1151 (1983). 31. A.J. Barrett, A new look at Pz-peptidase, Biol. Chem. Hoppe-Seyler 371: Suppl:311 (1990). 32. M. Kikuchi, K. Fukuyama, and W.L. Epstein, Purification and characterization of bradykininhydrolyzing enzyme from 2-day-rat epidermis, Biochim. Biophys. Acta 965: 176 (1988). 33. A.C.M. de Camargo, E.B. Oliveira, O. Toffoletto, K.M. Metters, J. Rossier, Brain endo-oligopeptidase A, a putative enkephalin converting enzyme, J. Neurochem. 48: 1258 (1987). 34. K.J. Conn, M. Pietropaolo, S.-T. Ju, and C.R. Abraham, Monoclonal antibodies against the human metalloprotease EC 3.4.24.15 label neurofibrillary tangles in Alzheimer's disease brain, J. Neurochem. 66:2011 (1996). 35. F. Slot, and C.R. Abraham, unpublished results 36. P.M. Dando, M.A. Brown, A.J. Barrett, Human thimet oligopeptidase, Bioch. J. 294(2):451 (1993). 37. S.R. Sahasrabude, A.M. Brown, J.D. Hulmes, J.S. Jacobsen, M.P. Vitek, A.J. Blume, J.S. Sonnenberg, Enzymatic generation of the amino terminus of the beta-amyloid peptide, J. Biol. Chem. 268: 16699 (1993). 38. M.S. Medeiros, N. Iazigi, A.C.M. Camargo, and E.B. Oliveira, Distribution and properties of endooligopeptidases A and B in the human neuroendocrine system, J. Endocrinol. 135:579 (1992). 39. E. S. Oliveira, P.E.P. Leite, M.G.Spillantini, A.C.M.Camargo, and S.P. Hunt, Localization of endooligopeptidase (EC 3.4.22.19) in the rat nervous tissue, J. Neurochem. 55:1114 (1990).
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Carmela R. Abrahamand Franchot Slot K. Sakyo, J. Kobayashi, A. Ito, and Y. Mori, Partial purification and characterization of gelatinase and metal dependent peptidase from rabbit uterus and their synergistic action on gelatin in vitro, J. Biochem 94:1913 (1983). U. Tisljar and A.J. Barrett, A distinct thimet peptidase from rat liver mitochondria, FEBS Lett. 264:84 (1990). B. A. Lessley and D.L. Garner, Purification and characterization of Pz-peptidase B, a neutral metalloendopeptidase from bovine spermatozoa, Biol. Reprod. 43:643 (1990). J.-M. Chen, A. Changco, M.A. Brown, and A.J. Barrett, Immunolocalization of thimet oligopeptidase in chicken embryonic fibroblasts, Exper. Cell Res. 216:80 (1995). A.J. Barrett, M.A. Brown, P.M. Dando, C.G. Knight, N. McKie, N.D. Rawlings, and A. Serizawa, Thimet oligopeptidase and oligopeptidase M or neurolysin, Methods Enzym. 248:529 (1995). A. J. Wolfson, C.N. Shrimpton, R.A. Lew, and A.I. Smith, Differential activation of endopeptidase EC 3.4.24.15 toward natural and synthetic substrates by metal ions, Biochem. Biophys. Res. Comm. 229:341 (1996). G. Bartzokis, D. Sultzer, J. Cummings, L.E. Holt, D.B. Hance, V.W. Henderson, J. Mintz, In vivo evaluation of brain iron in Alzheimer disease using magnetic resonance imaging, Arch. Gen. Psychiatry 57:47 (2000). G.A. Trapp, G.D. Miner, R.L. Zimmerman, A.R. Mastri, L.L. Heston, Aluminum levels in brain in Alzheimer’s disease, Biol. Psych. 13:709 (1978). M. Orlowski, C. Michaud, and C.J. Molineaux, Substrate-related potent inhibitors of brain metalloendopeptidase, Biochem. 27:597 (1988). J. Jiracek, A. Yiotakis, B. Vincent, A. Lecoq, A. Nicolaou, F. Checler, V. Dive, Development of highly potent and selective phosphinic peptide inhibitors of zinc endopeptidase 24-1 5 using combinatorial chemistry, J. Biol. Chem. 270:2 1701 (1995). A.C.M. Camargo, M.D. Gomes, A.P. Reichl, E.S. Ferro, S. Jacchieri, I.Y. Hirata, and L. Juliano, Structural features that make oligopeptides susceptible substrates for hydrolysis by recombinant thimet oligopeptidase, Biochem J. 324:517 (1997). P.M. Cummins, A. Pabon, E.H. Margulies, M.J. Glucksman, Zinc coordination and substrate catalysis within the neuropeptide enzyme endopeptidase EC 3.4.24.15. Identification of active site histidine and glutamate residues, J. Biol. Chem. 274: 16003 (1999). B. Meckelein, H.A.R. de Silva, A.D. Roses, P.N. Rao, M.J. Pettenati, P.-T. Xu, R. Hodge, M.J. Glucksman, and C.R. Abraham, Human endopeptidase (THOP1) is localized on chromosome 19 within the linkage region for the late-onset alzheimer disease AD2 locus, Genomics 3 1:246 (1996). F. Slot, A.S. Olsen, P.N. Rao, and C.R. Abraham, Localization of human endopeptidase EC 3.4.24.15 (THOP1) to chromosome 19p13.3, Alzheim. Rep. 1:327 (1998). A. Kato, N. Sugiura, Y. Saruta, T. Hosoiri, H. Yasue, and S. Hirose, Targeting of endopeptidase 24.16 to different subcellular compartments by alternative promoter usage, J. Biol. Chem. 272: 153 13 (1997). A. Pierotti, K.-W. Dong, M.J. Glucksman, M. Orlowski, and J.L. Roberts, Molecular cloning and primary structure of rat testes metalloendopeptidase 3.4.24.15, Biochem 29:10323-10329 (1990). D. P. Healy and M. Orlowski, Immunocytochemical localization of endopeptidase 24.15 in rat brain, Brain Res. 571:121 (1992). M. Buchler, U. Tisljar, and D.H. Wolf, Proteinase yscD (oligopeptidase yscD). Structure, function and relationship of the yeast enzyme with mammalian thimet oligopeptidase (metalloendopeptidase, EP 24.15), Eur. J. Biochem. 219:627 (1994). J.F. Woessner Jr., Collagen remodeling in chick skin embroygenesis, in: Chemistry and Molecular Biology of the Intercellular Matrix, Vol. 3, E.A. Balazs, ed., Academic Press, New York (1970). E. Keiditsch and L. Strauch, Peptidase and collegenase activities in invasion zones of tumors of the brease, in: Chemistry and Molecular Biology of the Intercellular Matrix, Vol. 3, E.A. Balazs, ed., Academic Press, New York (1970). A. Serizawa, P.M. Dando, and A.J. Barrett, Characterization of a mitochondrial metallopeptidase reveals neurolysin as a homologue of thimet oligopeptidase, J. Biol. Chem. 270:2092 (1995). C.L. Silva, F.C.V. Portaro, V.L.D Bonato, A.C.M. de Camargo, and E.S. Ferro, Thimet oligopeptidase (EC 3.4.24.15), a novel protein on the route of MHC class I antigen presentation, Biochem. Biophys. Res. Comm. 255:591 (1999). L.J. Launer, L. Fratiglioni, K. Andersen, M.M. B. Breteler, R.J.M. Copeland, J.-F. Dartigues, A. Lobo, J. Martinez-Lage, H. Soininen, and A. Hofman, in: Alzheimer’s disease and Related Disorders, Iqbal et al. Eds., John Wiley and Sons, LTD. (1999). A. Alzheimer, Ueber eine eigenartige Erkrankung der himrinde. Algemeine Zeitschrift fuer Psychiatrie und Psychisch-Gerichtliche Medizin. 64:146 (1907).
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84b. B. Kest, M. Orlowski, R.J. Bodnar, Increases in opioid-mediated swim antinociception following endopeptidase 24.15 inhibition, Physiol. Behav. 50:843 (1991). 85. R. Mentlein, P. Dahms, Endopeptidases 24.16 and 24.15 are responsible for the degradation of somatostatin, neurotensin, and other neuropeptides by cultivated rat cortical astrocytes, J. Neurochem 62:27 (1994). 86. L.F. Lue, Y.M. Kuo, A.E. Roher, L. Brachova, Y. Shen, L. Sue, T. Beach, J. H. Kurth, R.E. Rydel, J. Rogers, Soluble amyloid beta peptide concentration as a predictor of synaptic change in Alzheimer's disease, Am. J. Pathol. 155:953 (1999). 87. R.B. Gibbs, Estrogen replacement enhances acquisition of a spatial memory task and reduces deficits associated with hippocampal muscarinic receptor inhibition, Horm. Behav. 36:222 (1 999). 88. B.V. R. Sastry, V.E. Jenson, L.K. Owens, and O.S. Tayeb, Enkephalin- and substance P-like immunoreactivities of mammalian spenn and accessory sex glands, Biochem. Pharm 31:3519 (1982). 89. A.J. Slooter, J. Bronzova, J.C. Witteman, C. Van Broeckhoven, A. Hofman, C.M. van Duijn, Estrogen use and early onset Alzheimer's disease: a population-based study, J. Neurol. Neurosurg. Psych. 67:779 (1999). 90. C. De Cree, Comment on health issues for women athletes: exercise-induced amenorrhea, J. Clin. Endocrinol. Metab. 84:4750 (1999). 91. G.A. Laughlin, S.S. Yen, Nutritional and endocrine-metabolic aberrations in amenorrheic athletes, J.Clin. Endocrinol. Metab. 81:4301 (1996). 92. C. Yamanaka, M.C. Lebrethon, E. Vandersmissen, A. Gerard, G. Purnelle, M. Lemaitre, S. Wilk, J.P. Bourguignon, Early prepubertal ontogeny of pulsatile gonadotropin-releasing hormone (GnRH) secretion: I. Inhibitory autofeedback control through prolyl endopeptidase degradation of GnRH, Endocrin. 140:4609 (1999). 93. M. Lesser, K. Fung, H.S. Choi, O.H. Yoo, C. Cardozo, Identification of two zinc metalloendopeptidases in alveolar macrophages of rats, guinea pigs, and human beings, J. Lab. Clin. Med. 120:597 (1992). 93a. J.C. Ansel, C.A. Armstrong, I. Song, K.L. Quinlan, J.E. Olerud, S.W. Caughman, N. W. Bunnett, Interactions of the skin and nervous system, J. Invest. Dermatol. Symp. Proc. 2(1):23 ( 1997). 94. T. Chikuma, Y. Ishii, T. Kato, H. Kodama, Y. Hakeda, M. Kumegawa, Effect of prostaglandin E2 on PZ-peptidase and several other peptidase activities in a clonal osteoblast-like cell line derived from newborn mouse calvaria, J. Biochem. Tokyo 97: 1533 (1985). 95. W.T. McGraw, R. Yamin, E.A. Berg, M. Gartner, S. Keve, E.M. Schaefer, R.E. Fine, and C.R. Abraham, Bleomycin Hydrolase Modulates the Maturation and Trafficking of the Amyloid Precursor Protein and the Secretion of Ab in a Dose-dependent Manner, (submitted). 96. M.A. Cicilini, M.J. Ribeiro, E.B. de Oliveira, R.A. Mortara, A.C. de Camargo, Endooligopeptidase A activity in rabbit heart: generation of enkephalin from enkephalin containing peptides, Peptides 9:945 (1988). 97. T. G. Chu, M. Orlowski, Soluble metalloendopeptidase from rat brain: action on enkephalin-containing peptides and other bioactive peptides, Endocrinology 116:1418 (1985). 98. J.L. Montiel, F. Cornille, B.P. Roques, F. Noble, Nociceptin/orphanin FQ metabolism: role of aminopeptidase and endopeptidase 24.15, J. Neurochem. 68:354 (1997). 99. G.R. Acker, C. Molineaux, M. Orlowski, Synaptosomal membrane-bound form of endopeptidase-24.15 generates Leu-enkephalin from dynorphin1-8, alpha- and beta-neoendorphin, and Met-enkephalin from Met-enkephalin-Arg6-Gly7-Leu8, J. Neurochem. 48:284 (1987). 100. C.J. Molineaux, A. Lasdun, C. Michaud, M. Orlowski, Endopeptidase-24.15 is the primary enzyme that degrades luteinizing hormone releasing hormone both in vitro and in vivo, J. Neurochem. 51:624 (1988). 101. M.G. Oakes, T.P. Davis, The ontogeny of enzymes involved in post-translational processing and of neuropeptides, Br. Res. Dev. Br. Res. 80: 127 (1994).
CYSTEINE PROTEASES, SYNAPTIC DEGENERATION AND NEURODEGENERATIVE DISORDERS
Mark P. Mattson and Sic L. Chan Laboratory of Neurosciences National Institute on Aging Baltimore, MD 21224
INTRODUCTION Neurons communicate with each other at highly specialized structures called synapses and, accordingly, receptors for neurotransmitters, neurotrophic factors and some cytokines are concentrated in synaptic terminals. Signaling at synapses plays or controls all of our behaviors including processes such as learning and memory, and is also critical for the growth and survival of neurons. Recent findings suggest that synapses are sites where the neurodegenerative process begins in disorders ranging from Alzheimer’s, Parkinson’s and Huntington’s diseases, to stroke. Apoptosis (a form of programmed cell death) and excitotoxicity (resulting from overactivation of glutamate receptors) may occur in neurons in such disorders. Recent findings indicate that apoptotic and excitotoxic biochemical cascades are activated in synaptic terminals in experimental models of neurodegenerative disorders. Proteases of the caspase and calpain families are implicated in the neurodegenerative process, as their activation can be triggered by calcium influx and oxidative stress. Caspases cleave a variety of substrates including cytoskeletal proteins, kinases, cell surface receptors, members of the Bcl-2 family of apoptosis-related proteins, preseniliis, amyloid precursor protein, and DNA-cleaving enzymes. Calpains degrade cytoskeletal and associated proteins, kinases and phosphatases, membrane receptors and transporters, and steroid receptors. Many of these substrates are located in pre- and/or postsynaptic compartments of neurons wherein they play roles in modulating synaptic transmission and plasticity. Emerging data suggest that excessive cleavage of synaptic proteins by cysteine proteases mediates synaptic degeneration and neuronal cell death in several different neurodegenerative disorders. Accordingly therapeutic strategies are being developed that are aimed at preventing caspase and/or calpain activation or inhibiting the activated proteases. CHARACTERISTICS OF CASPASES Caspase-like proteases were first discovered in studies of genes that regulate programmed cell death in the nematode C. elegans1. The first mammalian caspase identified was interleukin-1b converting enzyme (ICE), a homolog of the C. elegans cell death protease CED-3, which cleaves the 31 kDa pro-form of interleukin-1b to produce a 18 kDa active form of interleukin- 1b2. ICE is now called caspase- 1, and is one member of a family of at least 14 cysteine proteases that share the general function of controlling the process of programmed cell death or apoptosis. As is the case with other cysteine proteases, the proteolytic activity of caspases is dependent on protein dimerization and a catalytic dyad Role of Proteases in the Pathophysiology of Neurodegenerative Diseases, edited by Lajtha and Banik. Kluwer Academic/Plenum Publishers, New York, 2001.
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composed of a cysteine residue in close proximity to a histidine residue. Amino acids from both caspase subunits contribute to this active site, and subunits of different caspase family members can combine to form novel tetramers with divergent substrate specificities3,4,5 Caspases 1-7 and 11-14 contain the highly conserved pentapeptide sequence QACRG at the catalytic dyad. Substrate recognition depends on the sequence of four amino acids Nterminal to the critical aspartate residue at the PI position of a consensus cleavage site in all known caspase substrates6. The phosphorylation of amino acids adjacent to the caspase cleavage site in substrate proteins can modify the ability of the protease to cleave the substrate7. The caspase family can be subdivided into the ICE-like proteases (caspases 1, 4, 5, 11, 13 and 14) which prefer substrates with bulky hydrophobic amino acids at P4 and have the consensus cleavage sequence (YWL)EHD. Caspases 2, 3 and 9 belong to the CPP32like subfamily, which shows preference for aspartate at the P4 position and the DXXD consensus sequence. The third subfamily (caspases 6, 8, 9 and 10) prefers branched-chain aliphatic amino acids at P4 and prefers the consensus sequence (IVL)(QE)XD. There is considerable overlap in the substrates cleaved by members of the different subfamilies of caspases, which may ensure rapid and effective substrate processing in cells that express multiple caspases. The pivotal role of the PI and P4 residues to substrate specificity of caspases has permitted the design of specific synthetic tri- and tetrapeptide aldehyde caspase inhibitors. Experiments with such caspase inhibitors have proven invaluable in establishing the roles of caspases in various physiological and pathological processes. For example, zVAD-fmk and YVAD-fmk peptides are capable of inhibiting most caspases, DEVD-fmk specifically inhibits caspases 3 and 7, and VEID-fmk inhibits caspase 68 . Although caspases are present at high levels in the cytoplasm, they can also be localized in the nucleus, mitochondria and endoplasmic reticulum9,10, The inactive procaspase forms of caspases I and 2 contain nuclear localization signals11,12, suggesting that they can be transported to the nucleus. Subcellular compartmentalization of caspases could be an important determinant not only of their activation, but also substrate specificity (see below) and the ultimate cellular response. Caspases may also be associated with membranes, and a membrane-associated form of procaspase-3 was recently described13. Moreover, some caspases may act extracellularly as indicated by their association with cell surfaces14. Different caspases exhibit different patterns of cellular expression, and increasing data suggest that caspase expression is regulated spatially and temporally depending on cell type and developmental stage. Levels of mRNAs encoding caspases 2,3,10 and 14 mRNAs are quite high in many different embryonic tissues including the brain, and then decrease markedly during postnatal development15,16,17,18 . In comparison with many other types of cells, mature neurons express lower levels of caspases 2,6, 7 and 8 under basal conditions. As is the case in most cells, levels of caspase mRNA and protein are increased in neurons in response to various environmental insults. Thus, levels of mRNAs for caspases 1, 2 and 3 are increased in cortical and hippocampal neurons following cerebral ischemia19,20,21 and traumatic brain injury22. Levels of caspase-3 mRNA are increased in cultured cerebellar granule neurons incubated in medium containing a low concentration of potassium (a nondepolarizing condition that induces apoptosis in these cells)17; inhibitors of RNA and protein synthesis can prevent apoptosis in this paradigm23,24 suggesting a role for increased caspase production in the cell death process. As is the case with other cell types, most neurons express multiple caspases that may act at different stages of the apoptotic process. Moreover, because one type of caspase can cleave and thereby activate proforms of other caspases, the presence of multiple caspases in a single cell may provide an amplification mechanism to ensure rapid death and elimination of the cell. Caspases are synthesized as an inactive polypeptide with one large and one small subunit joined by a small spacer and a variable N-terminal prodomain which varies in size and sequence. The prodomain serves the function of preventing unwanted protease activation in healthy cells and controls protease activation by interacting with cofactors during apoptosis. When a cell receives an apoptotic stimulus, autoproteolytic processing removes both the spacer and prodomain resulting in a heterodimer comprised of small and large subunits. X-ray crystallographic analyses have revealed the structures of caspases 1 and 3 in complex with peptide-based inhibitors; in both cases the active protease is a tetrameric complex formed by two self-associating heterodimers interacting via the small subunits25,26. This tetrameric structure of the active enzyme complex suggests a requirement
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for multimerization of pro-caspase molecules during processing and activationz27. Additional studies have shown that the long prodomains in caspases 8, 9 and 10 are able to mediate dimerization of procaspase molecules and promote autoprocessing28. Accordingly, overexpression of these pro-caspases induces cell death that is dependent on their autocatalytic activity. Co-factors that modulate pro-caspase multimerization may therefore act as regulators of apoptosis29.
REGULATION OF CASPASE ACTIVITY There appear to be many different mechanisms whereby cells regulate caspase activity, positively or negatively. From the perspective of their positioning in apoptotic biochemical cascades, caspases can be classified as either initiator caspases (caspases 2, 8, 9, and 10) or effector caspases (caspases 3,4, 5, 6,7, 11, 12, and 13). One mechanism for positive modulation of caspase activity involves autocatalysis which was first suggested by the observation that initiator caspases have substrate specificities that are similar to caspase recognition sites present in their own sequence. Several pathways for activation of initiator caspases have been identified (Fig. 1). One pathway involves receptor-mediated activation of Fas-activated death domain (FADD)-associated caspase 8, which is activated by trimerization of death receptors such as Fas and tumor necrosis factor receptors (TNFR). Receptor engagement results in recruitment of death effector domain (DED)-containing procaspases (caspases 8 and 10) into a complex with the death receptor via the adapter protein FADD/MORT. This results in multimerization of pro-caspases followed by autoprocessing via transproteolysis. Activation of caspases 8 and 10 by this mechanism thus initiates an autocatalytic cascade because both of these caspases are capable of activating themselves and other caspases30. A second pathway of caspase activation involves postmitochondrial (cytochrome c-mediated) activation of caspase-9, a death-receptor independent process. Data suggest that cytochrome c, together with Apaf- I and cas ase-9, mediates the proteolytic activation of caspase-3 in an ATP-dependent manner31,32,33. The amount of caspase-8 generated at the receptor, which may vary between cell types, determines whether a mitochondria-dependent pathway is required for amplification of the caspase cascade34. Cytochrome c release is amplified by a caspase-8 dependent mechanism that involves cleavage and translocation of a protein called Bid to mitochondria35. Two prominent triggers of neuronal apoptosis that may play central roles in an array of neurodegenerative disorders are reactive oxygen species and calcium36,37. Exposure of cultured neurons to agents that induce calcium influx through plasma membrane channels (e.g., glutamate) or calcium release from endoplasmic reticulum (eg, thapsigargin) can induce apoptosis, and drugs that suppress calcium influx can prevent neuronal apoptosis38,39. Similarly, agents that induce oxidative stress (e.g., Fe2+ and amyloid b-peptide) can induce neuronal apoptosis, and antioxidants prevent such cell death40. Several endogenous caspase inhibitors have been identified including FADD-like ice (FLICE)-inhibitory proteins (FLIPS), inhibitor of apoptosis proteins (IAPs), CrmA and Bcl2. FLIPS are cytosolic caspase-like proteins that have two death domains at their aminoterminus and a caspase-like domain with significant homology to caspase-841. FLIPs inhibit caspases 8 and 10 by binding to the death domains of the caspase which interferes with the recruitment of caspases 8 and 10 to FADD. FLIPS are differentially expressed among cell types, and their expression can be regulated in ways suggesting an important role for changes in FLIP expression in differential susceptibility to apoptosis mediated by death receptors42,43,44. Another mechanism of caspase inhibition involves roduction of different splice variants of caspases that serve to inhibit caspase activity 45,46,3,47,18. For example, a form of caspase-9 which lacks the large subunit containing the catalytic domain functions as an anti-apoptotic protein by specifically blocking the Apaf-I-mediated activation of caspase 348. This form of caspase-9 may also 49form a heterodimeric complex with caspase-9 that would result in a nonfunctional enzyme . Short forms of caspases 2 and 6 can protect transfected cells from apoptosis by acting as dominant inhibitors of caspase activity45,4. Interestingly, analyses of mice lacking caspase-2 have shown that germ cells, but not motor or sympathetic neurons, are resistant to apoptosiss50, suggesting that activity of specific caspase splice variants modulates cell death and survival in the absence of caspase-251.
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Figure 1. Mechanisms of caspase activation and inactivation. Engagement of "death receptors" such as those for CD95 and tumor necrosis factor (TNF) receptor 1 stimulates the Fas-activated death domain (FADD), which then recruits caspases 8 and 10. The latter caspases, which contain death domains, are then proteolytically activated and released from the receptors and are then able to activate effector caspases such as caspases 3 and 7. Caspase 8 can also cleave Bid (l), which then translocates to mitochondria and induces cytochrome c release3. Cytochrome c combines with Apaf- I to activate caspase 95, which then cleaves downstream procaspases 3 and 7. Receptor-independent apoptotic signals, such as calcium and reactive oxygen species (ROS), can also induce cytochrome release (2) or can act in the nucleus to release yet unknown apoptogenic factors (7). Bc1-2 and Bc1-XL can block caspase activation at various steps. Calcium induces a conformational change in calpains, which subsequently translocate to the plasma membrane where both subunits undergo autocatalytic conversion at the N-termini to form the 76- and 18-kDa subunits. Activated calpain can interact with calpastatin in the presence of calcium and become inactivated. Caspases can cleave calpastatin which results in increased calpain proteolytic activity.
Members of the Bcl-2 and IAP families of apoptosis-regulating proteins can interact with caspases. Bcl-2 is an anti-apoptotic protein that can protect neurons against different apoptosis-inducing stimuli includin tro hic factor withdrawal, glutamate, oxidative insults and DNA-damaging agents52,53,54,39,40. Bcl-2 acts at a premitochondrial stage to prevent apoptosis and may do so by either preventing cytochrome c release from mitochondria or by binding to Apaf-1 (a protein that activates caspase-9) and thereby reventing multimerization of Apaf-I28. In addition, Bcl-2 blocks activation of nuclear55 and membrane-bound13 caspase-3. On the other hand, Bcl-2 is itself a substrate for caspase-3 and cleavage of Bcl-2 by caspase-3 may convert Bcl-2 to a pro-apoptotic protein56,57. IAPs are a family of caspase inhibitors that were identified based on homology to baculovirus. IAPs may suppress apoptosis, in part by inhibiting the activation of pro-caspase 958 and the activities of caspases 3 and 759. Many viruses produce proteins that inhibit caspases. For example, overexpression of p35 and CrmA in sympathetic neurons prevents cell death induced by trophic factor deprivation, staurosporine and Fas60,61,62. The mechanism whereby p35 suppresses apoptosis involves inhibition of caspases 1, 2, 3, 4, 6 and 1063,64, whereas CrmA inhibits caspases 1, 3, 7 and 865,66, CrmA and p35 act as competitive substrate
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inhibitors of caspases such that following cleavage their fragments remain bound to the active site of the caspase67,66. As is the case with many other proteins that regulate physiological processes such as apoptosis, caspase activity is subject to modulation by phosphorylation. For example, caspase-9 is inactivated when phosphorylated by Akt, a kinase that mediates anti-apoptotic actions of some growth factors68. Evidence suggests that several other kinases also modulate the activity of one or more caspases; the list of such kinases includes mitogenactivated protein kinase kinase69,70,71, protein kinase C72 and Akt71. Nitric oxide and other reactive oxygen species can also affect caspase activity73,74. NO inhibits apoptosis in some cell types75,76,77,78 by a mechanism involving reversible inhibition of caspases by direct Snitrosylation of the catalytic cysteine residue that is essential for enzyme activity75,79 . On the other hand, oxidative stress and nitric oxide can induce apoptosis in other cell types including neurons, and caspases mediate such oxidative stress-induced apoptosis 80,81,40,82,83. Table 1. Examples of caspase and calpain substrates that may mediate effects of the enzymes in synaptic plasticity and cell death.
Caspase substrates E-Catenin DII Spectrin (D -Fodrin) Actin Amyloid precursor protein Bcl-2/Bl-xL DNA-dependent protein kinase catalytic subunit (DNA-PKcs) DNA-repair enzyme PAPR Focal adhesion kinase (pp125FAK) Gelsolin Glutamate receptor (AMPA subunits) Inositol trisphosphate receptors (IP3R1 and IP3R2) Lamins A, B 1, C NFκB p50 and p65 subunits Presenilins 1 and 2 Protein kinase B Protein kinases G Protein phosphatase A2 (PP2a)
Calpain substrates Actin Amyloid precursor protein Bax Ca2+ATPase Focal adhesion kinase Glucocorticoid receptor IκBD Ionotropic glutamate receptors L-type Ca2+ channel MAP2 P53 Phospholipase C (PLC) Protein kinase A Protein kinase C Ryanodine receptors Spectrin (DII and EII) Tau
There are many different substrates cleaved by caspases, and their specific roles in apoptosis are being revealed (Table 1). Because nuclear DNA condensation and fragmentation are not observed in cells of caspase-3 knockout mice, and caspase-3 inhibitors also prevent DNA fragmentation, substrates cleaved by this enzyme are required for the alterations in nuclear DNA84. The nuclear envelope protein lamin and the actin-severing protein gelsolin play a role in mediating these nuclear fragmentation events. An enzyme called caspase-activated DNAase is activated by caspase-3 and effects internucleosomal cleavage of DNA leading to DNA laddering, a hallmark of apoptosis85. The activity of the DNAase is also dependent on caspase 3-mediated processing of an inhibitory protein associated with the DNAase86, which is a homologue of human DNA defragmentation factors87. Other nuclear proteins that are substrates for caspases include poly ADP-ribose polymerase (PAW), DNA-dependent protein kinase, and Ku protein88,89,90. Caspases can also proteolyze cell cycle-related proteins including cyclin D, several protein components of the RNA splicing complex, and the retinoblastoma tumor suppressor protein91-92. Some caspase substrates are activated upon cleavage. For example, several stress response proteins are activated by caspases and may play a role in triggering apoptosis93. Overexpression of the caspase-3-generated catalytically active fragments of PKC or PAK alone contribute to certain features of the apoptotic phenotype. It has been suggested that cleaved signaling proteins act to turn on death-promoting and/or turn off survival pathways
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that may differ from one cell type to another. Caspase-3-mediated cleavage of MAPK/ERK kinase kinase I (MEKKI) leads to the constitutive activation of Jun no-terminal kinase (JNK)/SAPK, which promotes the death pathway induced by a variety of cellular stresses including ultraviolet and J-irradiation, heat-shock, and camptothecin94. In addition, antiapoptotic signaling cascades may be turned off by caspases. For example, caspases can cleave and inactivate) the p50 and p65 subunits of NF-kB95, an anti-apoptotic transcription factor36,96, and caspases also cleave AKUPKB, a kinase that normally inactivates the proapoptotic Bad protein17. PAK2 has been shown to activate the JNK pathway and may be involved in JNK activation during apoptosis signaling. Phosphorylation of c-jun increases the transcriptional activity of the AP-I complex and is essential for apoptosis in cerebellar granule neurons and differentiated PC- 12 cells following withdrawal of surviving factors97. Interestingly, an aldehydic product of membrane lipid peroxidation called 4-hydroxynonenal, which is implicated in various experimental models of neurodegenerative disorders37, activates AP-1 by a mechanism involving caspase activation98. Cleavage of the regulatory subunit of protein phosphatase 2A (PP2A) increases its activity and leads to altered phosphorylation states of several substrates including MAP kinase, whose activity is dependent on phosphorylation. These studies suggest that caspases induce cell death not only by protein degradation but also by proteolytic activation of specific downstream effector molecules. The data further suggest roles for caspases in modulating these signaling kinases and transcription factors under physiological (nonapoptotic) conditions. Other cell survival proteins such as anti-apoptotic members of the Bcl-2 family (Bcl-2, and Bcl-xL) are cleaved by caspase-3 at functional domains that reverse their roles so as to promote cell death rather than survival56,57. This may result in the opening of the mitochondrial permeability transition (PT) pore because Bcl-2 has been reported to associate with the PT pore constituents and regulate the function of this entity in both isolated mitochondria and intact cells99,100,10.
CASPASES IN NEURONAL APOPTOSIS AND NEURODEGENERATIVE DISORDERS Three major lines of evidence support roles for caspases in neuronal apoptosis that occurs during normal development of the nervous system and in neurodegenerative disorders. The first line of evidence is that caspase activation is increased in neurons prior to their demise during development of the nervous system in mammals, and in humans with neurodegenerative disorders. The second line of evidence is that caspase activation occurs prior to, and in association with, neuronal death in cell culture and in vivo models of neurodegenerative disorders. The third line of evidence is that caspase inhibitors or genetic “knockout” of caspases prevent neuronal death. Caspase activity is typically assessed by determining whether caspase substrates are cleaved, and by employing “reporter” caspase substrates in either in vitro or in situ assays101,102. Analyses of neuronal populations in which programmed cell death occurs during development have revealed evidence for caspase activation. For example, caspase-3 is activated in CNS neurons that undergo apoptosis in developing mice103. Examination of tissue sections from brains of Alzheimer’s patients immunostained with an antibody that specifically recognizes activated caspase-3 reveals evidence for caspase activation in vulnerable neuronal populations in the hippocampus and cerebral cortex. Masliah et al.104 stained brain sections from Alzheimer’s disease and age-matched control patients with antibodies against activated caspase-3 and reported that, compared to agematched controls, Alzheimer’s patients exhibited greatly increased numbers of neurons with caspase-3 immunoreactivity. Chan and coworkers102 showed that overall levels of activated caspase- 1 activity are increased in hippocampal tissue from Alzheimer patients and that many neurons exhibit immunoreactivity with an antibody against activated caspase-3. The latter study also provided evidence for caspase-mediated degradation of the AMPA-type glutamate receptor subunits in Alzheimer’s brain tissue and in cultured neurons exposed to amyloid bpeptide. Immunostaining of brain sections from Alzheimer’s disease and Down Syndrome patients using an antibody against activated caspases revealed labeling of approximately 0.02 —0.1 % of neurons, while no neurons were labeled in tissue from control patients105. The involvement of individual caspases in programmed death of neurons has been studied in mice using caspase inhibitors and gene-targeting methods to specifically disrupt a caspase gene. Overexpression of the caspase-1 inhibitor crmA prevents programmed cell
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death of dorsal root ganglion neurons in the developing chick60. Examination of caspase knockout mice has revealed interesting tissue-specific roles for individual caspases106,107,108,50. For example, mice lacking caspase-3 have severe defects in nervous system development, but no apparent abnormalities in apoptosis of lymphocytes. Caspase-3 knockout mice die within five weeks of birth and display profound abnormalities in the cerebral cortex and forebrain owing to failed apoptosis in the proliferative Caspase-9 knockout mice exhibit a phenotype similar to that of neuroepithelium106,109. caspase 3-deficient mice, but die at a younger age. Cells lacking caspase-9 are highly resistant to apoptosis induced by radiation, but are susceptible to death induced by Fas ligation107. Mice lacking caspase-8 suffer early lethality and are resistant to Fas- and TNFinduced cell death, indicating that despite the association of these receptors with caspase 10, they depend on caspase 8 for cell death induction in vivo110. Caspase-1 knockout mice have no major defects in apoptosis and are developmentally normal, indicating that this caspase does not play a critical role in cell death during development111. Nevertheless, mice lacking caspase- 1 do have a defect in interleukin-lb (IL- 1b) processing in response to lipopolysaccharide and are resistant to endotoxic shock112. Moreover, the production of inflammatory cytokines (TNF, IL-Ib and IL-6) is impaired consistent with a role for caspase1 in inflammation113. Thus, caspase activation does not necessarily result in apoptosis, as cytokine processing is observed during a normal immune response without apoptosis of the secreting cells114. Studies of experimental models of neurodegenerative disorders have provided further evidence for a causal role for caspase activation in the neurodegenerative process (Fig. 2). Exposure of cultured hippocampal neurons to amyloid β-peptide induces caspase-3 activation, and treatment of the neurons with caspase inhibitors protects them against apoptosis induced by amyloid E-peptide115,116. When expressed in knockin mice, mutations in presenilin- 1 that cause early-onset inherited Alzheimer’s disease result in increased neuronal vulnerability to apoptosis which is associated with increased caspase activation117.
Figure 2. Roles of caspases and calpains in neuronal degeneration in Alzheimer's disease. See text for discussion. Ab, amyloid b-peptide; ApoE, apolipoprotein E; APP, amyloid precursor protein; ER, endoplasmic reticulum; PAW, poly-ADP-ribose polymerase; ROS, reactive oxygen species; RyR, ryanodine receptor; VDCC, voltage-dependent calcium channel.
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Transgenic mice expressing exon 1 of the huntingin gene with an expanded polyglutamine repeat, a model of Huntington's disease, exhibit increased caspase- 1 activation in association with progressive neurodegeneration. Expression of a dominant negative caspase-1 mutant delays development of neurodegenerative changes and motor dysfunction118. Degeneration of dopaminergic neurons in the substantia nigra of rats induced by 6-hydroxydopamine, a model of Parkinson's disease, is greatly reduced in animals pretreated with the caspase inhibitor zVAD-fmk119.
CHARACTERISTICS AND REGULATION OF CALPAINS Calpains are calcium-activated neutral (cysteinyl/thiol) proteases that are widely expressed in tissues throughout the nervous system. The two major isoforms of calpain that are widely expressed in mammals and have been most intensively studied are µ-calpain and m-calpain120,121. µ-calpain (also called calpain-I or CANP- I) binds Ca2+ with relatively high affinity (micromolar), whereas m-calpain (Calpain-II or CANP-11) binds Ca2+ with relatively low affinity (millimolar). Information on the structure and biochemical properties have been thoroughly reviewed previously122.121 and will therefore not be covered in the present article. Calpains I and II have distinct subcellular distributions suggesting different physiological roles for these two enzymes123. Within the central nervous system calpain I is localized mainly in neurons wherein its levels are higher in dendrites and the cell body than in axons. In contrast, calpain II is present at higher levels in axons and glial cell124,125 Whereas calpain I exhibits a relatively high level of basal activity, calpain II is generally responsive to stimuli, such as glutamate, that elevate intracellular Ca2+ levels. Calpains are expressed as proenzyme heterodimers consisting of an 80-kDa catalytic subunit, unique to each isozyme and encoded by a separate ene, and a 30-kDa regulatory subunit shared by both isozymes126,121. As with other Ca 2+-binding proteins, each subunit of the calpain heterodimer contains an EF hand Ca2+-binding domain127. The 30-kDa subunit also contains a hydrophobic glycine-rich domain that allows the enzyme to associate with cell membranes, whereas the catalytic site containing the critical cysteine and histidine residues is located on the 80-kDa subunit. Calpains are activated in response to increased levels of intracellular Ca2+ and inhibited by binding to the protein calpastatin. Calpain-I is activated in the cytosol or when bound to the cell membrane, whereas calpain-II activation occurs primarily at membrane sites. When calpains bind to Ca2+ they undergo a conformational change and translocate to phospholipid membranes where limited autolysis of the N-terminus of both subunits occurs. Attachment to plasma membrane sites serves to increase Ca2+ sensitivity, facilitating autocatalytic conversion of calpain at physiological concentrations of Ca2+ (0.1 – 1 µM). During this process, the 80-kDa subunit is processed to a stable 76-kDa form through a 78kDa intermediate, while the 30-kDa subunit is cleaved to an 18-kDa subunit128. An important consequence of autocatalytic proteolysis is that the cleaved protease requires a lower Ca2+ concentration for its activation, and is able to degrade membrane proteins directly or is freed to the cytoplasm where it may further proteolyze cytosolic substrates or become inactivated by combining with calpastatin. Calpastatin is a 110 kDa protein that is the only known endogenous inhibitor of calpains129. Calpastatin is widely expressed122 and is generally found at a higher concentration than calpains130,131. It contains four 140 amino acid repeat calpain inhibitory domains132. Calpastatin may be regulated by environmental signals because it can be phosphorylated by several different kinases including cyclic AMPdependent kinase133. Phosphorylation of calpastatin has been shown to alter its interactions with µ and m forms of calpain134, suggesting that phosphorylation of calpastatin may be a mechanism for modulating calpain activity.
CALPAINS IN NEURODEGENERATIVE DISORDERS The involvement of calpains in neurodegenerative conditions has been most intensively studied in experimental models of excitotoxic and ischemic brain injury135,136. It is well-established the disruption of neuronal Ca2+ homeostasis occurs as the result of acute ischemic insults, epileptic seizures and traumatic brain injury. Data suggest that calpains are activated in vulnerable neuronal populations in each of these conditions137,135,138.
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Administration of calpain inhibitors to animals in stroke models reduces neuronal damage and improves functional outcome139,135. Several chronic neurodegenerative disorders are also characterized by altered levels of calpain and/or calpastatin in vulnerable neuronal populations, including Alzheimer's In the case of disease140, amyotrophic lateral sclerosis141 and Parkinson's disease142, Alzheimer's disease, considerable data indicate that perturbed cellular calcium homeostasis contributes to the neurodegenerative process. In addition to evidence for activation of calpains in neurons in Alzheimer's patients, experimental studies have shown that: amyloid ß-peptide increases intracellular calcium levels in cultured hippocampal and cortical neurons and thereby promotes apoptosis and excitotoxicity143,144; mutations in the amyloid precursor protein that are responsible for some cases of Alzheimer's disease result in increased production of neurotoxic amyloid ß-peptide and decreased production of the neuroprotective (and calcium-stabilizing) secreted form of amyloid precursor protein (Fig. 2)145; mutations in presenilin- 1 that are responsible for many cases of early-onset inherited Alzheimer's disease perturb endoplasmic reticulum calcium homeostasis and thereby increase vulnerability of neurons to apoptosis and excitotoxicity39,146,117. It was recently shown that µ-calpain interacts with presenilin-2147, further suggesting a contribution of calpains to the pathogenic mechanism of presenilin mutations. While the majority of data suggest that calpains may contribute to the neurodegenerative process in Alzheimer's disease, it was shown that calpain inhibitors may alter proteolytic processing of the amyloid precursor protein in a manner that increases amyloid ß-peptide production148, suggesting that the contributions of calpains to the neurodegenerative process in Alzheimer's disease may be quite complex. Calpains are believed to play important roles in both apoptosis and necrosis in neurons149. Treatment with calpain inhibitors can protect neurons against apoptosis and necrosis in several experimental models150. In cultures of ciliary neurons deprived of trophic factors, inhibitors of calpain are at least as effective as caspase inhibitors in preventing DNA fragmentation and neuronal death151. However, because calpains normally subserve a variety of important physiological functions (see below), it is unclear how these proteases are recruited to the tightly regulated process of apoptosis. Calpains may work in concert with, or independently of, the process of apoptosis.
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Receptors for neurotransmitters, neurotrophic factors and cell adhesion proteins are concentrated in synaptic terminals. Accordingly, synapses are sites at which signal transduction pathways are highly activated. Many of these same signal transduction pathways that mediate adaptive changes in neuronal structure and function are also involved in degeneration of synapses and neuronal cell death during development and in pathological settings152. The two synaptic signaling pathways that have been most extensively studied from the perspectives of neuronal plasticity and death are those activated by the excitatory neurotransmitter glutamate and by neurotrophic factors152. Glutamate is the major excitatory neurotransmitter in the central nervous system, and activation of glutamate receptors is required for both short- and long-term changes in the structure and function of neuronal circuits153. For example, glutamate regulates neurite outgrowth and synaptogenesis in the developing hippocampus153, and long-term potentiation of synaptic transmission (a correlated of learning and memory) in the adult hippocampus154. Activation of glutamate receptors and reduced activation of neurotrophic factor receptors can induce caspase activation in neurons36,37. Recent findings suggest that caspases and calpains, in addition to their roles in neuronal death, play important roles in synaptic plasticity. Before considering the roles of caspases and calpains in synaptic plasticity and degeneration, it is of obvious importance to establish that these enzymes are present in synaptic compartments. Immunohistochemical studies of hippocampal neurons in cell culture and in vivo have shown that both calpains and some caspases (caspases 3 and 8) are present in dendrites and axons155,36,37. Experiments performed in cerebrocortical synaptosomes have shown that caspase-3 can be activated in synaptic terminals, and can mediate apoptotic changes in those terminals including mitochondrial alterations36,37. Excitotoxic and ischemic insults can induce calpain activation in dendrites following
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excitotoxic in cell culture and in vivo156,157, and calpains can also be activated in synaptosomes wherein they proteolyze spectrin155. In addition to their being activated in response to apoptotic and/or excitotoxic insults, caspases and calpains can also be activated by physiological activity in neurons. For example, caspases can be activated in response to membrane depolarization in cultured hippocampal neurons (M. P. Mattson, unpublished data) and calpains are activated in response to stimulation of hippocampal slices at frequencies that induce long-term potentiation200. Intraventricular administration of caspase inhibitors to adult rats impairs their performance in a water maze spatial learning task (Fig, 3), suggesting that caspases may normally play a role in this form of synaptic plasticity.
Figure 3. Caspases are activated in neurons in response to electrical activity, and may serve important functions in learning and memory processes. A. Rat hippocampal neurons in culture were exposed to saline (control), 30 mM KC1, or 100 mM KC1 for the indicated time periods and levels of caspase-3 activity in neuronal cell bodies were then quantified. Values are the mean and SEM. B. Saline (control) or the caspase inhibitor zVAD-fmk (10 pg) was administered to adult male rats via injection into the lateral ventricles, and goal latencies were determined in the Morris water maze one hour later as described previously. Values are the mean and SEM (n=4).
What are the protein substrates of caspases and calpains that might mediate effects of these enzymes on synaptic plasticity? A large number of caspase substrates have been identified and can be placed into several categories including structural proteins, proteins involved in signal transduction pathways, and nuclear proteins. Many cytoskeletal proteins are cleaved by caspases including those that form dynamic complexes involving actin filaments, membranes, and cell adhesion proteins. Interactions of actin and spectrin with membranes play major roles in regulating growth cone behaviors in developing neurons and synaptic structures such as dendritic spines in mature neuronal circuits. The cleavage of such cytoskeletal substrates likely contributes to the alterations in cell morphology that occur in cells undergoing apoptosis (cell rounding and membrane blebbing). Calpains plays a role in calcium-mediated changes in the actin-spectrin system199 and caspases, which are also responsive to elevations of intracellular calcium levels158,159, may also effect cytoskeletal changes when calcium levels are increased. One function of caspase and/or calpain-mediated cleavage of actin and spectrin may be to regulate neuronal calcium homeostasis. For example, it has been shown that actin-depolymerizing agents such as cytochalasin D reduce calcium influx through voltale-dependent calcium channels and N-methyl-D-aspartate (NMDA) receptor channels160,161,162. Studies of gelsolin knockout mice have shown that this calcium-activated actin-severing protein plays an important role in modulating the activity of voltage-dependent calcium channels and NMDA receptors162. Geloslin itself is a caspase substrate, and it will therefore be of considerable interest to elucidate the role of its cleavage in synaptic plasticity. Calpain activity may regulate the shape of dendritic spines in hippocampal neurons163, and data further suggest that NMDA and AMPA (a-amino-3hydroxy-5-methyl-4-isoxazole propionate) receptor subunits are substrates for calpains164,165,166, and that AMPA receptor subunits are substrates for caspases102. Cleavage
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of AMPA receptor subunits by caspases reduces AMPA responses, and may be a mechanism to prevent excitotoxic necrosis102. Proteins involved in signal transduction pathways are substrates for caspases and/or calpains. For example, several different membrane-associated and soluble kinases are cleaved by calpain including focal adhesion kinase and protein kinase C (PKC)167,168,169. Similarly, caspases can cleave focal adhesion proteins, certain PKC isozymes, and mitogenactivated protein kinases93,170,171,172,173. Phosphatases such as calcineurin are also substrates for cysteine proteases174. Physiological roles for cleavage of such substrates remain to be established, but seem very likely based on the wide array of physiological processes (ion channel activity, transcription, neurotransmitter release, etc.) that are regulated by phosphorylation and dephosphorylation. Indeed, long-term potentiation of synaptic transmission involves several caspase substrates including calcineurin, calcium/calmodulindependent protein kinase and protein kinase C. Both calpain inhibitors175 and caspase inhibitors (Fig. 3) have been reported to impair LTP and/or spatial learning. Cleavage of kinases and phosphatases by caspases and calpains may also influence neuronal cell death by altering neuroprotective signaling pathways. Synapses are sites where focal adhesion complexes are concentrated in neurons. Adhesion complexes consist of integral membrane proteins, such as neural cell adhesion molecules, cadherins and integrins that interact with similar cell adhesion molecules on other cells or the extracellular matrix; integrins also interact with cytoskeletal proteins on the cytoplasmic side of the membrane176. Integrins appear to play roles in modulating synaptic plasticity in the hippoccampus177,178 and integrin-mediated signaling may promote cell survival through anti-apoptotic signaling upon activation by ECM proteins179. Recent studies suggest that integrin engagement by laminin activates a neuroprotective signaling pathway involving PI3 kinase and Akt kinase in hippocampal neurons180. Several proteins in the integrin signaling pathway are substrates for caspases including Akt181. On the other hand, activation of the PI3 kinase – Akt athway can suppress caspase activation in several different paradigms of apoptosis182,183. The collective data therefore suggest intriguing reciprocal interactions between caspases and integrin signaling pathways exist that are presumably involved in controlling the various physiological processes regulated by integrin signaling. Studies of the molecular and cellular underpinnings of Alzheimer's disease have revealed additional caspase substrates that may play roles in regulating synaptic plasticity and degeneration. The ß-amyloid precursor protein (APP) is an axonally transported integral membrane protein that is present in synaptic terminals145. APP contains a 42-amino acid peptide called amyloid b-peptide, which is the major component of the insoluble amyloid "plaques" that accumulate in the brains of Alzheimer's patients. Amyloid b-peptide is liberated from APP by enzymatic cleavages at each end of the peptide. An alternative processing pathway, effected by an enzyme activity called (a-secretase, cuts in the middle of the amyloid ß-peptide and releases a large extracellular domain called sAPPa from synaptic terminals184,145. Studies of synaptic plasticity in hippocampal slices have shown that sAPPa can shift the frequency dependence for induction of long-term depression, and can enhance sAPPa can also protect neurons against apoptosis and long-term potentiation185. excitotoxicit in various cell culture and in vivo models186,187. APP is a substrate for caspases188,189 and calpains190,192, and it will be of considerable interest to determine the consequences of such cleavage on APP processing and the normal functions of APP. On the other hand, amyloid b-peptide can induce caspase activation in dendrites and synaptic terminals116, whereas sAPPa can stabilize intracellular calcium levels and suppress activation of apoptotic pathways that involve caspases186,201. Thus, changes in APP processing can indirectly affect caspase activity levels. Many cases of early-onset inherited forms of Alzheimer's disease are caused by mutations in the presenilin-1 gene191. Presenilin-1, an integral membrane protein with 8 transmembrane domains localized primarily in the endoplasmic reticulum, plays important roles in development and appears to interact with the Notch signaling pathway. Neurons expressing presenilin-I mutations exhibit increased vulnerability to apoptosis and excitotoxicity146,117 which appears to result from an adverse effect of the mutations on endoplasmic reticulum calcium regulation192. Presenilin-I is a substrate for caspase-3193,194, and its cleavage could conceivably contribute to neuronal apoptosis in Alzheimer's disease. Because calcium regulation by the endoplasmic reticulum is increasingly recognized as playing a role in synaptic plasticity195, caspase-mediated cleavage of presenilin-I might also serve a physiological role in synapses. In addition to APP and presenilin-1, several other
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proteins linked to neurode enerative disorders are caspase substrates. For example, the Huntingtin gene product196, the dentatorubral pallidoluysian atrophy (DRPLA) protein197, and the androgen receptor198 are each caspase substrates. The latter proteins contain elongated polyglutamine repeats caused by CAG expansion, which upon cleavage by caspase-3 generate fragments that aggregate intracellularly198.
CONCLUSIONS Caspases and calpains act on many different protein substrates in neurons, and cleavage of these substrates results in a variety of physiological and pathophysiological changes in the structure and function of neuronal circuits. In addition to playing central roles in the process of neuronal apoptosis, caspases appear to regulate synaptic plasticity and may be involved in synaptic degeneration and remodeling. The calcium sensitivity of calpains suggests that they are important effectors of changes in neurons brought about by calcium influx, an important physiological and pathological signal in neurons. Many different neurodegenerative disorders involve excessive activation of caspases and calpains including Alzheimer’s, Parkinson’s, Huntington’s diseases and stroke. Experimental findings suggest that caspase and/or calpain inhibitors can attenuate neuronal degeneration in models of these neurodegenerative disorders.
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THE UBIQUITIN/PROTEASOME PATHWAY IN NEUROLOGICAL DISORDERS
Maria E. Figueiredo-Pereira and Patricia Rockwell Department of Biological Sciences Hunter College of the City University of New York New York, NY 10021
INTRODUCTION Proteolysis is an important cellular event involving tightly regulated removal of unwanted proteins and retention of those that are essential. The ubiquitin/proteasome pathway plays a major role in the quality control process by eliminating mutated or abnormally folded proteins by degradation to prevent their accumulation as aggregates that often form intracellular inclusions. In many neurological disorders, aggregates of ubiquitin protein conjugates are detected in neuronal inclusions but their role in neurodegeneration remains to be defined. However, it has become increasingly evident that functional changes in the ubiquitin/proteasome pathway are critical to the neurodegenerative process. The aims of this chapter are to provide an overview of the: (1) components of the ubiquitin/proteasome pathway, (2) relationship between the ubiquitin/proteasome pathway and mechanisms such as oxidative stress, inflammation, and apoptosis, which are thought to participate in neurodegeneration, (3) recent information suggesting that the ubiquitin/proteasome pathway plays a role in hereditary forms of neurodegenerative disorders, and (4) current knowledge on the biogenesis of ubiquitin protein inclusions (aggresomes).
THE UBIQUITIN/PROTEASOME PATHWAY The ubiquitin/proteasome pathway is a proteolytic mechanism with broad specificity, cleaving peptide bonds after basic, acidic and hydrophobic amino acids. To function efficiently, this pathway requires proteins to be tagged by ubiquitin to target them for degradation. Therefore, proteolysis by the ubiquitin/proteasome pathway involves two major steps: ubiquitination followed by degradation. A de-ubiquitination step also plays an important role in this pathway.
Ubiquitination/de-ubiquitination Ubiquitin (Ub) is a small protein of 76 amino acids which can form polyubiquitin chains by the successive attachment of monomers. These are linked by an isopeptide bond most frequently formed between the side chain of Lys48 in one ubiquitin molecule and the carboxyl group of the C-terminal Gly76 in another ubiquitin molecule. Polyubiquitin chains thus formed are attached to lysine residues on a protein substrate resulting in at least a 1 0-fold increase in its degradation rate1. Polyubiquitin chains with linkages involving lysine residues on Ub other than Lys48 were found to play distinct roles2. In humans, there Role of Proteases in the Pathophysiology of Neurodegenerative Diseases, edited by Lajtha and Banik. Kluwer Academic/Plenum Publishers, New York, 2001.
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are three Ub genes. Two of these contain heat-shock promoters, namely polyubiquitin B and C that code for four and nine copies of ubiquitin, respectively. The ubiquitin A gene codes for Ub fused to ribosomal proteins, and its function is not well understood 3
.
Figure 1. Protein ubiquitination. First, a high energy thioester bond is formed between ubiquitin (Ub) and a ubiquitin-activating enzyme (El). This reaction requires ATP hydrolysis. Secondly, the activated ubiquitin is transferred to a ubiquitin conjugating enzyme (E2). Thirdly, the activated ubiquitin is ligated, via an isopeptide bond, to the protein substrate by a ubiquitin ligase (E3). Lastly, the ubiquitin chain is elongated, by an ubiquitin-chain elongating factor (E4) which drives polyubiquitin chain (poly Ub) assembly.
Ubiquitination of proteins (Figure 1) is a complex process involving the following sequence of events: (1) formation of a high energy thioester bond between Ub and a ubiquitin-activating enzyme (El) in a reaction that requires ATP hydrolysis; (2) formation of a thioester bond between the activated ubiquitin and ubiquitin-conjugating enzymes (E2); (3) covalent attachment of the carboxyl terminal of ubiquitin, usually to the H-amino group of a lysine residue on protein substrates via an isopeptide bond mediated by ubiquitin ligases (E3); and (4) assembly of polyubiquitin chains carried out by a novel family of ubiquitination factors (E4) which promote the production of longer Ub-chains4. In some cases, ubiquitin can be transferred directly to the protein substrate by ubiquitinconjugating enzymes (E2). There are many different E2 and E3 enzymes, indicating that this pathway may operate through selective proteolysis5. There are at least 30 E2s identified in humans. They share a common 150-amino acid catalytic core, whereas each subfamily possesses affinity for a different class of E3 enzymes. E3s recognize specific protein substrates for ubiquitination, and at least four classes have been described. (D enzymes bind protein substrates with basic or hydrophobic N-terminal amino acids5. The HECT-E3s (h omologous to E6AP carboxyl-terminus) form ubiquitin-thioester intermediates and ubiquitinate substrates directly. So far, 20 members of this class (HECT-E3s) have been identified6. Other E3 classes, such as Skpl-Cullin-F box complexes (SCF) and anaphase pomoting complexes (APC) do not form a ubiquitinthioester intermediate6. Several RING-finger-containing proteins were found to be E2dependent ubiquitin ligases (E3)7,8. Ubiquitin is removed from ubiquitinated proteins by de-ubiquitinating enzymes which also disassemble polyubiquitin chains. More than 90 genes coding for de-ubiquitinating enzymes have been identified, making them the largest family of enzymes involved in the ubiquitin pathway9. There are two major classes of de-ubiquitinating enzymes: (1) Ubiquitin carboxyl-terminal hydrolases (UCHs) that remove small amides, esters, peptides and small proteins at the carboxyl terminus of ubiquitin, and (2) ubiquitin-specific processing proteases (UBPs) which disassemble the polyubiquitin chains and may edit the ubiquitination state of proteins10.
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Proteasome degradation Covalent binding of ubiquitin to proteins marks them for degradation by the 26S proteasome (Figure 2), an enzymatic complex with a native molecular weight of approximately 2,000 kDa11. The 26S proteasome includes two major particles: a 20S particle, known as the 20S proteasome, which is the catalytic core, and a 19S particle, known as PA700, which is the regulatory component. Association between the two particles in the cell is a dynamic process and requires ATP-hydrolysis. The 20S proteasome can associate with other regulatory members, such as PA28, but this combination is not known to degrade ubiquitinated proteins11.
Figure 2. The 26S proteasome. Its two major particles, the 20S particle (20s proteasome) which is the catalytic core, and the 19S particle (PA700) which is the regulatory component, require ATP hydrolysis to assemble into the 26S proteasome. The PA700 lid confers ubiquitin/ATP-dependency to proteasome proteolysis. The PA700 base has ATPase and chaperone-like activity. The midlongitudinal view of the 20S proteasome was drawn from Iryp.pdb12.
The 20S particle is composed of 28 subunits arranged in four heptameric-stacked rings forming a cylindrical structure with a hollow center in which proteolysis takes place12. The 20S proteasome hydrolyses most peptide bonds present in a protein13, and the rate of this hydrolysis is influenced by proteasome subunit composition14. Assembly of this particle from precursor subunits is a complex process and was shown to require the assistance of a short-lived chaperone15. The 19S particle (PA700) contains at least 17 subunits, including ATPases, a deubiquitinating enzyme and polyubiquitin-binding subunits. It confers ubiquitid/ATPdependency to proteolysis by the 26S proteasome11. PA700 can also stimulate proteasomal degradation of non-ubiquitinated proteins such as ornithine decarboxylase, which requires only ATP hydrolysis for its proteasomal breakdown16. The subunits in PA700 are distributed into a lid and base arrangement, with the lid required for ubiquitin/ATPdependent proteolysis17. The base, containing the ATPases, exhibits chaperone-like activity18. The 26S proteasome is found in the cytosol next to intermediate filaments of the cytoskeleton19. It also resides in the nucleus and in association with the cytosolic side of ER membranes20,21. Localization studies with fluorescently labeled subunits of the 20S and 19S particles demonstrated that proteasomal proteolysis occurs mainly at the nuclear envelope/rough ER site22. An important function of such proteolysis is to eliminate abnormal secretory proteins residing in an EWpre-Golgi compartment11. Functionally inefficient, misfolded or unassembled ER proteins leave this intracellular compartment by retrograde transport through the Sec61 translocation channel, whereupon, they are ubiquitinated by ubiquitin-conjugating enzymes associated with the cytosolic side of the ER membrane, and then degraded by the cytosolic 26S proteasome23. Although this ER degradation pathway appears to be non-essential for viability, its importance is underscored by its evolutionary preservation "despite strong negative selection" since disruption of this mechanism seems to be associated with many diseased states23. The 20S proteasome was detected in all areas of the rat CNS, but higher levels were found in pyramidal cortical neurons of layer 5 in the brain and the motor neurons of the ventral horn of the spinal cord24.
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Substrate recognition Three key characteristics target proteins for ubiquitination/degradation: (1) misfolding due to mutation or damaging events; (2) constitutively active ubiquitination signals; and (3) post-translational modifications such as phosphorylation/dephosphorylation events or co-factor binding25. The unfolding of normal substrates precedes their degradation. This step is required to allow entry into the proteolytic chamber of the 20S proteasome through its narrow openings26. Unfolding activities may be provided by ATPase subunits in the PA700 base or by extraproteasomal chaperones. Degradation of ubiquitinated proteins is enhanced when more than one ubiquitin is attached to the target protein. The minimal signal for efficient degradation is a tetraubiquitin chain26. Removal of two ubiquitins from a tetraubiquitinated substrate by deubiquitinating enzymes, such as UCH37, can decrease substrate/26S proteasome affmity by approximately 100-fold, allowing the substrate to escape degradation. Longer chains do not increase substrate/26S proteasome affinity, but optimize their interaction time26. The interaction of the polyubiquitin chain with the 26S proteasome involves hydrophobic patches on the surface of the tetraubiquitin chain, generated by Leu8, I1e44, and Val70 in each ubiquitin moiety, and two hydrophobic sequences with the motif LeuAlaLeuAlaLeu in the PA700 subunit S5a27. Additional ubiquitin-binding subunits on the 26S proteasome must exist since S5a is not an essential protein in yeast27. The rate at which protein substrates of this pathway are degraded depends on the interplay between their deubiquitination and their unfolding26.
Ubiquitin-like proteins Two types of ubiquitin-like (Ubl) proteins have been identified: type 1 and type-2 Ubls28. Type 1 Ubls, such as SUMO1 (small Ub-related modifier) and NEDD8 (neural precursor cell-expressed developmentally down-regulated gene), are small and are covalently attached to proteins in a manner similar to ubiquitination, although they require their own enzymatic components29. Some SUMO1-modified proteins seem to assist nuclear translocation of other proteins28. NEDD8-protein interaction is important in cell cycle regulation28. Type-2 Ubls, such as RAD23, Parkin and ElonginB, are not ligated to other proteins. Instead, they occur as fusion proteins with a ubiquitin-like domain located at their N-terminus, in the central portion, or at the C-terminus. The physiological significance of these fusion proteins remains uncertain, although they may function in DNA repair (RAD23) or as ubiquitin ligases (ElonginB).28
THE UBIQUITIN/ROTEASOME PATHWAY AND NEURODEGENERATIONINDUCING MECHANISMS Although selective sets of neurons are affected in different neurodegenerative disorders most of them share an intriguing morphological feature, namely, the accumulation of ubiquitinated proteins30. These diseases are, therefore, associated with an inability of the neuron to degrade ubiquitinated proteins, and may be classified as ubiquitinopathies31. In general, high levels of ubiquitinated proteins do not accumulate in healthy cells as they are rapidly degraded by the ubiquitin/proteasome pathway. The inability to eliminate these modified proteins may result from a functional failure of the ubiquitin/proteasome pathway or from structural changes in the protein substrates which render them inaccessible to the degradation component. The ubiquitin/proteasome pathway may, therefore, play a role in mechanisms such as oxidative stress, inflammation and apoptosis, all of which are implicated as mediators of abnormal protein deposition and cell death in neurodegeneration.
Oxidative Stress The involvement of oxidative stress in neurodegeneration has gained support from increasing evidence of its role in neuronal death in disorders such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). Studies with autopsied brains of AD patients showed a co-localization of high levels of oxidative stress products with neurofibrillary tangles and senile plaques32. Signs of oxidative stress, such as lipid peroxidation and a decline in
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reduced glutathione, were also detected in the substantia nigra in brains of PD patients33 The production of free radicals and lipid peroxidation by oxidative stress promotes partial unfolding of cellular proteins, resulting in exposure of previously buried hydrophobic domains to proteolytic enzymes34,35, and to ubiquitin-conjugating enzymes.36 Therefore, one important cellular anti-oxidant mechanism is an increase in intracellular proteolysis by the ubiquitin/proteasome pathway. Oxidative stress can affect components of the ubiquitin/proteasome pathway as well as its substrates, leading to an increase in the intracellular levels of ubiquitinated proteins. Covalent attachment of the lipid peroxidation product 4-hydroxy-2-noneal (HNE) to the 20S proteasome decreases its activity37,38. The chaperone HSP90, however, can prevent the inactivation of the proteasome under such conditions39 . In addition, HNE-modification of proteins results in their accumulation as ubiquitin-conjugates, confirming that the metabolism of HNE-altered proteins involves the ubiquitin/proteasome pathway40. Cadmium- or zinc-induced oxidative stress results in protein thiolation and inhibits the activity of the ubiquitin/proteasome pathway41,42. The increased levels of protein mixed disulfides produced in cadmium- and zinc-treated neuronal cells was found to be accompanied by an accumulation of ubiquitinated proteins, suggesting that thiol-modified proteins are broken down by the ubiquitin/proteasome pathway. Oxidative stress induced by hydrogen peroxide also affects the ubiquitin/proteasome pathway, either by directly decreasing the activity of the 20S or 26S proteasome43-45 or by increasing the expression and activity of at least two members of the ubiquitination machinery, namely E1 and E2 enzymes46. In addition, H2O2-induced oxidative stress increases intracellular levels of protein-bound carbonyls. Such modified proteins are removed by the proteasome47. The H2O2-effects are dependent on changes in the redox status of the cell, manifested by a decrease in reduced glutathione (GSH) and an increase in oxidized glutathione (GSSG). Reestablishment of the GSSG:GSH ratio allows recovery from the oxidative stress insult. The stability and, therefore, the activity of two transcription factors namely iron regulatory protein2 (IRP2)48 and hypoxia-inducible factor1 alpha (HIF1 D were found to be dependent on the oxidation level of iron- or oxygen-degradation-dependent domains on each protein, respectively. In iron- or oxygen-replete cells, IRP2 and HIF1 D are rapidly and selectively turned over by the ubiquitin/proteasome pathway. However, they are transcriptionally active only under conditions of iron depletion, in the case of IRP2, or oxygen deprivation, in the case of HIF1 D Degradation of these transcription factors by the ubiquitin/proteasome pathway appears to involve recognition of specific oxidatively modified amino acids on these proteins by ubiquitin protein ligases. Just as phosphorylation of selective amino acids dictates turnover of proteins like ,N%D so may oxidation of particular amino acids target proteins, such as IRP2 and HIF1D for degradation. Furthermore, stabilization of HIF1D is not only elicited by hypoxia but also by transition metals, iron chelators, and several anti-oxidants50. Together, these results indicate that the ubiquitin/proteasome pathway plays a key role in the intracellular antioxidant defense mechanism, because it removes oxidatively damaged proteins and modulates the activity of oxidation-dependent transcription factors.
Inflammation Many neurodegenerative disorders, including AD, are associated with chronic inflammation, as shown by the presence of more than 40 immunoprotective proteins in AD brains at autopsy 51 . These immunoprotective proteins cannot be detected in normal brains. In addition, epidemiological studies involving 1686 participants in the Baltimore Longitudinal Study of Aging demonstrated that the use of non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, decreases the relative risk for AD, and that this decrease is proportional to the duration of the treatment.52 The protective effect of NSAIDs may correlate with their inhibition of the enzymatic activity of a pro-inflammatory and inducible cyclooxygenase, known as COX-2. Up-regulation of this enzyme causes tissue damage through prostaglandin and reactive oxygen species production53. While COX-2 protein levels are almost undetectable in normal brains, its expression increases after focal ischemia in infarcted human brains52 . This enzyme was shown to be an immediate early gene transiently induced in hippocampal neurons after injection of the excitotoxin kainic acid into rat brains54. In patients with Fukuyama-type congenital muscular dystrophy, a
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neurodegenerative disorder transmitted through autosomal recessive inheritance, upregulation of COX-2 precedes appearance of neurofibrillary tangles (NFT)-containing neurons and neurodegeneration55. No E-amyloid deposits or senile plaques are detected in this disorder, but COX-2 immunoreactivity was found to co-localize with NFT-containing neurons. Similarly, both NFT-containing and damaged neurons in Down's syndrome and AD were found to exhibit high expression of COX-256 These findings suggest that the spatial and temporal association of COX-2 with neuropathological changes, such as NFTformation, correlates with neurodegeneration in these diseases. Recent studies with neuronal cells also provided evidence that a relationship may exist between COX-2 induction and the accumulation of ubiquitinated proteins. Neuronal cell death resulting from inhibition of the ubiquitin/proteasome pathway was preceded by an accumulation of ubiquitinated proteins in conjunction with increased expression levels of the stress-inducible protein HSP70, COX-2 and its pro-inflammatory product, prostaglandin PGE257. In addition, these studies showed that COX-2 turnover was mediated by the ubiquitin/proteasome pathway. Other investigations demonstrated that prostaglandins act as neurotoxins by increasing the levels of ubiquitin-conjugates and Eamyloid production in differentiated neuroblastoma PC 12 cells58 . Thus, the metabolic products induced by pro-inflammatory responses in neuronal cells may create a mechanism of self-destruction via an autotoxic loop59. This event could intensify the fundamental pathology of some neurodegenerative disorders such as AD.
Apoptosis The causes of neuronal cell death in many neurodegenerative disorders remain unclear. Hereditary forms of neurodegeneration can be attributed to specific gene mutations, but the underlying mechanisms responsible for loss of selective neuronal populations in these diseases have yet to be identified, although apoptosis appears to play a role60 . In particular, members of a family of proteases known as caspases, specifically caspase 1 and 3, were implicated as mediators of neuronal apoptosis61. In primary neuronal cultures, proteasome inhibitors were found to induce apoptosis, manifested by activation of caspase 3 proteases, disruption of mitochondrial membrane potential, and release of mitochondrial cytochrome C into the cytosol62. Stimuli-induced Bcl-2 turnover by the ubiquitin/proteasome pathway was shown to be preceded by its dephosphorylation63,64. It is now clear that the ubiquitin/proteasome pathway plays an important role in apoptosis, upstream of the caspase cascade, because it regulates the levels of the anti-apoptotic protein Bcl-2 (B-cell lymphoma-related protein).65 This view is supported by the finding that proteasome inhibitors prevent cerebellar granule neuronal death caused by a reduction in extracellular potassium, if they are administered before the onset of this process66.
POSSIBLE ROLE OF THE UBIQUITIN/PROTEASOME PATHWAY IN HEREDITARY FORMS OF NEURODEGENERATIVE DISORDERS The neurofibrillary tangles (NFT) in AD were the first neuropathological intracellular lesions found to immunostain with antibodies against ubiquitin conjugates67. Since then, ubiquitin conjugates were identified in innumerable neuronal inclusions68. The discovery that many neurodegenerative disorders are associated with mutations in genes other than those linked to the ubiquitin/proteasome pathway, suggested that the causal relationship between ubiquitin conjugate deposition and neurodegeneration is indirect. However, recent findings that mutations in ubiquitin and other components of the ubiquitin/proteasome pathway are associated with certain neurodegenerative diseases, indicate that the ubiquitin aggregates may hold a clue to the pathological process in neurodegeneration.
Alzheimer's disease Most cases of AD result from sporadic changes in neuronal cell metabolism whereas a small percentage (up to 10%) is genetic and occurs as autosomal dominant mutations. Familial AD is associated with mutations in the amyloid precursor protein (APP) and
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presenilins but the exact functions of these proteins are not known. In sporadic cases, individuals carrying two copies of the allele 4 of apolipoprotein E (ApoE4) have an increased risk of contracting the disease.69 The accumulation of dysfunctional frameshift proteins in neurons is fairly common and may occur as a result of incorrect editing of RNA transcripts. Frameshift mutants of ubiquitin B (UbB+l) and APP (APP+l) were found to co-localize with NFT and senile plaques in the cerebral cortex of patients with sporadic AD. The genes encoding the two proteins contain one or more GAGAG motifs that are prone to GA deletions during transcription. A single dinucleotide deletion (GA) in the first GAGAG motif of UbB mRNA, produces UbB+1, which lacks the C-terminal glycine, an amino acid essential for ubiquitination31. UbB+1 molecules may impair degradation of ubiquitinated proteins by competing with wild type ubiquitin for the interaction with the 26S proteasome. Presenilins (PS) are transmembrane proteins. They may regulate APP maturation, as certain mutations in PSs seem to increase production of one of the products of APP, Aβ, a peptide present in senile plaques of AD brains and postulated to be neurotoxic70. One of the presenilins, PS1, was found to interact directly with subunits of the 20S proteasome71. The demonstration that proteasome inhibitors promote PS1 accumulation as highmolecular weight ubiquitin conjugates provided evidence that the ubiquitin/proteasome pathway degrades this presenilin.71,72 The ubiquitin/proteasome pathway, therefore, seems to play an important role in PS1 turnover. APP and ubiquitin were found to co-localize in AD brain extracts subjected to electrophoresis on non-denaturing gels, suggesting that their interaction is non-covalent73. In addition, in vitro studies demonstrate that Aβ,1-40, but not its reverse peptide Aβ40-1, can enter the catalytic chamber of the 20S proteasome and inhibit the degradation of ubiquitinated proteins.74,75 The in vivo importance of these interactions remains to be established. Neurofibrillary tangles (NFT) in AD brains contain not only ubiquitin conjugates, but also neurofilaments and tau, a cytoskeleton protein required for stabilization of microtubules in the polymerized state. In NFTs, tau is hyperphosphorylated and forms paired helical filaments, losing its microtubule-stabilizing properties. Mutations in the tau gene, some of which lead to an increase in intracellular levels of normal tau, were found to cause frontotemporal dementia and parkinsonism linked to chromosome 1776,77. Transgenic mice overexpressing the four-repeat human tau in neurons, mimicking tau mutations in intron 10, developed axonal degeneration, astrogliosis and accumulation of ubiquitinated proteins in a transgene-dose-dependent fashion. These effects appeared without formation of intraneuronal neurofibrillary tangles78. Transgenic mice overexpressing the smallest tau isoform developed inclusions mostly in the spinal cord, but these inclusions lacked detectable ubiquitin/conjugates79. These findings indicate that higher than normal levels of a protein become cytotoxic when they accumulate in aggregates, if their rate of synthesis far exceeds their rate of degradation.
Amyotrophic lateral sclerosis Amyotrophic lateral sclerosis is the dominant motor neuron disease. Familial ALS is associated with mutations in the human Cu/Zn superoxide dismutase gene (SOD1).80 Transgenic mice expressing certain SOD mutations develop a motor neuron disease (MND) that is phenotipically similar to ALS. Lewy body-like inclusions containing crosslinked neurofilaments and ubiquitinated proteins are detected in motor neuron of FALS patients and MND mice.80 Both protein modifications are thought to result from oxidative stress induced by the SOD-mutations.80 Exacerbation of this insult in affected motor neurons may impair degradation of the modified proteins by the ubiquitin/proteasome pathway. Spinal cord injury in humans and spinal cord compression injury in rats are followed by accumulation of ubiquitinated proteins and of neuronal PGP9.5, an ubiquitin C-terminal hydrolase. These findings implicate a role for the ubiquitin/proteasome pathway in the recovery process.81
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Angelman's syndrome Sporadic and familial cases of Angelman's syndrome (AS) are associated with severe motor dysfunction and mental retardation82 . AS is one of the first hereditary disorders in humans shown to result directly from a loss of the maternal copy of a gene that codes for a component of the ubiquitin/proteasome pathway and which is located on chromosome 1583-85. Thus, positional cloning revealed that loss-of-function mutations in ube3, which encodes the E3 ubiquitin ligase E6AP, are the cause of the disease84,85. E6AP belongs to the HECT class of E3s. The crystal structure of its catalytic HECT domain revealed that most AS mutations map to the catalytic cleft and affect ubiquitin-thioester bond formation.6 While the protein substrates critical for AS have yet to be identified it is known that E6AP ubiquitinates p53, several Src family protein kinases, the human homologue of Rad23 and MCM7, a protein that plays a role in chromosomal replication.6 In a mouse model of AS, animals inheriting E6AP mutations in the maternal chromosome, have a severely impaired long-term potentiation (LTP), although their baseline synaptic transmission and neuroanatomy remain normal86. These findings suggest a role for E6AP-mediated ubiquitination in LTP. Interestingly, PGP9.5, a neuronal ubiquitin C-terminal hydrolase, was found to be an immediate early gene product involved in long-term facilitation (LTF) in the marine snail, Aplysia87. LTF in Aplysia involves prolonged activation of the CAMP-dependent protein kinase, which in turn requires the ubiquitin/proteasome pathway for degradation of its regulatory subunit.88 Persistent kinase activity is needed to induce the CREB-mediated transcriptional cascade for synthesis of selective proteins that participate in new synapse growth.87 The Aplysia transcription factor ApC/EBP, active early in LTF, was also found to be degraded by the ubiquitin/proteasome pathway only when dephosphorylated.89 In neuroblastoma cells, proteasome inhibitors promote neuritogenesis and NGF-treatment causes an increase in the levels of ubiquitinated proteins and of Ub-E1 and Ub-E2 thioesters.90,91 These data suggest that stabilization, rather than degradation of substrates of the ubiquitin/proteasome pathway must play a role in neurite outgrowth. Other studies showed that differentiation induced by retinoic acid in human neuronal progenital cells resulted in changes in proteasome activity and composition92.
CAG/polyglutamine expansion diseases At least eight neurodegenerative diseases are caused by polyglutamine (polyQ) repeats in specific proteins: DRPLA (dentatorubral pallidolusian atrophy), HD (Huntington's disease), SBMA (spinal and bulbar muscular atrophy), and the spinocerebellar ataxias SCA1, SCA2, SCA3, SCA6, and SCA7.93 The expanded polyQs of the mutant proteins participate in the formation of toxic intranuclear inclusions within the neuron. This may lead to cell death and neurodegeneration.94 In HD, mutant huntingtin, with 36 to 120 glutamine(Q)-repeats at its N-terminus, forms intranuclear inclusions containing ubiquitin-conjugates in affected brain regions, such as the striatum and the cerebral cortex.95 Wild type huntingtin, with only 6 to 34 Qrepeats at its N-terminus, is localized in the cytosol. The toxic nature of the intranuclear inclusions is a controversial issue. The first HD transgenic mouse model was established by inserting 141-157 CAG/glutamine repeats into exon 1 of the human huntingtin gene.96 The onset of the disease in these mice is at approximately 8 weeks of age, and prior to this stage, no ubiquitin staining could be detected in the neuropil aggregates. An inclusion analysis demonstrated that intranuclear lesions were formed in these mice by selfaggregation of the mutant protein into amyloid-like fibrils prior to the onset of symptoms, possibly triggering manifestations of the disease.93 Only upon onset of the disease could ubiquitin-aggregates be detected, suggesting that ubiquitination represents a final attempt to remove the aggregates by proteolysis,96,97 Abundant nuclear inclusions in cellular models of HD do not correlate with cell death.98-100 These investigations, however, test only the short-term effects of overexpressed mutant huntingtin, and may not mimic the diseased state. 100 Other studies associate toxicity with mutant huntingtin only when it is expressed with a nuclear targeting signal and not with a nuclear export signal, suggesting that only nuclear mutant huntingtin plays a role in the pathogenesis of the disease.95,101,102 Properties of mutant huntingtin independent of its potential for aggregation may be more directly linked to the disease. The function of the normal huntingtin protein is not
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well understood, but it seems to interact with cytoskeleton elements and to be required for neurogenesis.103 Huntingtin interacts with the ubiquitin-conjugating enzyme E225K, also known as Hip-2 (huntingtin-interacting protein), and its interaction occurs independently of the number of polyQ repeats in huntingtin.103,104 Studies on mouse brain development indicate that the mRNA expression of huntingtin and E225K is spatiotemporally related during neuronal maturation, suggesting that the interaction between the two proteins is required for normal development and the onset of HD.105 Huntingtin turnover is carried-out by the ubiquitin/proteasome pathway.103,104 Mutant huntingtin, however, is ubiquitinated but not degraded, suggesting that polyQ repeats may block protein binding or access to the proteolytic core of the 26S proteasome for degradation. 103 Like huntingtin, the mutant proteins ataxin1 and ataxin3 associated with spinocerebellar ataxias SCA1 and SCA3 (also known as Machado-Joseph disease), exhibit polyQ repeats and accumulate in intranuclear inclusions containing ubiquitin-conjugates. In both diseases, the nuclear environment is essential for aggregate toxicity. These aggregates also stain positive for components of the 20S and 19S particles of the 26S proteasome, a finding that strongly suggests a role for the ubiquitin/proteasome pathway in aggregate biogenesis.106-108 Further studies support this view, since proteasome secific inhibitors in cellular models of SCA3 were shown to increase aggregate formation.109 Both wild type ataxin1 with two glutamines (Q) and mutant ataxin1 with 92 Qs were found to be polyubiquitinated, but proteasomal degradation of mutant ataxin1 was significantly impaired.110 In addition, transgenic mice expressing mutant ataxin1 and lacking the ubiquitin ligase E6AP, do not develop nuclear inclusions but exhibit accelerated SCA1 pathology.110 Although nuclear inclusions may not be essential to trigger neurodegeneration in these diseases, a loss of selective proteasomal degradation by a lack of E6AP-activity may be pivotal to the process. 111 This may be the case in SCA6, an ataxia due to a small polyQ expansion on a calcium channel which does not form detectable ubiquitin intranuclear inclusions. 109
Parkinson's disease The etiology of Parkinson's disease remains unknown. In the substantia nigra of Parkinson's diseased brains, ubiquitin-conjugates accumulate in cytosolic inclusions known as Lewy bodies. These inclusions also contain a protein of unknown function, Dsynuclein, an ubiquitin C-terminal hydrolase, UCH-L1 also known as PGP9.5, and proteasome subunits. These findings clearly implicate the ubiquitin/proteasome pathway in the etiology of this disease.112 Mutations in D-synuclein were found to cause familial PD in four different families.113 In vitro studies demonstrated that mutant D-synuclein (A53T) is cleaved by the ubiquitin/proteasome pathway at a slower rate than wild type, an event that provides a basis for its aggregation in intracellular inclusions.112 Deletions in the exon regions of the parkin gene were found to be associated with an autosomal recessive juvenile parkinsonism.114 Parkin, the protein product, is abundant in the brain and its N-terminal sequence is moderately similar to ubiquitin. The function of this newly identified ubiquitin-like protein remains unknown. A missence mutation (Ile93Met) in the uch-l1 gene, which codes for a deubiquitinating enzyme, was identified in a German family with PD.115 This ubiquitin Cterminal hydrolase is very abundant in the brain and the mutation described was shown to decrease its catalytic activity.115 Genetic analysis in other Caucasian families failed to detect a similar mutation.116-118 An in-frame deletion including exons 7 and 8 of the uch-l1 gene was described as the cause of gracile axonal dystrophy (gad ) in mice. This mutation results in a truncated protein lacking 42 amino acids including a possible active site histidine119. This genetic model is characterized by a retrograde accumulation of amyloid E-protein and ubiquitinconjugates in sensory and motor neurons, as seen in certain inherited human neurodegenerative diseases119,120. The gad mouse is the first mammalian model of a hereditary neurodegenerative disorder that results from a mutation in a component of the ubiquitin/proteasome pathway. Future studies may reveal defects in other proteins that function in the ubiquitin/proteasome pathway as further evidence is acquired to substantiate the role of this pathway in pathogenesis of neurodegeneration.
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Prion diseases Prion diseases occur as either genetic disorders or as sporadic forms some of which are acquired by infection. Both types share a central pathogenic event in which a conformational change is elicited in the wild type prion protein (PrPc), a 209- amino acid glycoprotein res linked to the plasma membrane121. This results in the conversion of PrPc to an isoform (PrP ) that is protease-resistant and, in some cases, transmissible by unknown mechanisms. Twenty three pathogenic mutations are thought to cause spontaneous conformational changes in PrPc and are manifested as three phenotypes: Creutzfeldt-Jakob disease (CJD), fatal familial insomnia (FFI), and Gerstmann-Straussler-Scheiker (GSS) syndrome121. One of the PrPc mutations associated with GSS involves replacement of tyrosine (TAT) at codon 145 with a stop codon (TAG), designated Y145stop, to yield a truncated PrP protein (PrP145). Neuroblastoma cells transfected with the gene encoding PrP145 showed expression of an unstable mutant protein that was rapidly degraded by the ubiquitin/proteasome pathway121. These studies are the first to show degradation of a prion protein by the ubiquitin/proteasome pathway. Proteasome inhibition led to accumulation of PrP145 in aggregates that could be extracted as detergent insoluble and soluble fractions, both forms displaying resistance to proteinase K treatment121. Zanusso et al121 suggest that decreases in proteasomal activity with advanced age are responsible for accumulation of the mutant protein. Hence, this age-related decrease in proteolysis may increase the levels of the highly amyloidogenic PrP fragments and cause the formation of amyloid deposits in cerebral parenchyma and vessels detected in the variant of the GSS disease expressing PrP145.
Wilson disease Copper is a trace element and maintenance of its homeostasis is essential for the nervous system to function properly122. Its importance is underscored by the discovery of Wilson disease, a hereditary human disorder caused by a deficiency in copper metabolism that leads to neurodegeneration and hepatic cirrhosis122. The Wilson protein is a coppertransporting ATPase which resides in the trans Golgi network and its absence or dysfunction causes neurodegeneration by disrupting copper homeostasis. Mutations in this protein result in its misfolding and retention in the ER, followed by a retrograde transport out of the ER and its degradation by the ubiquitin/proteasome pathway23.
BIOGENESIS OF UBIQUITIN PROTEIN INCLUSIONS (AGGRESOMES) The hallmark of many neurodegenerative diseases is the presence of intraneuronal inclusions consisting of ubiquitin protein conjugates. The mechanisms leading to formation of such abnormal aggregates remain unclear and their role in the progression of the disease has yet to be elucidated123. It is possible that inclusions arise from a cellular attempt to compartmentalize accumulated proteins, and prevent their interference with normal cell function. Their presence may also confer cytotoxic effects that can contribute to cellular damage associated with neurodegeneration. Aggregate size may be a pivotal determinant in their toxicity92. As the ubiquitin protein aggregates expand they may confer fatal effects by chokin the cell as the cytosolic or nuclear space is ultimately filled by the abnormal aggregates 124 . The fact that many components of the ubiquitin/proteasome pathway, such ubiquitin C-terminal hydrolases and 26S proteasome subunits, are found together with ubiquitin conjugates within inclusions strongly supports a role for this pathway in inclusion biogenesis. A broad range of diseases, including cystic fibrosis and neurodegenerative disorders such as prion diseases, Wilson disease, and AD, are associated with defective proteins lacking the capability for correct ER processing125. Gene mutations in a transmembrane conductor regulator, a prion glycoprotein (PrP) associated with the plasma membrane, and a copper-transporting ATPase, are responsible for such disorders as cystic fibrosis, GSS syndrome and Wilson disease, respectively. The improper transport of these mutant proteins results in their accumulation in the ER, followed by their targeting for cytosolic
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degradation by the ubiquitin/proteasome pathway. It is postulated that in AD there is an association between deficiencies in APP processing in the ER and presenilin mutations and tau hypophosphorylation, the latter being a component of NFTs. All these deficiencies may contribute to aggregate formation125. Centrosomes, which are microtubule-organizing centers, were found to be deposition sites for ubiquitinated proteins that escape degradation by the ubiquitin/proteasome pathway, and were named accordingly as "aggresomes"126. Ubiquitin protein aggregates shown to be deposited in the aggresome resulted from either overexpression of mutant cystic fibrosis transmembrane conductor regulator (CFTR) or presenilin1(PS1) or from impaired protein degradation induced by treating cells with proteasome-specific inhibitors126-128. Centrosomes were shown to be associated with high levels of 26S proteasomes and also with de-ubiquitination activity129. While some studies demonstrated that the retrograde transport of ubiquitin protein aggregates to the centrosome is dependent on the integrity of microtubules126,126 , others found that this process does not require intact microtubules129. The mechanism by which ubiquitin protein aggregates are deposited in the aggresome may mimic the formation of intraneuronal inclusions found in many neurodegenerative diseases. It is unclear why neurodegeneration associated with hereditary forms of neurodegenerative disorders only becomes symptomatic in the adult or at an advanced age despite the congenital presence of specific mutant proteins. Zanusso et al121 postulate that the ubiquitin/proteasome pathway degrades mutant proteins shortly after they are produced, thus preventing their aggregation. Malfunction of this pathway caused by harmful conditions, such as oxidative stress or inflammation, may mediate a decrease in the degradation rate of abnormal proteins, bringing about their accumulation as protein aggregates which may form inclusions. An aging-induced decrease in proteasome function may also contribute to stabilization and aggregation of mutant proteins that are normally turned over by the ubiquitin/proteasome pathway. A micro-array analysis of the expression of 6347 genes in mouse skeletal muscle revealed age-dependent decreases in the expression of genes encoding stress factors and proteins involved in the ubiquitin/proteasome pathway, including 26S proteasome subunits and ubiquitin thioesterases130. Most of the identified changes could be reversed by caloric restriction diets130. These findings strongly support the influence of the aging process on the regulation of transcriptional activation of genes involved in the turnover of damaged and misfolded proteins, such as those encoding components of the ubiquitin/proteasome pathway.
CONCLUSIONS Recent advancements in gene cloning techniques and gene expression analyses provide compelling evidence linking the ubiquitin/proteasome pathway with the turnover of many proteins required to maintain neuronal homeostasis. These findings address new and exciting questions concerning the impact of a deregulation in proteolysis on cellular function and its causal relationship to the intracellular deposition of ubiquitin protein conjugates in neurodegeneration. Genetic data revealed that components of the ubiquitin/proteasome system are far more complex in number and function than previously thought. Findings from studies on hereditary forms of neurodegeneration, such as Angelman's disease in humans and the gad phenotype in mice, provide direct evidence that the manifestation of these disorders results from genetic defects in enzymes that are essential components of the ubiquitin/proteasome pathway. Consequently, these findings support the notion that malfunctions in this system may be critical events that trigger the initiation of the neuodegenerative process. Under harmful conditions, such as those induced by oxidative stress, inflammation, and genetic mutations, the cell may rely on the ubiquitin/proteasome pathway to remove abnormal proteins produced under such conditions, thus promoting neuronal homeostasis. An age-dependent decline in the activities of this pathway may be critical to the neurodegeneration process. This explanation provides an alternate interpretation to the view that ubiquitin conjugate deposition is merely an indirect consequence induced by other factors involved in the disease. The need for more research to identify and define components associated with the ubiquitin/proteasome pathway underscores the potential for new targets of therapeutic
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intervention in neuronal diseases as well as diagnostic markers for individuals at risk for these disorders
ACKNOWLEDGMENTS We thank Ms. Romia Bull for editorial comments and Ms. Tine Herreman for preparing the 20S proteasome structure shown in Figure 2. National Institutes of Health Grants NS34018 (to M.E.F.-P.) and RR03037 (Research Centers in Minority Institutions) supported this work.
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107. M.K. Perez, H.L. Paulson, S.J. Pendse, S.J. Saionz, N.M. Bonini and R.N. Pittman, Recruitment and the role of nuclear localization in polyglutamine- mediated aggregation, J Cell Biol, 143: 1457 (1998). 108. C.J. Cummings, H.T. Orr and H.Y. Zoghbi, Progress in pathogenesis studies of spinocerebellar ataxia type 1, Philos Trans R SOC Lond B Biol Sci, 354:1079 (1999). 109. Y. Chai, S.L. Koppenhafer; S.J. Shoesmith, M.K. Perez and H.L. Paulson, Evidence for proteasome involvement in polyglutamine disease: localization to nuclear inclusions in SCA3/MJD and suppression of polyglutamine aggregation in vitro, Hum Mol Genet, 8:673 (1999). 110. C.J. Cummings, E. Reinstein, Y. Sun, B. Antalffy, Y. Jiang, A. Ciechanover, H.T. Orr, A.L. Beaudet and H.Y. Zoghbi, Mutation of the E6-AP ubiquitin ligase reduces nuclear inclusion frequency while accelerating polyglutamine-induced pathology in SCA1 mice [In Process Citation], Neuron, 24:879 (1999). 111. J.A. Floyd and B.A. Hamilton, Intranuclear inclusions and the ubiquitin-proteasome pathway: digestion of a red herring? [In Process Citation], Neuron, 24:765 (1999). 112. M.C. Bennett, J.F. Bishop, Y. Leng, P.B. Chock, T.N. Chase and M.M. Mouradian, Degradation of alpha-Synuclein by Proteasome, J Biol Chem, 274:33855 (1999). 113. M. Goedert, Familial Parkinson's disease. The awakening of alpha-synuclein [news], Nature, 388:232 (1997). 114. T. Kitada, S. Asakawa, N. Hattori, H. Matsumine, Y. Yamamura, S. Minoshima, M. Yokochi, Y. Mizuno and N. Shimizu, Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism [see comments], Nature, 392:605 (1998). 115. E. Leroy, R. Boyer, G. Auburger, B. Leube, G. Ulm, E. Mezey, G. Harta, M.J. Brownstein, S. Jonnalagada, T. Chernova, A. Dehejia, C. Lavedan, T. Gasser, P.J. Steinbach, K.D. Wilkinson and M.H. Polymeropoulos, The ubiquitin pathway in Parkinson's disease [letter], Nature, 395:45 1 (1 998). 116. S. Lincoln, J. Vaughan, N. Wood, M. Baker, J. Adamson, K. Gwinn-Hardy, T. Lynch, J. Hardy and M. Farrer, Low frequency of pathogenic mutations in the ubiquitin carboxy-terminal hydrolase gene in familial Parkinson's disease. Neuroreport, 10:427 (1999). 117. B.S. Harhangi, M.J. Farrer, S. Lincoln, V. Bonifati, G. Meco, G. De Michele, A. Brice, A. Durr, M. Martinez, T. Gasser, B. Bereznai, J.R. Vaughan, N.W. Wood, J. Hardy, B.A. Oostra and M.M. Breteler, The Ile93Met mutation in the ubiquitin carboxy-terminal-hydrolase-L1 gene is not observed in European cases with familial Parkinson's disease, Neurosci Lett, 270:1 (1999). 118. D.M. Maraganore, M.J. Farrer, J.A. Hardy, S.J. Lincoln, S.K. McDonnell and W.A. Rocca, Casecontrol study of the ubiquitin carboxy-terminal hydrolase L1 gene in Parkinson's disease, Neurology, 53:1858 (1999). 119. K. Saigoh, Y.L. Wang, J.G. Suh, T. Yamanishi, Y. Sakai, H. Kiyosawa, T. Harada, N. Ichihara, S. Wakana, T. Kikuchi and K. Wada, Intragenic deletion in the gene encoding ubiquitin carboxyterminal hydrolase in gad mice [see comments], Nat Genet, 23:47 (1999). 120. M.E. MacDonald, Gadzooks! [news; comment], Nat Genet, 23:10 (1999). 121. G. Zanusso, R.B. Petersen, T. Jin, Y. Jing, R. Kanoush, S. Ferrari, P. Gambetti andN. Singh, Proteasomal degradation and N-terminal protease resistance of the codon 145 mutant prion protein, J Biol Chem, 274:23396 (1999). 122. D.J. Waggoner, T.B. Bartnikas and J.D. Gitlin, The role of copper in neurodegenerative disease, Neurobiol Dis ,6:221 (1999). 123. P.B. Tran and R.J. Miller, Aggregates in neurodegenerative disease: crowds and power? [In Process Citation], Trends Neurosci, 22:194 (1999). 124. E. Mezey, A. Dehejia, G. Harta, M.I. Papp, M.H. Polymeropoulos and M.J. Brownstein, Alpha synuclein in neurodegenerative disorders: murderer or accomplice? [In Process Citation], Nat Med, 4:755 (1998). 125. M. Aridor and W.E. Balch, Integration of endoplasmic reticulum signaling in health and disease, Nat Med, 5:745 (1999). 126. J.A. Johnston, C.L. Ward and R.R. Kopito, Aggresomes: a cellular response to misfolded proteins, J Cell Biol, 143:1883 (1998). 127. W.C. Wigley, R.P. Fabunmi, M.G. Lee, C.R. Marino, S. Muallem, G.N. DeMartino and P.J. Thomas, Dynamic association of proteasomal machinery with the centrosome, J Cell Biol, 145:481 (1999). 128. R. Garcia-Mata, Z. Bebok, E.J. Sorscher and E.S. Sztul, Characterization and dynamics of aggresome formation by a cytosolic GFP- chimera, J Cell Biol, 146:1239 (1999). 129. R.P. Fabunmi, W.C. Wigley, P.J. Thomas and G.N. DeMartino, Activity and Regulation of the Centrosome-associated Proteasome, J Biol Chem, 275:409 (2000). 130. C.K. Lee, R.G. Klopp, R. Weindruch and T.A. Prolla, Gene expression profile of aging and its retardation by caloric restriction, Science, 285: 1390 (1999).
AMYLOID (TACE, BACE) AND PRESENILIN PROTEASES ASSOCIATED WITH ALZHEIMER'S DISEASE
Neville Marks1,2 and Martin J. Berg1 Center for Neurochemistry Department of Psychiatry New York University Nathan S. Kline Institute for Psychiatric Research Orangeburg, NY 10962 1
2
INTRODUCTION This overview emphasizes recent findings on proteolysis of amyloid precursor protein (APP) and presenilins (PS1/PS2). Interest in these components stems from their association with AD/FAD resulting in the overproduction of fibril forming amyloid peptides (Aβ). These accumulate in AD neuritic plaques and are thought to be etiological factors in neurodegeneration. Aβ is formed on shedding of an APP ectodomain followed by cleavage of cell-associated fragments by 'secretases', a term denoting enzymes for secretion of soluble metabolites. Until recently secretases were not available in purified form and data were descriptive and inferential. Recent isolation of Asp-proteases and metalloendoproteases provide new insights in mechanisms involved in APP turnover and a basis for synthesis of inhibitors or probes with therapeutic potential. Mutated presenilins differentially increase the secretion of C-terminally extended forms of Aβ (Aβx) indicating a shift in processing to account for altered composition of neuritic plaques in familial AD (FAD-PS) and in transgenic brains co-expressing FADPS/APPs. Cultured cells expressing FAD genes provide in vitro assays to monitor this genetic autosomal dominant gain-of-function. Converging on these themes are questions on the functional significance of PS itself since this component was discovered and then named only in the context of presenile pathologies. Clues for function arise from morphological similarities between phenotypes for Notch or PS knockouts, and from complementation assays using mutant flies or worms deficient in PS-homologs. These provide evidence for PS playing roles in Notch-signaling pathways including eye/wing maturation in Dps mutant Drosophila, or egg laying in sel-1/hop-1 deficient nematodes1-10 , and form a basis to examine effects of PS in turnover of other proteins containing a single TM domain. An example considered here is Notch-receptor (Notch-r), a Type-1 protein having its C-terminus in the cytosol, that is processed following binding to ligands. Other examples include protein components of the unfolding protein response (UPR) signaling
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pathway Type-1 IreI, and ATF6, a Type-2 protein having N-terminus in the cytosol. Because of space limitations we emphasize here recent developments since earlier findings are extensively documented elsewhere11-13. PROCESSING ENZYMES AND COMMENTS ON STRUCTURE OF APP/PS Structure and post-translational modifications are factors relevant to processing putative membrane components by tissue hydrolases (Table 1). APPs occur as glycosylated isoforms with Mr ~100-140 kDa formed by alternative splicing with some having a serine protease inhibitory sequence (KPI) and other domains, although several of these are absent in the major neuronal form, APP695 (Fig. 1). The variable sugar content and other post-translational modifications contribute to APP heterogeneity, and probably markedly, to their turnover as illustrated for catabolism of soluble rAPP versus axolemmal-bound precursor by purified brain cathepsin B14. Therefore it is necessary to evaluate action of putative secretases on membrane-bound precursor in addition to the use of shorter peptide surrogates. Earlier studies on isolated cells or crude membranes show sequential breakdown of APP with removal of soluble ectodomains followed by cleavage of cell-associated products (Fig. 2). Importantly, cleavage at K16L17 by D-secretase is nonamyloidogenic since this effectively destroys the fibrillar sequence. Cleavage of the residual fragment C-89 in this case results in formation of P3 fragment rather than AE, and the labile C-7 product that is undetected in tissues. The K16L17 and M-1D1 D- and E- sites are accessible to soluble hydrolases/proteases since they are downstream from the putative
Table 1. Secretases. Dresenilinases. and type-1/2 substrates discussed in this review
a, IC3, batimisat, marimastat, GI-120471, SE-205, TAPI, KD-1X-73-4, chelators42-44 b, Used for affinity purification19; c, ALLN, MG132 (calpain I), ALLM (calpain II); d, lactacystin, Abbr.: TACE, Tumor Necrosis Factor-α-Converting Enzyme; ADAM, A Disintegrin and Metalloendopeptidase; KUZ, Kuzbanian Protease; BACE, β-Amyloid Converting Enzyme; UPR, Unfolding Protein Response; A23187, Ca2+-ionophore; α-1PDX, α1-antitrypsin; L685,458, (1 S-benzyl-4R-(1-( 1Scarbamoyl-2-phenylethylcarbamoyl)- 1S-3-methyl-butylcarbamoyl)-2R-hydroxy-5-phenylpentyl) carbamic acid tert butyl ester, a transition state aspartyl-like inhibitor.
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Fig. 1: Domain structure of APP770. KPI ( ) and OX-2 ( ) domains deleted in neuronal APP695. Large open arrowheads show sites of major secretase cleavage in Aβ (bold type), with minor cleavage at Y10E11 shown with smaller arrowhead. Familial mutations are indicated in Italics and sites of substitution by arrowheads. The TM domain is shown within Sites of glycosylation shown by .
Fig. 2: Pattern for fragmentation of APP by secretases yielding soluble or cell-associated fragments. Numbers at bottom refer to AE sequence shown in Fig. 1.
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TM domain (Fig. 1). In contrast, formation of the Aβ moiety requires cleavage within the TM-domain implicating action by a membrane-bound TM-endoprotease referred to as J _secretase. Presenilins (PS) exist as two genes coding for proteins with ~70% homology (hPS1, 467 residues maps to chr 14q.24.3; hPS2, 448 residues maps to chr lq.42.1). Most mutations are found for PS1 (~50) with fewer (2) for PS2. Together these mutated genes comprise most of the reported cases for early-onset FAD or 10% of all cases of AD-like neurodegeneration. Presenilins are rapidly processed with residual full-length protein associated with the ER and NTF (28-34 kDa) and complementary CTF (18-20 kDa) fragments with Golgi membranes45. The deduced sequence contains 10 putative hydrophobic regions (HR) with 6-8 as TM4,46-48: the model of Nakai proposes 7 TM and one intramembranal HR48 and that of Li and Greenwald has 8 TM with the C-terminus as cytosolic4 as illustrated in Fig. 3A (solid black and dashed gray lines respectively). Hydropathy plots resemble the nematode homolog sel-12, a feature that may be consistent with comparable functional properties (Fig 3B). The NTF and CTF are formed via primary cleavage within the cytosolic loop along with minor metabolites as shown in Fig. ) although the "pressenilinase' enzyme(s) responsible have not been , 3A ( ' identified. It is unknown if one or more proteases account for variability of Mr and Ctermini of fragments. Importantly, FAD-mutations do not significantly alter turnover rates except for the PSl' exon9 lacking the 290-319 domain spanning the cleavage sites (Fig. 3A). The fact this mutant retains potency indicates breakdown is not obligatory for gain-offunction. Location of aspartyl residues postulated to participate in PS-mediated proteolysis are indicated in Fig. 3A (gray boxed).
DSECRETASES (Kl6L17 CLEAVAGE) Tissues contain metalloendopeptidases of the disintegrin family (ADAM) with putative α-secretase properties (Table 1). ADAM family members are widely distributed in tissues, and play diverse roles in a number of tissue functions by shedding ectodomains from a variety of components including TGFα, EGF, proTNFα, Fas-L, TNFR, L-selectin, ACE, Delta, Notch, erbβ4/HER4, and interleukin-6, thus acquiring the term 'sheddases' 1517,39,49-56 . Two groups independently identified TACE (ADAM-17) as a 501 polypeptide for conversion of 26 kDa ProTNFa to form active 17 kDa cytokine, later shown also to shed the APP ectodomain by cleavage at K16L15,16,39,50. Black et al.15 purified this enzyme from detergent-extracts of stimulated human monocyte cell line THP-1 cells and assayed fractions with Ac-SPLAQAVRSSR-amide, and Moss, et al.16 using an affinity derivative termed GW9471 purified enzyme from porcine spleen cleaving N-flagged ProTNFα and also the same peptide surrogate. Buxbaum et al.50 provided evidence for cleavage by TACE at the relevant APP site for the surrogate Ac-VHHQKLVFFA-amide, and release of sAPPα or Aβ by CHO cells expressing APP751. Cleavage was blocked by the inhibitor designated IC3, along with reduced secretion of sAPPβ for fibroblasts from TACE knockout mice. The most compelling evidence for ADAM-17 acting as a putative αsecretase is reduction of sAPPα secretion on transfection of K293 cells with a dominant negative (DN) mutant lacking the Zn-binding motif17. The deduced sequence of ADAMs show existence of the Zn-binding motif HEXXH (see Fig 4), providing a potential target for design of anti-inflammatory agents notably hydroxamates (Table 1), and the creation of the DN mutant. Comparison of the hydroxamate inhibitor batimastat shows 100-700 fold higher potency towards TACE and collagenase compared to ACE secretase and a-secretase. The analog marimastat is ~4 fold more potent towards APP than ACE but still retains considerable potency towards
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Fig. 4: Domain structure of h TNFa Convertin Enzyme (TACE) zymogen (see GenBank Accession Nos. U69611, U86755)15,I6. Cleavage by Furin at R 211VKRR214 leads to release of active enzyme through removal of proenzyme domain containing motif PKVC 184 GYL forming a 'cysteine switch' coordinately binding to and inhibiting the metalloendopeptidase catalytic center H405ELGH. For other details see Fig. 1 and text.
collagenase and thus is not specific for α-secretase42,44. Studies on COS and neuroblastoma cells show hydroxamates affect stimulated cells more potently than basal APP turnover although in the case of PKC the effects are unrelated to APP phosphorylation suggesting it acts on other processing events43,57. CHO cells expressing APPSW treated with phorbol esters and the hydroxamate TAPI show α- competes with βsecretase-like activity in the TGN providing another example of reciprocal relationships between these processing enzymes (see comments on BACE localization below)58. ADAM-10 cleaves Aβ11-28 or APP at the K16L17 site with reduced hydrolysis for the peptide substrate bearing the non-AD Dutch mutation although mutations associated directly with FAD were not examined (Fig. 1)17. TACE and ADAM-10 act at K16L17 compared to MCD9 (γ-meltrin) which cleaves at H14Q15 that may be the preferred site in hippocampal neurons59,60. MCD9 has α-secretase-like properties on transfection in phorbol ester-treated COS and K293 cells61. While there is scope to develop inhibitors62, since α-secretase destroys the putative amyloidogenic domain, activation rather than inhibition is more desirable provided this does not alter other secretases (see above for effects of TAPI)58. Testosterone-treated N2A cells or rat primary neurons increases the secretion of sAPPα and secretion of Aβ63. There is evidence for participation of other factors for conversion of APP or ProTNFα in -/stimulated TACE-/- fibroblasts, or after their fusion with PKC CHO cells64,65. ProTACE itself requires activation by a furin-like convertase and the removal of a Cys residue coordinately blocking the active Zn2+ motif ('cysteine switch', see Fig. 4)66. Overexpression in K293 cells of the prohormone convertase PC7, a furin enzyme, increases sAPPα thereby lowering Aβ via a pathway inhibited by α-1-PDX67.
Yeast Asp-proteases (Yapsins) Similarities between APP turnover in insect or yeast cells suggest one or more aspartyl proteases act as putative α-secretases68-70. Interestingly, these were discovered using an approach similar to that for identifying mammalian furins by use of yeast Kex mRNA probes, a method also found useful for recent purification of β-secretase71. Asp proteases Yap3 and McK7 (Yapsins) increase in yeast strains defective in vacuolar transport providing a basis for their characterization. Yapsins restore APP processing to sec 17 or 18 deficient mutants providing evidence for their α-secretase-like properties. Interestingly, some assays utilize an internally quenched fluorescent substrate AcRE(Edans)VHHQKLVPFK-(dabcyl) based on the α-secretase site. The relevance of yeast Yapsins or if these are expressed in mammalian tissues remains to be clarified.
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E-SECRETASE (M1D1 CLEAVAGE) Tissues contain acidic Asp-like proteases with putative β-secretase properties (BACE1/2, Asp 1-4, memapsins 1/2) although there is no agreed terminology. These resemble, enzymes predicted earlier as present in cells, membranes, extracts, or inferences based on analyses of body fluids (Table 2). Such studies provided evidence for processing in acidic compartments, roles for endocytosis, and with higher activity in neurons compared to astrocytes. Effects of serine protease inhibitors are questionable because of high concentrations used72.
Table 2. A. Examples of β-secretase type cleavage in diverse cell lines.
,
,
I
*Transfection with APPwt unless as noted. a , CM; conditioned medium b, NSE; neuronal-specific enolase c , GFAP; glial fibrillary acidic protein
Aspartyl proteinases Vasser et al.18 constructed a directional cDNA expression library using a CMV promoter to transfect HEK 293 cells overexpressing APP to identify a 501 polypeptide sharing homology to pepsin-like aspartyl proteases (Fig. 5). BACE has a 21 mer signal peptide (SP), a 24 mer pro-domain (Pro Pep), a lumenal 414 mer catalytic domain, a single 17 TM plus a 24 mer cytoplasmic tail. A furin-like convertase cleavage may be responsible for maturation of the BACE proenzyme83.
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Fig. 5. Domain structure of β-APP Converting Enzyme (BACE-l/Asp-2) (see GenBank Accession No. NM 012104 for sequence). Glycosylation sites ( ), Cys residues( )form intralumenal disulfides capable of for altering the conformation of the (Asp)-catalytic centers.
Lack of inhibition by pepstatin indicates presence of a unique lumenal catalytic center D*S/TGS/T 84-86 with six cysteines forming three potential intramolecular disulfides. This enzyme acts also at Glu11, consistent with earlier predictions on whole cells or membranes78,87, while cleavage at Val-3 and Ile-6 reported for intact cells probably represents processing by other tissue proteases/peptidases12. hBACE- 1 mRNA is present in all adult peripheral tissues and especially the pancreas. In brain, detection in hippocampus and cortical membranes is of interest to pathology since these regions are vulnerable to neuronal loss. Also high levels of BACE-1 in Golgi, TGN, and secretory vesicles occur at sites linked to APP processing: this was confirmed by co-localization of a hemagglutinin-tagged enzyme with sAPPβ and C99 in cells overexpressing APP. BACE-1 overexpression results in cleavage at Met-1 and Tyr10: antisense probes in APPSW expressing cells reduce formation of products associated with β-secretase. Specificity studies show BACE-1 cleaves the 30 mer T-21-K8(dnp)G9 at the MD site. BACE-1-IgG fusion protein catalyzed APPSW better than wt or was blocked using the M-1/V mutant. There will be interest in knockouts to evaluate roles of this unique Asp protease in APP turnover and on morphology. Sinha et al.19 designed transition-state analogs to directly purify from human brain a similar 501 polypeptide with pH maxima 5.5. The inhibitor used for affinity chromatography was prepared using an APP sequence with the Swedish mutation and Leu at P1: KTEEISEVNLstatineVAEF, ID50 of 30 nM (Table 1). Enzyme converted C-125-MBP fusion protein at the M-1D1 site. Sequence analysis and lack of effects by inhibitors indicated a unique aspartyl protease lacking typical serine or cysteinyl catalytic centers. Acetylation of the statine hydroxyl or replacement of (S)- with (R)-statine enantiomer blocked action of the inhibitors. The enzyme is largely neuronal, and not readily detected in peripheral tissues. Co-transfection with APPwt or APPsw increased formation of Aβ and SAPPβ. Yan et al.21 scanned the genome of C. elegans to isolate candidate protease genes to subsequently isolate mammalian homologs. Four new human sequences termed Asp- 1 to – 4 were identified with -3 and -4 comparable to napsins 1 and 288. Asp-l/2 have C-terminal extensions containing a single TM domain. Asp-2 (BACE-1) maps to chr 11q23.2 while Asp-1 (BACE-2) maps to chr 21 .q22.2-.3 linked to Down's syndrome89-91: co-localization of BACE-2 with the trisomic region of chr2 1 may have implications in Down's pathology. There is a 52% homology between BACE-1 and -2, with both containing a single TM and requisite Aspartyl motifs; they are divergent only at the C and N-termini. BACE-1 is the major β-secretase in HEK 293 cells since BACE-2 does not compensate for loss of BACE1 in these cells which can be blocked by antisense probes. This is consistent with low expression of BACE-2 mRNA in most human tissues, and especially fetal and adult brain, along with a distribution that does not match that predicted for β-Secretase92. In IMR-32 neuroblastoma cells, antisense probes reduce Asp-2 mRNAs and formation of C9921. Murine Asp-2 cDNA is 98% homologous to hBACE- 1. Enzyme acts on SEVKMDAEFR
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and more avidly on peptide bearing the Swedish KM/NL mutation. Pepstatin, leupeptin, E64, or EDTA do not inhibit this aspartyl-like protease. BACE is a potential target for drug design since inhibition prevents formation of intermediates containing Aβ. It will be of interest if BACE knockouts can be created and the morphological or behavioral consequences on overexpression of APPs. Hussain et al.22 obtained a cDNA clone from a proprietary EST database termed βsecretase Asp-2, but did not divulge methods of cloning although the enzyme appears to be homologous to BACE- 1. Transient expression of Asp-2 in different cell lines increases Aβx secretion confirming its role as a β-secretase. This is supported by Asp site mutations at the catalytic center (Asp-Ser/Thr-Gly-Ser/Thr)84 abrogating secretase properties. Tissues also contain alternative membrane bound Asp proteinases (memapsins 1/2) with memapsin 2 having putative β-secretase activiy towards wt or Swedish APP in cotransfected HeLa cells93. The PS1 mutation (V1717I, see Fig. 3) enhances formation also of N-truncated AEpyroGlu3-42 and 4-42 in situ. Unless these 'alternative' Esecretases are definitively characterized, such fragments may arise by N-terminal trimming by aminopeptidases or suggest PS mutations influence sites cleaved by BACE94. Scope exists to examine localization of BACE in endosomal or other pathways as factors in APP turnover95,96. Comments on cathepsins or other candidates Transfection of cathepsin D, a prototypic aspartyl protease, does not promote Aβ z secretion in cells 2. Since Aβ secretion by cathepsin D knockouts continues, this lysosomal aspartic protease is not essential for its formation97. Nevertheless, cathepsin D polymorphisms may constitute a risk factor for AD but play an alternative role in the clearance or turnover of other neurodegenerative proteins98-100. Cathepsin D is reported present in AD neuritic plaques, but its function is unknown especially since these deposits lack APP holoprotein or its CTFs101. Cathepsin S or the serine protease cathepsin G active at physiological pH also degrade APP but do not generate directly Aβ11,102-104. Similarly, the neuronal metalloendopeptidases such as phosphoramidon-sensitive 24.1 1 or insensitive 24.15 do not have secretase properties14,105, although this property is reported for platelet or leukocyte derived enzymes106. Overexpression of thimet oligopeptidase in COS cells is reported to enhance secretion of sAPPβ107. Roles are implicated for a GPI-anchor or caveolin-3 to facilitate β-secretase activity108,109. One study shows a 68 kDa serine proteinase processes lymphocyte but not brain APP, and attributes the differences to states of glycosylation110. γ-SECRETASES (VV40IA42A43TVIV) In cell models, processing of APP-CTFs (C-89/-99) within the TM results in release of P3 along with labile C-7 fragments (Fig. 1, Table 1)111-115 representing an example of regulated intramembrane proteolysis (RIP), applicable to several animal and bacterial proteins116. A common feature is removal of the bulk of the extracytosolic domain prior to action within the TM. Depending on the Typel/2 protein used as substrate, cleavage occurs in the ER-lumen, in a post-ER compartment, or at the cell surface. The putative γsecretase does not resemble the 'site-2 ‘ enzyme converting Sterol Regulatory Element Binding Protein (SREBP) acting within the TM since CHO cells deficient in this enzyme still secrete Aβ117,118 Similarly, putative γ-secretases do not resemble catabolism of APP by other lysosomal hydrolases119. Purification of γ-secretases is awaited to establish their localization, properties, specificity, and number120. There currently is little coherence on
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effects of site-specific agents as a method for identification (Table 1): this is attributable to overlap in specificity of such agents and discordant results especially on comparing effects on stimulated versus basal secretion for Aβ27,121-123. Rank order potencies for putative calpain and proteosome inhibitors on HeLa-pNAN8 cells expressing an APP C-103–YCFA construct suggest a single γ-secretase accounts for the secretion of Aβ/Aβx123. This also appears to be the case for a 2.0 x 106 kDa complex extracted from HeLa cells using CHAPS or CHAPS0 containing iR N- and C-termini of PS1: these degrade a Met-C-100-flag (DYKDDPPK) fusion protein to release Aβ1-40 and 142 by a mechanism sensitive to pepstatin, the transition-state inhibitor L685,458 containing a hydroxyethylene dipeptide isostere, but with a conformation opposite to HIV protease inhibitors, or other similar anologs23,124-127 . However, a recent comparison using E64 and peptidyl aldehydes proposes cysteine proteinases account for Aβ40 while calpains account for Aβx122. A lower Aβ/Aβx ratio for cells exposed to ALLN or MG132 (calpain I) or ALLM (calpain II) points to a Ca2+-activated protease for conversion Aβ/TM 26-57 domain see Fig. 1) flanked by N-hemagglutinin and C-c-myc epitopes when used as a substrate26. Effects of leupeptin, pepstatin, phosphoramidon, Z-LL-CHO, Z-VL-CHO, ZLL-leucinal, and lactacystin in vitro yield conflicting data (Table 1). Studies using APPs or PS1 constructs in transfected cells are difficult to interpret because of presence of multiple proteases/peptidases yielding major and minor products112.120. This is illustrated for APPSW with I637 F/P substitutions favoring cleavage at G38V, G37G, and V40I. In contrast, insertion of a repeat G625AII sequence favors cleavage at G33L or G38G. Deletion of this tetrapeptide altogether from the native holoprotein favors formation of C-terminally extended products112. In another study, transfection of a Leu-Glu-C-99 fusion protein in COS-7 cells shows T43A-V46F or T43G-V46F yields Aβx, while I45E increases the Aβx/Aβ ratio by 34 fold compared to V44F that yields AE38111. Processing of a C100 carrying a trans-Golgi sorting signal to yield Aβ40/42 was increased whereas this fragment bearing an ER sorting signal was decreased in FAD-PS 1 transfected N2A cells by a pathway inhibited by brefeldin but not monensin128.
PRESENILINS Mutated presenilins co-segregate with the majority of early onset presenile dementias and lead to a differential increase in Aβ42/43 in neuritic plaques. Replication of this feature in transgenics or isolated cells co-expressing FAD-PS/APPs points to a shift in γ-secretase processing to favor C-terminally extended forms11,129,130 . However, whether an increase of 0.5-1.0 fold in secretion of Aβx versus Aβ in isolated cells is sufficient for AD pathology in situ is debatable, although this is the basis of assays to monitor gain-of function. Transfection leading to overexpression of PS/APPs in cells is likely to distort the endogenous pools of metabolites13, thus complicating the interpretation. Overproduction of Aβx ma be pathogenic since this fragment readily aggregates in solution, and in vitro is cytotoxic12,311.
Presenilinase Presenilins are labile and occur as NTF/CTF fragments in Golgi membranes or as residual holoprotein in ER45. The enzyme(s) responsible for forming major metabolites are unknown although tissues contain candidates that may account for minor products. Surprisingly, labile mutants retain toxicity comparable to those of stable mutants or constructs. The Mr and immunoreactive profiles indicate cleavage in the cytosolic loop at Met292 of PS1 or Met298 of PS2 to account for the formation of NTF (~30 kDa) and CTF
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(~20 kDa) in a stoichiometric ratio of 1:1 (Fig 3)30,132,133 . Sites of cleavage are confirmed by metabolic stability following Met292 substitutions of a FAD-PS1 . Interestingly, this construct retains Aβ toxicity on transfection in APPSW expressing HEK 293 cells132. In contrast, the comparable stable PS2-M298D construct lacks potency in neuroblastoma cells, but rescues egg laying in sel-12 defective worms133. This disparity has not been explained but may reflect different athogenicities for the two PS genes or lack of concordance between assay procedures3,134. Stable hPS1 D257 or D385, hPS2 D263 or D366, and zebrafish PS1 D374 mutants lack potency leading to the hypothesis that the Asp groups facilitate catalysis31,135,136. This hypothesis is supported by studies showing that co-transfection of PS1 and PS2 Aspdeficient mutants reduced Aβ secretion in CHO cells expressing hAPP, or lowered Notch cleavage and translocation to the nucleus135,137,138. These data suggest there is no PSindependent pathway for production of Aβ/Aβx. PSI or 2 can substitute for each other for restoration of secretion of sAPPβ on transfection in PS1-/- fibroblasts139. Mutagenesis of putative catalytic Asp residues, plausibly, may be beneficial by reducing processing of APP intermediates. Binding of APP to PS in ER, followed by PS and C83/C99 in Golgi/TGN points to processing within these compartments for vectorial transport and production of Aβ/Aβx140. N2a cells doubly transfected with PS1' exon 9 and APPSW results in co-localization of Aβ42 with rab8, a marker of TGN vesicles141; also, PSI N-terminal binds rab GDI (GTP dissociation inhibitor), a component that decreases 2-fold in PSdeficient neurons142, Processing of APP lacking the C-terminal consensus sequence for internalization by PS1-transfected CHO cells indicates endocytosis is not obligatory (Fig 2)143.
Properties of PS fragments and complexes The ~10 fold increase in half life from 1.5 to 12-24 h for NTF/CTFs compared to holoprotein suggests stabilization of fragments on binding to cell accessory proteins144. Binding proteins themselves may be rate limiting since overexpression of PS in cells or in transgenics, leads to accumulation of holoprotein145. Also lack of toxicity on transfection of FAD-NTF ± the complementary CTF provides clues on events that occur prior to binding146. Current data on PS suggest N- and C- fragments share the consensus sequence required for toxicity. Structure-activity relationships show C-terminal third of PS2 (FADN141I) is critical since its removal, or modifications by addition of five His residues, or replacement of hydrophobic residues, reduce toxicity while the N-terminal 25-75 residues are dispensable147,148. Chimeras consisting of PS1/2 fragments also are active, in line with contribution from the N and C-termini146. The C-terminus of PS2 may contain a signal for entry into the processing pathway149. Complexes with Mr of ~150-250 kDa vary with cell-type and methods of extraction using detergents. Such complexes contain ir-NTF/CTF and one or more of the following components: catenins, Ca2+ - binding cadherins, calsenilins or other calcium-binding proteins, bcl-2/bclx, cavelolin-3, or syntaxin 1A among others109,150-163 . Recently, a 2.0 x 106 kDa complex from HeLa cells containing iR N- and C-terminal fragments of PS1 was shown to degrade an APP C-100 fusion protein to yield Aβ40 and 42 in a process inhibited by aspartyl protease inhibitors124. In HEK 293 or other cells, PS1 binds to C-APPs by a process facilitated by a 708-mer polypeptide Nicastrin164: it is speculated that nicastrin promotes pseudocatalytic properties of PS165. The conspicuous absence of mixed PS 1/PS2 heterodimers suggests catabolism of PS holoprotein occurs only on binding to access0 proteins thereby providing a 'scaffold' to retain the pathogenic signature of mPS146. Mutations may alter PS configuration in a subtle manner to account for the genetic gain-offunction although this remains conjectural. Other binding components include 42-mer
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armadillo repeat proteins (ARD) p0071 and neuronal B6P-plakophilin, capable of binding to the 372-399 region of PS1-CTF, but their role(s) in pathogenesis is unclear154,166. PS processing by caspases and proteosomes. Caspases are conserved Asp-specific cysteine proteases linked to apoptotic cell death. In peripheral cells expressing FasL/TNFα receptors, interaction with antigens recruits proximal caspases and adaptor proteins containing ‘death-effector domains’ for activation of distal effector forms167. Alternative pathways in non-mitotic neurons result in release of cytochrome c from mitochondria, and conversion of proximal caspase-9 for sequential processing of distal procaspases. These degrade PSI at D345 (PS1) or D329 (PS2) to yield alternative NTFs/CTFs compared to other putative presenilinases32,33. Stability of mutant PSID326/PS2-D329 or effects of N- and C-blocked tetrapeptide inhibitors -DEVD-, -YVAD-,or . the -D345SYD- recognition sequence of PS1 provides evidence for cleavage32. Caspase activation resulting from induction of apoptosis probably precedes formation of Aβ and its cytotoxic actions. In H4 neuroglioma, treatment with etoposide or staurosporine to activate caspases results in degradation of PS2 to form a ~20 kDa CTF33. Caspases differ in rates of hydrolysis of an ENDD329 PS1 sequence without effect by five PS missense mutations168. Independent1y, caspases degrade the CTF by a mechanism that is decreased via phosphorylation of Ser169,170. Caspase-12 present in ER, on activation is thought to contribute to turnover of amyloid peptides: mice knockouts resist apoptosis resulting from ER-stress [see section ‘Notch-signaling and the Unfolded Protein Response (UPR) ’ below] 171 . Proteosomes, a family of cytosolic ~700 kDa proteases, with mixed chymotryptic-, tryptic-, and ostglutamyl-like protease activities also degrade PS to yield alternative metabolites36,172. Cleavage occurs within the Met288-Glu299 domain using a purified 20 S proteosome and a synthetic substrate, but is inhibited by lactacystin or other similar agents36,173. However, roles for proteosomes are considered unlikely since they require ubiquinated substrates, harsh conditions for activation, and in situ a variety of accessory factors172.
NOTCH-SIGNALING AND THE UNFOLDED PROTEIN RESPONSE (UPR) Presenilins recently were found to influence turnover of Notch-r and components of the ‘Unfolded Protein Response’ thus reinforcing their roles as putative Asp-proteases (see Fig. 6). While they are spatially separated in cells, the binding of PS to Notch at the ER membrane provides a potential pathway for targeting to the plasma membrane, the site for Notch processing. Mutation of PS aspartyl residues, while not blocking trafficking, prevents Notch processing174,175. Notch-r is formed by proteolytic processing of a large multidomain precursor several fold larger than APP (Fig 6). Like APP it contains a single TM, and, in addition, 29-36 epidermal growth factor (EGF) repeats, 3 copies of a Lin-12/Notch/Glp motif in the ectodomain, and six CDC10/Ankyrin repeats among other motifs (Fig. 6). The shedding of the ectodomain by furin and/or ADAMs ( KUZ/TACE ) yields funtional receptors38,39,176 for binding ligands, resulting in further proteolysis at or within the TM55,177 to release a downstream activator (NICD) translocated to the nucleus where it acts on C/S/L genes (CBF-1/Suppressor of Hairless/LAG-1)178. Ligands identified in Drosophila include Delta and Serrate, in C. elegans are LAG-2 and APX-1 , and in rodents and humans are Delta1 and Jagged1/2179. NICD in neuronal nuclei influences rates of differentiation; this pathway is downregulated in DN mutants. Developing cortical neurons also express inhibitors of Notch-r of the Numb family (Numb , Numb-like, Deltex) influencing neuronal differentiation180. These resemble adaptor/scaffold proteins and include a phosphotyrosine
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binding (PTB) domain and proline rich (PRR) C-terminal containing Src and EH binding motifs181 . Interestingly, Notch-3 mutations are associated with CADASIL ( cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) and those of Jagged linked to Alagille syndrome, a mild mental retardation associated with multiple developmental disorders178,182. Recent studies implicate wt/mPS in the turnover of TM-containing UPR components Ire1α and ATF640,183,184, and reinforces roles for PS acting as or in concert with a transmembrane protease. UPR results from ER-stress triggering synthesis of hsp proteins GRP (glucose regulated protein) 78/94, and a Growth Arrest of DNA damage product GAD D153, also known as CHOP (c/ERP homologous protein)185,186. ER-stress results in phosphorylation of components within the UPR signaling pathways, in some cases resulting in apoptosis. GRP78 increases on exposing isolated hippocampal neurons to Glu excitotoxicity, Fe2+, or Aβ, and in NG108-15 exposed to ethanol as examples187,188. Antisense GRP78 promotes cell death of NGF-deprived PC-12 cells. This is reduced by dantrolene, an agent blocking ER-Ca2+ release, or by Z-VAD-fmk, a pan-caspase inhibitor187. Mammalian cells contain UPR ER-resident proteins Ire1α and β (Ern I and 2), and PERK, an interferon-induced PKR-like ER kinase186. Ire1 D is a type- 1 protein containing a sensor domain responsive to calcium ionophores, or thapsigargin that releases stores of ER Ca2+, or tunicamycin, an inhibitor of glycosylation. These induce Ire1 D phosphorylation and dimerization with cleavage at the TM domain to release a fragment having dual kinase and nuclease activity (K/N in Fig. 6). This fragment on translocation to the nucleus results in synthesis of GRP78/94, calreticulin, and protein disulfide isomerase to counteract ER protein misfolding (Fig.6)40,189,190. ER-stress also results in phosphorylation but not cleavage of PERK via a parallel pathway with phosphorylation of eIF2 α kinases ; this reduces protein synthesis and alleviates ER protein overload186. Roles for UPR in pathology are suggested by decrease in GRP78 mRNA on mPSl transfection for SK-N-SH neuroblastoma, and their enhanced response to tunicamycin by processes reversible on overexpression of this chaperone183. In PS1-/- HeLa cells, defective IreI processing points to significant roles for PS in conversion40. Significantly, a higher level of GRP78 in CNS neurons spared in AD suggests this chaperone is neuroprotective191. In line with this property, GRP78 levels are lower in temporal cortex of sporadic AD and FAD-PS1 brains183. The binding of ER-Ire1 to mPSl may prevent synthesis of GRP78 and protein refolding. GRP78 (T37G), a tightly binding ATPase mutant192 on transfection in HEK 293 overexpressing APP, reduces secretion of sAPP and Aβ40/42 points to novel therapeutic applications. Pathways in yeast for UPR show a requirement for DNA-binding protein hac1p acting as a co-transcriptional activator for protein folding. The use of yeast ER-Stress Response Element (ERSE) CCAATN9CCAGG recently lead to purification of ATF6, a Type-2 90kDa mammalian homolog that responds to ER-stress via proteolytic cleavage within the TM. This releases the biologically active N-terminal 50 kDa fragment containing a leucine-zipper domain (bZIP) binding to the ERSE and acting as a co-transcriptional activator (Fig. 6)184. Among many unresolved questions are how J-secretases act within TM, and the role of adaptor or chaperone proteins. Generally, scissile bonds are protected by hydrogen bonding within an α-helical conformation. However the removal of extracytosolic domains by D-type secretases (sheddases) followed by interaction with PS may facilitate unfolding of D-helices, and formation of random coils that may render these more susceptible to proteolysis, resulting in enhanced generation of fibril-forming or aggregated Thus, processing of UPR components may be relevant to AE peptides116. folding/misfolding of APP or intermediates implicated in AD/FAD pathology.
Fig. 6.: Postulated roles for PS modulating a TM-endopeptidase (J -secretase). See text for abbreviations and other details.
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CONCLUDING COMMENTS Interest in secretases arises from their roles in turnover of APP or intermediates to form senile plaque Aβx. Rapid advances within the past year have lead to successful purification of a new class of novel aspartyl proteases including BACE-1 (Asp-2 ) having Esecretase specificity at pH 4.5-5.5. Metalloendopeptidases of the disintegrin family ADAM-17 (TACE ), and ADAM-10 (KUZ ) act as putative D-secretases, but cleavage within the fibril-forming domain of APP yields non-amyloidogenic products. There has been less progress in characterizing γ-secretase(s) essential for final processing of C-terminal APP to form Aβ/Aβx found in AD deposits. Interest in PS stems from an autosomal dominant gain-of-function conferred by single point or other mutations: these promote generation of Aβx by shifting sites targeted by γsecretase. Presenilins are labile although enzymes forming major metabolites remain to be characterized. Proteolysis is not mandatory since FAD-PS or constructs lacking key protease sites retain toxicity. Gain of function for labile PS mutants may arise by binding of fragments to accessory proteins to form ‘stabilized’ complexes that retain a pathological signature. Recent studies implicate PS for conversion of other TM proteins including Notch-r, and UPR (unfolding protein response) components Ire1 D and ATF6. Potential roles for PS itself as J-secretase with unique aspartyl protease properties, or as an adjunct protein necessary for its activity remain to be explored, and this may require novel concepts that cannot be explained by classical enzymology.
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VanderVere, M. L. Bayne, C. D. Strader, J. M. Rommens, P. E. Fraser, and P. St.George-Hyslop, Presenilins interact with armadillo proteins including neural-specific plakophilin-related protein and beta-catenin, J. Neurochem. 72:999 (1999). 155. M. Nishimura, G. Yu, G. Levesque, D. M. Zhang, L. Ruel, F. Chen, P. Milman, E. Holmes, Y. Liang, T. Kawarai, E. Jo, A. Supala, E. Rogaeva, D. M. Xu, C. Janus, L. Levesque, Q. Bi, M. Duthie, R. Rozmahel, K. Mattila, L. Lannfelt, D. Westaway, H. T. Mount, J. Woodgett, and P. St George-Hyslop, Presenilin mutations associated with Alzheimer disease cause defective intracellular trafficking of beta-catenin, a component of the presenilin protein complex, Nat. Med. 5:164 (1999). 156. B. Stahl, A. Diehlmann, and T. C. Sudhof, Direct interaction of Alzheimer's disease-related presenilin 1 with armadillo protein p0071, J. Biol. Chem. 274:9141 (1999). 157. S. M. Stabler, L. L. Ostrowski, S. M. Janicki, and M. J. 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CASPASES IN NEURODEGENERATION
Jörg B. Schulz1, Michael A. Moskowitz2 Neurodegeneration Laboratory Department of Neurology University of Tübingen D-72076 Tübingen, Germany 2Stroke and Neurovascular Regulation Laboratory Neurology and Neurosurgery Service Massachusetts General Hospital Harvard Medical School Charlestown, MA 02129 1
INTRODUCTION Caspases are the mammalian cell-death-effector proteins. They may have an important role in acute and chronic neurodegenerative diseases, exemplified by stroke, head trauma, Huntington's, Parkinson's and Alzheimer's disease. They execute cell death but may also be linked to the initiation of chronic neurodegenerative diseases. Peptide or protein inhibitors of caspases protect neurons in vitro or in animal models of neurological disorders. Although preclinical results are promising, clinical studies have not been performed because of the lack of synthetic caspase inhibitors that cross the blood brain barrier. Such agents are a major focus in current programs of drug development and will hopefully become available soon.
APOPTOSIS Apoptosis is an important form of cell death characterized by a series of distinct morphological and biochemical alterations suggesting the presence of a common execution machinery in different cells. Condensation and fragmentation of nuclear chromatin, compaction of cytoplasmic organelles, a decrease in cell volume and alterations to the plasma membrane are classically observed resulting in the recognition and phagocytosis of apoptotic cells. The nuclear alterations are often associated with internucleosomal cleavage of DNA, recognized as DNA laddering on conventional agarose gel electrophoresis. Internucleosomal cleavage of DNA is a relatively late event in the apoptotic process, which in some models of neuronal cell death may be dissociated from early critical steps1-3. In fact, apoptosis is not restricted to nucleated cells4. Nevertheless, detecting DNA fragmentation is simple and often used as a criterion to determine whether or not a cell is dying by apoptosis. Unfortunately, it is often overinterpreted and not without shortcomings.
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CASPASES Caspases are the major executioners of apoptosis but some caspases are also involved in cytokine processing and inflammation. A family of at least 14 related cysteine proteases are known, named caspase-1 to caspase-14, depending upon their sequence of discovery. The family includes two murine homologues (caspase-11 and – 12) that have no known human counterparts yet. Caspases are synthesized and stored as inactive proenzymes. They contain an N-terminal prodomain together with one large (p17 to p20) and one small (p10 to p12) subunit. The activation of caspases requires cleavage (usually by other caspases) to liberate one large and one small subunit, which associate into a heterotetramer, containing two small and two large subunits5. Due to their differential substrate specificities they may be divided into three major groups , which also provides insight into their biological roles in inflammation and apoptosis6,7. The three groups can be largely distinguished by their P4 preferences, a crucial determinant in caspase specificity. Group-I enzymes (caspases-1, -4, -5 and -13) prefer hydrophobic residues at P4 and are involved in the maturation of multiple proinflammatory cytokines. Group-II enzymes (caspases-2, -3, and-7 and) have a strict requirement for Asp at P4 and will cleave DxxD apoptotic substrates. The cleaved substrates will disable cellular repair, halt cell cycle progression, inactivate inhibitors of DNA fragmentation, dismantle structural elements and mark dying cells for engulfment. GroupIII enzymes (caspases-6, -8, -9, -10) prefer branched-chain aliphatic amino acids in the position P4 and will activate group-II caspases and other group-III caspases.
EVIDENCE FOR APOPTOSIS AND CASPASE ACTIVATION IN HUMAN DISEASES The development of therapeutic targets for acute and chronic neurodegenerative diseases depends in part upon identifying specific mechanisms of cell death in humans and animal models. In sporadic and inherited neur egenerative disorders like Huntington's disease (HD) and Alzheimer's disease (AD) 8,9 , the presence of chromatin condensation and DNA fragmentation suggests that cells are dying by an apoptotic-like mechanism. The results are more controversial for Parkinson's disease (PD): two studies reported that 5-8% of neurons in the substantia nigra pars compacta (SNpc) of PD patients show DNA-end labeling, a third study reported 6% of the melanin-containing neurons with chromatin changes upon electron microscopy 10-12 On the other hand, others have failed to detect apoptotic changes in the SNpc13-15, possibly because apoptotic DNA fragments have a relatively short half-life. While the significance of morphologic features suggestive of apoptosis remains controversial in human postmortum tissue, the detection of molecular apoptotic markers in human brain tissue and in animal models supports the pathological evidence. In PD16, HD17 and AD18 , activation of caspases as well as appearance of substrate cleavage products support the hypothesis that apoptosis and processed caspases are important mediators of neuronal cell death in neurodegenerative diseases. In brain tissue taken during surgical decompression for acute intracranial hypertension following trauma, cleavage of caspase- 1, upregulation and cleavage of caspase-3 were found along with DNA fragmentation with both apoptotic and necrotic morphologies19. Evidence for apoptotic cell death in human stroke is scant at the present time. In two autopsy cases, a significant number of TUNEL-positive granule cells were found in the cerebellum after global ischemia20 ; the importance of such changes to postmortem interval was not clarified. Unlike rodents, human cerebral neurons reportedly exhibit little or no caspase-3 immunoreactivity under normal conditions21. However, during ischemic degeneration, caspase-3 protein expression increases.
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Figure 1: cascade of apoptotic events in acute and chronic neurodegenerative diseases. The panel depicts our current understanding of intracellular events leading to the activation of effector caspases, e.g. caspase3. Different apoptosis-triggering pathways employ distinct signal transduction pathways that culminate in the release of cytochrome c from mitochondria. Alternatively, caspase-8 which contains two death effector domain-like molecules (DED), reacts with FADD (Fas-associating protein with death domain), and is recruited for activation at either the CD95 death-inducing signaling complex (DISC) or the tumor necrosis factor-α receptor-1 (TNF-R). However this mechanism has not clearly been show to occur in mature, differentiated neurons (Induction phase ). Two general mechanisms for release of cytochrome c (or other caspase-activating proteins) have been proposed: one involves osmotic disequilibrium leading to an expansion of the matrix space, organellar swelling, and subsequent rupture of the outer membrane; the other envisions opening of channels in the outer membrane, thus releasing cytochrome c from the intermembrane space of mitochondria into the cytosol. Members of the Bcl-2 family may perform double duty, controlling cytochrome c release from mitochondria and also possibly binding Apaf-1. Cytochrome c activates caspases by binding to Apaf-1 causing it to associate with initiator procaspases (e.g., procaspase9). Apaf-1 shares sequence similarity with the prodomain of Ced-3 and other initiator caspases with long prodomains including caspase-1, -2, -8, -9 and -10. This domain may serve as a caspase recruitment domain (CARD complex) by binding to caspases that have similar CARDS at their NH2 termini. Upon reception of a death stimulus, the complex might dissociate, freeing Apaf-1 and thereby triggering the activation of initiator caspases (Propagation phase ). Active initiator caspases may activate effector caspases (e.g. caspase-3), initiating the proteolytic cascade that culminates in apoptosis (Execution phase ). Potential sides for peptide (zIETD-fmk, caspase-8 inhibitor; zLEHD-fmk, caspase-9 inhibitor; DEVD-fmk, caspase-3 inhibitor) and protein inhibitors (IAP, p35) of caspases to interfere with this pathway are noted.
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T-cell mediated inflammation may play a key role in the pathogenic mechanism sustaining multiple sclerosis. At nearly all stages of multiple sclerosis, apoptoti cells bearing myelin markers, presumably oligodendrocytes, are present in brain 22,23. Multiple sclerosis plaques show a pronounced expression of Fas/Apo-1/CD95 and Fas ligand death signaling molecules on glia cells, including oligodendrocytes, suggesting that the Fas signaling pathway may be pathogenetically relevant to multiple sclerosis23,24.
STUDIES IN ANIMAL MODELS Because post mortem brains often contain artifacts due to autopsy delay, and typically show end stage disease rather than an evolving disease process, the best clues to mechanisms underlying neurodegenerative diseases come from animal studies.
Stroke Morphological and biochemical characterization of central neurons following global or focal ischemia suggests that apoptosis contributes to ischemic death of neuron25,26. Two lines of evidence indicate that caspase-3 activation plays a key role following transient forebrain ischemia. Firstly, immunohistochemical and biochemical studies show that caspase-3 activation occurs in susceptible cortical and hippocampal neurons following temporary (2 hr) middle cerebral artery occlusion produced by filament insertion into the carotid artery or four vessel occlusion for 12 min and global ischemia, respectively27,28. Secondly, intracerebral administration of selective caspase peptide inhibitors reduce cellular and behavioral deficits following transient focal (30 min to 2 h filament insertion into the carotid artery) or global ischemia (bilateral carotid artery occlusion for 5 min)29-31. Moreover, neuroprotection can still be achieved when intracerebral administration of a caspase-3-specific (DEVD-cmk) or a pan-specific (zVAD-fmk)-caspase inhibitor was delayed by 6-9 hr after mild transient (30 min) focal ischemia29,32 or after chemically-induced hypoxia33. In both models the N-methy-Daspartic acid receptor antagonist dizolcipine (MK-801) is only efficacious when administered less than 1 hour after the initial insult. The prolonged therapeutic window makes caspase inhibitors particularly attractive for the treatment of stroke. The observation that distinct mechanisms of cell protection reduce neuronal injury in ischemia suggests the possibility that, when combined, these treatments may act in synergy. Pretreatment with subthreshold doses of MK-801, and delayed treatment with subthreshold doses of zVAD-fmk, provide synergistic protection compared with either treatment alone. Moreover, both treatment xtend the therapeutic window for caspase inhibition for an additional 2 to 3 hours33,34. The data suggest the potential value of combining treatment strategies to reduce potential side effects and to extend the treatment window in cerebral ischemia. Inhibitors of apoptosis proteins (IAPs) are a family of proteins which confer resistance to neuronal apoptosis35 by caspase inhibition36,37. Adenovirally-mediated overexpression of neuronal apoptosis inhibitory protein (NAIP) and of X-chromosomal IAP (XIAP) attenuates ischemic damage in the hippocampus er global ischemia induced by four vessel occlusion for 12 min and behavioral deficits 27,38 . Spinal cord ischemia activates caspases-8 and -3 which colocalize in neurons with cells showing DNA fragmentation. The Fas receptor expressed in neurons coexpressing caspase-8, may provide one upstream mechanism for caspase activation39.
Trauma The inhibition of caspases may offer therapeutic potential in the treatment of traumatic brain or spinal cord injury. Caspases-1 and -3 are cleaved and activated after fluid percussion-, impact- or cold injury-induced brain trauma40-42 and impact spinal trauma43 in neurons and oligodendrocytes. Intracerebroventricular injection of zVADfmk, a panspecific caspase inhibitor, zDEVD-fmk, a caspase-3 specific inhibitor, or YVAD- fmk, a caspase-1 specific inhibitor, markedly reduces posttraumatic apoptotic
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cell death and significantly enhances neurological recovery40-42. Intraocular application of caspase inhibitors reduces delayed cell death of retinal ganglion cells caused by transection of the optical nerve44.
Multiple Sclerosis Inhibition of oligodendrocyte apoptosis in autoimmune demyelinating diseases may block or attenuate the neurological manifestations of the disorder. Experimental autoimmune encephalomyelitis (EAE) is a rodent model of multiple sclerosis. Oligodendrocytes from transgenic mice that express the baculovirus anti-apoptotic protein p35 (inhibits multiple caspases), were resistant to cell death induced by TNF-D agonistic anti-Fas antibody and INF-J Further, cre/p35 transgenic mice were resistant to EAE induction by immunization with the myelin oligodendrocyte glycoprotein. The numbers of infiltrating T cells and macrophages/microglia in the EAE lesions were significantly reduced, as wer the numbers of apoptotic oligodendrocytes expressing the activated form of caspase-345.
Huntington’s diseases This disease is characterized by the presence of mutated Huntingtin protein containing extended repeats of the amino acid glutamine; this mutated protein appears to be neurotoxic, but proteolytic cleavage may be needed to generate a neurotoxic fragment from the full-length, mututated Huntingtin protein. Caspase-3 cleaves Huntingtin in vitro and in apoptotic cells46,47, although an in vivo role for caspase-3 in generating Huntingtin fragments has not yet been established. Caspases are synthesized as pro-enzymes that are activated by proteolytic cleavage. According to conventional theory, procaspases are not active; recently, however, several groups have shown that procaspases may also have catalytic activity, albeit at a level much lower than that of active caspases48. Mutant, full-length Huntingtin with extended polyglutamine tracts may provide a suitable substrate for basal procaspase activity in the absence of apoptosis generating neurotoxic Huntingtin fragments. Ona and colleagues have shown recently, that a dominant-negative mutant of interleukin1-1 E-converting enzyme (caspase-1), delays the onset and progression of pathology in a transgenic model of Huntington’s disease expressing a mutant human Huntingtin exon 1 encoding an expanded polyglutamine repeat49. Further, intracerebroventricular administration of zVAD-fmk delays mortality in this model.
Parkinson’s disease 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces clinical, biochemical and neuropathologic changes reminiscent of those occuring+ in idiopathic Parkinson’s disease (PD). The toxicity of its active metabolite MPP involves the activation of caspases in vitro 50 and in vivo51. In mice chronic administration of MPTP induces apoptotic cell death in dopaminergic substantia nigra neurons. Transgenic mice expressing a dominant-negative m tant of interleukin-1 E converting enzyme are relatively resistant to MPTP toxicity52. Further, the overexpression of the antiapoptic protein, Bcl-2, prevents activation of caspases and provides protection against MPTP toxicity51.
CASPASE INHIBITION AND INFLAMMATION Until recently, the brain was considered immunologically privileged and unable to develop inflammation unless the blood-brain barrier was disrupted. We now know that the brain is capable of sustaining its own endogenous inflammatory reaction, and the evidence in Alzheimer’s disease is particularly strong53, but information occurs in other neurodegenerative diseases as well. It has been hypothesized that this reaction contributes heavily to progressive neuronal death.
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Non-specific caspase inhibitors block group-II and III caspases involved in apoptosis, and caspase- 1, the enzyme that cleaves pro-interleukin- 1E to mature interleukin-1E Since interleukin-1 receptor antagonists prevent damage after focal ischemia induced by permanent middle cerebral artery occlusion54, the first study using pan-caspase inhibitors was in ended to show that blocking ICE activity prevents ischemic damage in the same model55. In fact, transgenic mice expressing a dominant negative caspase-1 are protected in animal models of stroke induced by 3 h of cerebral artery occlusion followed by 24 h of reperfusion56, impact-induced head trauma 41, Parkinson’s disease52, amyotrophic lateral sclerosis57 and Huntington’s disease58. It remains to be elucidated whether both, inhibition of inflammation and inhibition of caspase-mediated neuronal apoptosis contribute to the protective effects of caspase inhibitors. Of note, murine caspase-11, with homology to human caspase-4, promotes both caspase-1 and caspase-3 processing, thereby enhancing both apoptosis and cytokine maturation59.
CASPASE INHIBITORS UNDER DEVELOPMENT The development of non-peptide selective caspase inhibitors which cross the blood brain barrier has become a major goal of drug discovery. One example, L-826791 is under development by Merck for the treatment of cerebrovascular ischemia60. L826791 has an IC50 value of only 8.0 nM and in vivo limits cerebral cortical damage following acute occlusion of the middle cerebral artery in rats. The caspase inhibitor IDN-6556 is under investigation by IDUN Pharmaceuticals for the treatment of alcoholic hepatitis, inflammation, neurodegenerative diseases and ischemia. Its IC50 values for maximal efficacy against recombinant activity and in cell culture are 0.5 nM and 1.8 µM, respectively 61. Cytovia is investigating caspase inhibitors for the potential treatment of degenerative diseases, hepatitis, sepsis and cerebral ischemia. The lead compound, CV1013 shows good efficacy in animal models and has low toxicity and favorable PK profile62. Other companies including Vertex Pharmaceuticals, Texas Biotechnology Corp., Novartis and Aventis are developing new and specific caspase inhibitors but no further published information is currently available.
LIMITATIONS AND CAUTIONS Caspases and related proteins are emerging as important therapeutic targets in a variety of acute and chronic CNS diseases. Preclinical evidence in stroke supports the need to investigate anti-apoptotic treatment strategies, particularly because caspase inhibitors reduce tissue injury when administered many hours after mild ischemia. In the clinical setting, anti-apoptotic strategies might become useful to treat brief episodes of brain ischemia or be given in advance of risky surgical procedures (e.g. cardiopulmonary by-pass) or combined with thrombolytics or agents in which synergy has been documented (e.g. glutamate receptor antagonists). Furthermore, caspase inhibitors may provide promising opportunities for other neurological conditions in which cell death is prominent. Although the results of treatment in animals with caspase inhibitors are promising, clinical studies have not yet been performed because of the lack of synthetic caspase inhibitors that cross the blood brain barrier. Such agents are a major focus in current programs of drug development and will hopefully become available soon. In addition, therapies that lead to the increased expression of IAPs are a potential avenue for treatment of chronic neurodegenerative disorders.
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THERAPEUTIC APPROACHES WITH PROTEASE INHIBITORS NEURODEGENERATIVE AND NEUROLOGICAL DISEASES
IN
Kevin K.W. Wang Department of Neuroscience Therapeutics Parke-Davis Pharmaceutical Research A Division of Warner-Lambert Company Ann Arbor, MI 48 105
INTRODCUTION TO THE CLASSES OF PROTEASES IMPLICATED IN NEURODEGENERATIVE AND NEUROLOGICAL DISEASES There are five major classes of mammalian proteases identified to date: serine proteases (EC 3.4.21), cysteine proteases (EC 3.4.22), aspartate proteases (EC 3.4.23) metalloproteases (EC 3.4.24) and theronine proteases (EC 3.4.25)1. Interestingly, members of all five protease classes have been implicated at contributing factors in various neurological or neurodegenerative disorders (Table 1). Due to the differences in how these proteases are activated and how they function, different strategies of inhibition are required. Cysteine proteases have cysteine; histidine and asparagine residues that form the catalytic triad involved in the hydrolysis of protein peptide bonds. Due to the requirement of the reduced cysteine sulfhydroyl group, a reducing intracellular environment is needed. Calpain is a heterodimeric cytosolic cysteine protease that is also regulated by free Ca2+ and it has been implicated in contributing to cell death in stroke, traumatic brain injury (TBI) as well as Alzheimer’s disease (AD)2,3 (Table 1). Another subfamily of cysteine proteases called caspases has been implicated in apoptotic neuronal death. Caspases-3 which is activated by caspase-8 via receptor-linked pathway (e.g. TNF-alpha receptor) or by caspase-9 via a mitochrondria-dependent pathway. The cytosolically located caspase-3 then goes on and attacks various cellular proteins and executes the programmed cell death (apoptosis)3-6 Thus, caspase-3, -8 and -9 contribute to the apoptotic cell death components in various neurological (stroke, TBI, spinal cord injury (SCI)) and neurodegenerative disorders (AD, amyotrophic lateral sclerosis (ALS)) (Table 1). Their related cousin Caspase-1 (interleukin converting enzyme or ICE), on the other hand, is more likely to be involved in the inflammatory responses upon neuronal injury. It does so by processing and activating the pro-inflammatory cytokine pro-interleukin beta to its mature form7. Cathepsin B is a lysosomal cysteine protease that is also implicated in ischemic strokes8,9 Its selective and cell-permeable inhibitor CA074Me10 is neuroprotective11,12 Both cathepsin B and cathepsin D immunostatining appear to intensified in AD brain13. Cathepsin D, unlike cathepsin B, is an aspartate protease which has been linked to cerbral ischemia14. It has also been implicated as a candidate beta-secretase of amyloid precursor protein (APP) 15-17. Also recently, a fury of activity in the literature have pointed to the identification of two potential asparate proteases as candidate beta-secretases (BACE or Asp-2 and BACE2 or Asp-118-21 (Table 1). They are unique in that they have
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transmembrane helix in the C-terminal which localized them to golgi and endosome membrane where APP protein is also located. Thrombosis is one of the major causes of ischemic stroke in human. Anticoagulants such as heparin have demonstrated beneficial effects in reducing brain edema and infarct volume in rats subjected to thrombotic middle cerebral artery occlusion (MCAO), a model for human thrombotic strokes22. Following the same rationale, thrombin (a serine protease) is also implicated in thrombotic stroke23- 25 . In a rat thrombotic MCAO model, where a platelet-rich thrombus was used for occlusion, a selective thrombin inhibitor argatroban, was found to decrease the size of the cerebral infarction and improved neurological deficits26. A recent study shows that post-ischemic subcutaneous injection of argatroban (5 mg/kg) significantly attenuated cell damages in the cerebral cortex, attenuated of brain edema, increased cortical cerebral blood flow after reperfusion and also attenuated during 14 days' observation27. But it is important to point out that antithrombotic agents are generally contraindicated in hemorrhagic stroke. Matrix metalloproteases (e.g. gelatinases, collagenases and stromelysins) are zincrequiring proteases that are secreted into intercellular space (MMP-1 through MMP-12)28. Their major targets are extracellular matrix (ECM) proteins, such as collagen. Some of them are inducible when the cells are stimulated. MMPs also have a reputation of capable of refolding back into active enzyme following removal of denaturing detergent such as SDS. A zymography technique is commonly used. MMP (such as MMP2 (gelatinase A) or MMP9 (gelatinase B) or MMP3 (stromeysin-1)) preferred substrates such as collagen, Because of their matrix-degradative capability, MMP-2, -3 -7 and -9 have been implicated in cerebral ischemia29-31 multiple sclerosis (MS) or in its animal model EAE (experimental autoimmune encephalomyelitis) (Table 1) 32-35. Proteasome is a very complex multi-subunit and large oligomeric proteases36. Its is composed of both small regulatory subunits and protease subunits (with distinct substrate specificity). It is thus sometimes called multi-catalytic protease (MCP). It also contains a regulatory component that confers its ATP-dependence as well as ubiquitin-dependence. Interestingly ATP is hydrolyzed during peptide hydrolysis. Also, protein substrate must first be conjugated at lysine-residues with multimers of a protein called ubiquitin before its is recognized by proteasome. All the proteasome catalytic subunits belong to a novel class of protease (theronine proteases). It utilizes two theronine residues at the N-terminal for the hydrolysis of peptide bonds. Proteasome has also been implicated in stroke)37 as well as MS38. Proteasome inhibitor (CVT-634) was recently found to be neuroprotective by suppressing the NF-kB activation pathway39 (Table 1). Table 1 Known proteases implicated in neurodegenerativeand neurological diseases and reference inhibitors Protease
Class
Disorder(s)
Ref. Inhibitors
Calpain
Cysteine
Stroke, TBI, AD
MDL28170, PD150606, SJA6017
Caspase-3, 8,9
Cysteine
Stroke, TBI, SCI, AD
Z-VAD, Z-D-DCB, Ac-DEVD-fink
ICE (Caspase-I)
Cysteine
Stroke
Ac-YVAD-CHO, VE-18858, VX-740
Cathepsin B
Cysteine
Stroke, AD
CA074-Me
Thrombin
Serine
Stroke, TBI
Argatroban
Cathepsin D
Aspartate
AD
Pepstatin A, CEL5-A, EA-1
MMP2 3,7 & 9
Metalllo -
MS
CP-4- 7 1,474 (broad-spectrum)
BACE
Aspartate
AD
KTEEISEVN(Statine)DAEF
Proteasome
Theronine
MS, stroke
lactacystin, MG132, PSI
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CURRENT INHIBITORS OF PROTEASES The use of inhibitors to study the role of a protease in a particular neurodegenerative and neurological disease is a very powerful technique. But the most common limitations are the lack of cell permeability and selectivity of the protease inhibitor. Sometimes, by applying higher concentration of a peptide inhibitor with low cell permeability, one can get sufficient amount of the inhibitor into the cell. Selectivity is a more important issue. For example, several calpain inhibiting peptide aldehydes (such as calpain inhibitor I and II) also cross-inhibit theronine protease proteasome. Yet, a calciumbinding site directed calpain inhibitor PD1 50606 (Calbiochem Co.) does not inhibit proteasome40 (Table 1). Lactacystine is naturally occurring beta-lactone that specifically and colvantly modifies Thr and is thus highly selective in inhibiting proteasome38. Other proteasome inhibitors including Cbz-Leu-Leu-Leu-CHO (MG132), and Cbz-IleGlu(OtBu)-Ala-Leu-H (PSI), are also more selective for proteasome than for calpain (Calbiochem Co.) (Table 1). Thus, the use of more than one inhibitor is strongly advised. Iodinated calpain inhibitors have been used successfully in labeling activated calpain in activated platelets41. Similarly, Biotin-conjugated cas ase inhibitor. Recently, two biotinlabeled caspase inhibitors (Ac-YVK(Biotin)D-amk42 and Cbz-VK(Biotin)D-fmk43 were utilized successfully to label caspase-3 in apoptotic cell lysate. For the caspases family, the apoptosis-effector caspases (caspase-3 and 7) are readily inhibited by Ac-DEVD-CHO or DEVD-fmk, while the apoptosis upstream caspases (caspase-8, -9 and -10) are readily inhibited by Z-VAD. Pan-specific caspases inhibitors (Z-D-DCB, Boc-Asp-fmk) also exist and are quite useful neuroprotectant in vitro and in vivo3,44-49. On the other hand, inflammation-linked ICE (Caspase-1) are selectively inhibited by Ac-YVAD-CHO and Ac-WVAD-CHO. Vertex pharmaceutical have also developed potential clinical ICE inhibitor candidates ( VE- 18858, VX-740)3,50,51. Among the cysteine protease cathepsins K, L and B, there are now documented inhibitors that show subclass selectivity52-54 (Table 1). Aspartic protease inhibitor pepstatin A has been used successfully in inhibiting cathepsin D activity in intact cells55. Bi et al.56 recently reported several potent cathepsin D inhibitors (CEL5-A, CEL5-G, EA-1). Lastly, argatroban is a well established selective thrombin inhibitor57.
PROTEASE TARGET VALIDATIONS Assuming that there are some data in the existing literature suggesting that a specific protease might be involved in a particular neurological disease, it is important to provide further “proof-of concept” type of compelling evidence that the protease target is in fact valid and relevant to the disease of interest. Table 2 outlines a number of evidence that have been used in the past. Co-localization of protease protein to the site of disorder (e.g. a specific CNS region or specific neuronal cell types) gives a positive correlation of the protease to the disorder. Similarly, increased mRNA an/or protein level of the protease of interest in the disease state compared to control is powerful evidence. Sometimes, the specific protease activity can be monitored either by direct assaying or by following the integrity of endogenous protein substrate(s) in animal disease model. Should this protease activity increased significantly in disease state, it is compelling evidence for the involvement of the protease. It also applies to cell culture models (if available) of a neurological disorder) if increase of a protease activity can be detected reproducibly. It is also common to approach target validation by suppression of the protease of interest or intervention of its processing / activation. This is most commonly achieved pharmacologically by applying selective inhibitors to influence the outcome in the animal disease or in neuronal cell culture (e.g. neuroprotection against ischemic injury in vivo or in situ). Alternatively, genetic manipulation could be employed to knockout the protease gene and then study the k/o mice to see if they show different susceptibility compared to control mice. Similarly, overexpressing a protease using CNS neuron-specific promoters such as PDGF-B, Thy-1, prion or elonase promoters in transgenic mice is also very powerful technique that might produce a phenotype which mimics or exaggerates the disease state of interest.
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Table 2 Target protease validation checklist Key studies or evidence Localization of protease to site of disorder/disease Increased mRNA and protease levels in animal disease model or in human patients Increased protease activity or protease substrate breakdown product accumulation in neuronal cell culture model of disease Increased protease activity or protease substrate breakdown product accumulation in animal disease model or in human patients Selective reference inhibitors improve outcome in animal disease model or cell culture model Protease knockout mice have improved outcome in mouse disease model Protease-overexpressing transgenic mice have worsened outcome in mouse disease model
PROTEASE INHIBITOR SCREENING AND DEVELOPMENT STRATEGY Generally, for a pharmaceutical institute to discover and develop a selective patent-able protease inhibitor as a small organic molecule drug to treat a neurodegenerative and or neurological disease, various steps are required.
1. Source of enzyme and in vitro protease assays After the “proof of concept” stage, one must have either (I) purified native proteases from human and/or other species or (II) clone and express recombinant proteases (Fig. 1). In the latter case, one can either express the full length protease or in some cases a truncated form that preserves the catalytic protease domain is sufficient. Of course, the recombinant protease must have activity and substrate specificity similar to the native enzyme counterpart. An in vitro robust protease assay must then be established to determine inhibitory potency of compounds, as measured by inhibitory concentration that causes 50% inhibition (IC50) or kinetically by inhibitory constant (Ki) (Fig. 1). This primary assay can then be optimized and sometimes miniaturized to run through high volume and high throughput screening of random compound library (# entries usually needed to be at least 200,000300,000 to be successful in identifying structural leads). This strategy is designed to identify novel patentable inhibitor(s) simply by chance. Once structural leads are identified, additional medicinal chemistry support is required to further structure-activity relationship (SAR) to improve potency. Alternatively, if the protease can be crystallized as a complex with a reference inhibitor in an early stage, then rational drug design by medicinal chemists in collaboration with computer aided design (CAD) specialists can be launched to discover new patentable inhibitors. It is extremely desirable to have certain selectivity assays using other related protease within the same class and in other protease classes. Sometimes, even non-protease enzymes as selectivity screens are also included (Fig. 1). The lack of selectivity of a protease inhibitor drug usually contribute to undesirable side effects in vivo.
(b) Cell-based assays An important screening step for intracellularly located protease is the establishment of a cell-based protease assay (Fig. 1). To truly test the permeability and efficacy of an inhibitor in cells, it is not good enough to assay cell lysate but rather, the inhibitor must be
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introduced to intact cell culture medium directly and examine if there are active in inhibiting its target protease in intact cells! A powerful means to monitor intracellular protease activity is in fact not to introduce an artificial substrate, but rather monitoring the integrity and/processing of the protease as well as its endogenous protein substrates. The general technique calls for standard cellular protein extraction, SDS-PAGE, Western blots and then the detection with a specific antibody (Fig. 1). Most proteases exist as zymogens that are proteolytically or autolytically activated (e.g. cathepsin B, L, D, calpains, caspases and matrix metalloproteases (MMPs))1. Similarly, distinct endogenous protein substrates have been identified for a number of intracellular proteases such as calpain (alphaspectrin), caspases (poly(ADP)ribose polymerase or PARP) and proteasome (e.g. ikappaBalpha)58. With calpain and caspase, the substrates are generally cleaved into fragments with smaller molecular weight, readily distinguished from the intact proteins. The proteasome generally degraded proteins into very small peptides. So the disappearance or intensity reduction of the intact protein band, rather than protein fragments, is expected. Also, inhibition of proteasome (e.g. lactacystin) would result in an accumulation of the high molecular weight ubiquitin-conjugates of its protein substrates59 since ubiquitinization is the biochemical step preceding proteasome-mediated degradation . For certain proteases, it is possible to introduce a small fluorogenic peptide that is permeable to cell membrane. Thus, once the peptide is introduced to the cells, the protease can be activated by the addition of stimulus and the protease activity is tracked either continuously or as end-point measurement using a fluorometer. The use of chromogenic substrates (e.g. peptide-p-nitroanalide) usually does not have a strong enough signal for detection and is therefore not recommended. The choice of cell type is obviously very important. The primary criterion is the presence of reasonably high level of the protease of interest. Other favorable considerations are the lack of other proteases that could hydrolyze the same substrate and low levels of endogenous inhibitor(s) of the protease of interest. (Such as calpastatin for calpain, serpin for chymase and other serine protease, Inhibitor of apoptosis proteins (IAPs) for caspase60, and tissue inhibitor of matrix metalloprotease (TIMPs) for matrix metalloproteases). The choice is peptide substrate is also very important. The primary goal is to select a substrate that would be (i) selectively hydrolyzed by the protease of interest and (ii) cell membrane permanent. An example is the study of the calcium-activated protease (calpain). A cell-permanent fluorogenic peptide substrate such as succinyl-Leu-Leu-Val-Tyr-7-amino-4-chloromethylcoumarin (SLLVY-AMC) can be introduced and then the protease is activated by a calcium channel opener maitotoxin or calcium ionophore (e.g. A23 187), the fluorescence derived from the release of AMC is monitored in a 12-well plate format40. A similar peptide (tbutoxycarbonyl-Leu-Met-AMC) which is conjugated with glutathione intracellularly (used as cell trap) can be used61. A secondary cell-based assay or a cell-based assay to track cytotoxicity is also important to give an indication of the selectivity of the compound. Also, it is sometimes possible to establish a tissue-based assay (e.g. hippocampal slices) to monitor protease activity or other functional endpoints that can be influenced by the inhibitors (e.g. cell viability or conductivity).
(c) In vivo studies A desirable pharmacokinetic (PK) profile often makes the difference in whether the inhibitor will eventually become a drug or not, thus it is important to profile PK at an early stage. Acceptable plasma level, rat of clearance, metabolic stability (based microsome studies), cell membrane permeability, oral bioavailabilty (e.g. for chronic disorders) as well as brain penetration are important features. It is sometimes possible for the medicinal chemists to modify the inhibitor structures to improve their PK profile while maintaining potency of the compounds. Drug formulation is also an important issue. Depending on the route or administration (i.v,, p.o.) the criteria involved are quite diverse. Some experimentation is usually required to find an optimal formulation for in vivo studies. Ideally, the same formulation to be used in animal studies can be eventually used in humans. Any excipients used in such formulations (co-solvents, stabilizers, etc.) and the quantities used must meet Food and Drug Administration (FDA) guidelines. The physical stability of the compound upon storage also needed to be determined and potentially optimized.
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For chronically administrated drug, it is desirable to test compound in a small-scale acute toxicity study to ensure that the compound is reasonably safe before investing too much efforts in other in vivo studies (Fig. 1). Usually, PK data can help determine or recommend a therapeutic dose range to be used in vivo. Here, we assume that an animal model for the disease of interest is available. If a biomarker that tracks with the protease activity can be identified (e.g. a specific protein substrate fragment), it is greatly desirable to perform an initial studies to examine if the inhibitor can suppress the target protease in vivo and at what dose (mechanistic endpoint). Once this is established, it is time to conduct inhibitor studies with the optimal dosing regime and to determine if it would alter the efficacy endpoint in a positive direction (e.g. neuroprotection). If a single dose of a drug proves to be efficacious, it is often important to repeat the efficacy studies and establish a dose-response (efficacy) relationship. The data obtained will be extremely helpful to guide pre-clinical toxicology studies as well as possible clinical trails for the neurological or neurodegenerative disease of interest.
Figure 1. Flow chart for protease inhibitor screening and discovery strategy
PERSPECTIVES In this chapter, we gave an overview on various classes of proteases that might contribute to one or more neurological or neurodegenerative disorders. We also discussed the practical issues regarding how to discover and characterize new and selective protease inhibitors as potential drugs. It was shown that the inhibitory compound not only has been a great inhibitor pharmacologically, it also must have good drug-like features regarding pharmacokinetical and safety profile. Lastly, the ultimate test is to see if the compound of interest is robustly efficacy in animal disease model.
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PATHOPHYSIOLOGY OF CENTRAL NERVOUS SYSTEM TRAUMA: PROTEOLYTIC MECHANISMS AND RELATED THERAPEUTIC APPROACHES
Swapan K. Ray, Denise C. Matzelle, Gloria G. Wilford, Lawrence F. Eng*, Edward L. Hogan, and Naren L. Banik Department of Neurology Medical University of South Carolina Charleston, SC 29425 *Pathology Research Service Veterans Administration Hospital Palo Alto, CA 94304
INTRODUCTION Injury to the central nervous system (CNS) [e.g., spinal cord injury (SCI) and traumatic brain injury (TBI)] is one of the main health problems in the United States as well as in the world. CNS injury is also a major killer in the United States. The majority of these injuries are caused by automobile accidents, assaults, guns, falls, sports, and other traumatic events. The extent of the loss of neurological function depends upon the severity of injury. Primary injury to the CNS causes vascular change beginning at the mechanical impact and followed by secondary pathophysiological processes. These processes eventually lead to cell demise and tissue destruction which may be devastating. The secondary injury process develops over a period of hours or days after the primary injury, i.e. initial impact to the spinal cord or brain. It is associated with synthesis or release of mediating neurochemicals which alter blood flow, ion homeostasis, and metabolism and which may also be neurodestructive agents in CNS trauma. Although the full extent and interplay of mechanisms that underlie CNS injuries are yet unknown, several factors have been implicated in the secondary injury cascade. Employing impact or compression injury models in spinal cord and the controlled cortical contusion injury model in brain, investigators have identified a number of pivotal factors, including free radicals, Ca2+ influx, proteinases and lipases, glutamate, cytokines and other mediators in the progression of secondary injury in CNS trauma1-4. The identification of these destructive agents and the timing of their pathological actions are important steps in advancing research upon CNS trauma and will ultimately lead to strategies for the treatment of CNS injury.
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One of the most devastating events in the secondary injury process of CNS trauma is the increased intracellular calcium concentrations in neurons. Calcium plays many roles in the cell, including activation of proteinases and lipases whose activities are significantly increased in CNS lesions following injury. Among these is calpain, a ubiquitous, Ca2+-activated neutral proteinase whose increased activity degrades cytoskeletal proteins in spinal cord injury, traumatic brain injury, and cerebral ischemia with concomitant loss of both cytoskeletal and myelin proteins. The loss of these proteins destablizes membrane and neuronal architecture and eventually leads to cell death. Since these proteins maintain the structure and function of neurons and their processes, it is a high priority to protect neurons by prevention of cytoskeletal protein degradation. The use of calpain inhibitors as therapeutic agents in animal trials has proven useful for this and several recent studies of treatment of injured animals with calpain inhibitors alone and/or in combination with other agents, have shown neuroprotective effects3,5-7. Although this chapter has an overall concern with several proteolytic enzymes known to be altered in CNS trauma, emphasis is put upon calpain and calpain-related therapeutic strategies. Readers should consult the literature for reviews on the role of other proteinases in CNS injury.
PROTEINS AND PROTEINASES INVOLVED IN BRAIN AND SPINAL CORD INJURY Proteins It is not possible to review the vast literature on brain and spinal cord proteins in this short chapter. Instead, we have focussed upon the examination of the finite number of CNS proteins which are endogenous substrates of the proteases in brain and spinal cord activated following injury. The degradation of cytoskeletal, axonal and myelin proteins will be taken as the index of proteolytic activity in CNS injury because these are the essential framework elements in CNS. The loss of these proteins alters the structural integrity of cells and accompanies the axonal degeneration and myelin vesiculation observed following trauma. Comprehensive pictures of brain and spinal cord proteins are in recent review articles. The majority of studies of lesions in both spinal cord injury (SCI) and traumatic brain injury (TBI) have examined endogenous cytoskeletal and axonal proteins [including microtubule associated proteins (MAP1, MAP2), fodrin (D-spectrin), neurofilament proteins (NFPs; 68kD, 150kD and 200kD)] and myelin proteins [including myelin basic protein (MBP), proteolipid protein (PLP), myelin associated glycoprotein (MAG), and the enzyme protein, 2’,3’-cyclic nucleotide 3’phosphohydrolase (CNPase)]. Degradation of cytoskeletal proteins which maintain the integrity and architecture of cells and their processes, axons, and dendrites, has been correlated with post-traumatic morphological alterations in cells and processes. Loss of neurofilament proteins in SCI and TBI is associated with axonal degeneration6,8,9. Other proteins such as MAP1 and MAP2 are also readily degraded after CNS injury. The degradation of 230kD fodrin is widely used to assess the proteolytic activities of calpain and caspase-3 which cleave fodrin at different sites producing two different peptides, a calpain specific 150kD fragment and a caspase-3 specific 120kD fragment10,11. Degradation of myelin proteins accompanies a splitting and vesiculation of myelin lamellae. The two major proteins of myelin, proteolipid protein (PLP) and myelin basic protein (MBP), constitute about 50% and 30% of the total myelin
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protein, respectively. The molecular weight of PLP is approximately 24kD while that of MBP ranges from 17.5-21kD. In rodents, there is also a small MBP with a molecular weight of 14kD. The major MBP (18kD) is the most susceptible to degradations. PLP is masked by being tightly bound (covalently) to or complexed with lipids and resists proteolysis, though it can be degraded in the presence of detergent12,l3. Other minor proteins of the myelin sheath include MOG (myelin oligodendrocyte-specific glycoprotein), MOBP (myelin oligodendrocyte-specific basic protein) DM-20 of the PLP family of proteins, CNPase, and other enzymes14.
Proteinases Proteolytic enzymes which are found in the liver and other organs are also present in brain15. Activities of brain proteinases were first demonstrated in the 1930s by Krebs16 and Kerekes et al.17 and in the 1940s by Kies and Schwimmer18. Acid proteinase was the first such activity found in brain, and then other lysosomal proteinases, cathepsins A, B, and D were isolated and purified from brain19,20. These cathepsins degrade MBP and NFPs. Cathepsin D specifically hydrolyzes the PhePhe linkage in MBP14. Acid proteinase activity has also been found in neurons21. By contrast to acid proteinases, the non-lysosomal neutral proteinases are unstable and labile, difficult to purify and assay. However, with development of purification and assay methods, Ansell and Richter19 and Marks and Lajtha20 were able to determine neutral proteinase activity in brain and spinal cord. Subsequently, several neutral proteinases have been identified and purified from spinal cord and brain, including a very high molecular weight roteosome (750kD) [multicatalytic proteinase complex (MPC)], calpain (Ca2+-activated neutral proteinase)22, metalloproteinase23 and matrix metalloproteinases24,25. MPC consists of 7- 13 subunits26,27 while calpain exists as two isoforms, µcalpain and mcalpain, each with two subunits, an 80kD catalytic and 30kD regulatory subunit. Both isoforms are absolutely dependent on calcium for activity requiring µM and mM calcium concentrations, respectively. Calpain and the metalloproteinases are also associated with myelin22,23 and degrade MBP as substrate and with enzyme activity being increased in pathophysiology in trauma and diseases6,28,29. For detailed information on CNS proteinases readers are directed to relevant recent sources4,30.
MORPHOLOGICAL AND BIOCHEMICAL CHANGES IN SPINAL CORD INJURY Spinal cord injury, depending upon the severity, disrupts the functional axonmyelin structural unit and this leads to paralysis and other neurological deficits. The primary injury to the spinal cord disrupts blood vessels and initiates many devastating secondary pathoph siological alterations in the lesion which lead to cell death and tissue destruction8,31,23. There are extensive morphological changes, similar to those found in brain trauma, including progressive granular degeneration of axons, accumulation of hydroxyapatite-calcium crystals, vesicular degeneration of myelin, and phagocytosis by infiltrating macrophages8,31-37. Early studies of SCI lesions at the light microscopic level showed necrosis of gray and central white matter with damage to surrounding areas of white matter. Features of the lesions include edema, inflammation, and hemorrhage with infiltration of inflammatory cells (neutrophils) at 6 to 8 hours after trauma. The extent of damage to the cord depends upon the severity of injury as well as the time following trauma8,31,32,38 . Electron microscopic studies in experimental SCI have revealed progressive ultrastructural
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changes in the lesion. As early as 15 minutes after injury axons are undergoing granular degeneration and there is loosening of the myelin lamellae concomitant with significant axonal protein degradation in the lesion39. At longer intervals there is granular degeneration followed by vesiculation of the myelin sheath and inflammatory cell-mediated phagocytosis8,31,32. A most striking change in the SCI lesion is the accumulation of calcium crystals in axons as well as mitochondrial calcification8,31. This important finding first suggested that this increased calcium is responsible for the activation of calcium-activated lipases and proteinase8,39. Studies in numerous laboratories have correlated the ultrastructural changes observed in the SCI lesion at intervals following the injury with biochemical alterations. Changes have been described in lipids, cytoskeletal and myelin proteins, and lipolytic and proteolytic enzymes in the lesion and compared to sham (uninjured) controls at different times following the injury8,31,32,38,40-45. An early but significant loss of axonal and myelin proteins, particularly neurofilament proteins (NFPs), microtubule associated protein (MAP2) and myelin basic protein (MBP) is evident at 15 to 30 minutes and progresses with time following trauma8,34,39,46,47. All three major classes of NFPs (200kD, 150kD, 68kD) are progressively degraded and they are completely broken down in the SCI lesion at 6 to 72 hours after trauma. Among the myelin proteins, MBP is more susceptible to degradation in the lesion than proteolipid protein (PLP), the major protein of CNS myelin. A substantial (3040%) loss of MBP is evident at 1 hour after trauma while the loss of this protein at 24 hours following injury amounted to 90% and more. In comparison to MBP, only 40% of PLP is lost in the lesion at 24 hours after trauma. The myelin associated glycoprotein (MAG) also has been found to be extensively degraded in the lesioned cord compared to control. This time-dependent degradation of both cytoskeletal and myelin proteins in the lesion correlates very well with the ultrastructural degeneration of the axon-myelin structural unit39,48. The loss of these proteins in the lesion indicates the crucial involvement of proteolytic enzymes in the demise of cells and destruction of lesioned cord following injury. The ultrastructural or morphological changes that occur in brain following TBI and ischemia are not discussed in detail in this chapter. Nonetheless, like SCI, damage to cells and myelinated axons and axon shearing has been reported by many laboratories49-52. Diffuse axonal injury is a common feature in white matter of brain and optic nerve due to TBI, anoxia and ischemia. These changes also correlate with alterations in proteins and roteinases. For further information, consult articles cited above or related reviews53-55.
PROTEINASES IN CNS TRAUMA In the mid 1970s and early 1980s the activities of various hydrolytic enzymes were determined in the lesions of spinal cord following injury. Changes in the activities of several other hydrolases, including N+-K+-ATPases, acetylcholinesterase, cytochrome reductase, and lysosomal hydrolases (e.g., acid phosphatase) have also been determined in the lesion. The activity of these enzymes 44 were altered in the lesion when compared to control . However, the increase in the activities of lysosomal enzymes in the lesion appears to contribute to tissue degeneration only at longer times after injury and not in the secondary injury cascade of the first day. Similar hydrolytic enzymes, including phospholipases and others were studied in TBI and their activities were also found to be altered in brain following injury44,56.
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Detailed studies on the kind of proteolytic enzymes that are involved in tissue destruction in CNS trauma were first examined in the lesion of experimental SCI in the early 1980s. Cytoskeletal and myelin proteins were found to be progressively degraded with time following injury in SCI lesions, which suggested that proteolytic enzymes may be one of the mediators of secondary injury in tissue destruction. Since various proteinases may be involved in this process, the activities of both extralysosomal neutral and lysosomal acidic proteinases were determined. Soluble fractions from lesioned and control spinal cords (autologous and sham) were isolated and incubated at different pHs (cathepsin D-like activity, pH 3.0; cathepsin B-like, pH 6.0; and uncharacterized neutral proteinase, pH 7.4) using purified MBP as a substrate. Using an indirect assay method, the activities of different enzymes were determined by assessing the extent of loss of MBP using SDS-PAGE. The degradation of MBP by neutral proteinases in the lesion was progressive, usually with concomitant production of MBP-breakdown products43. In comparison to neutral proteinase, MBP breakdown by cathepsin B and cathepsin D-like proteases was negligible. In addition, the specific inhibitor of cathepsin D, pepstatin, did not inhibit MBP degradation while this breakdown of MBP was significantly prevented by leupeptin, a neutral proteinase and cathepsin B inhibitor. Leupeptin, on the other hand, did not prevent MBP breakdown by cathepsin B (at pH 6.0). These studies revealed that in the lesion, the neutral proteinase activity was much greater than that of the cathepsins and was primarily responsible for MBP degradation. In addition, neutral proteinase activity progressively increased with time following injury leading to a 300% increase in activity in the lesion at longer intervals (24 hours) following injury compared to autologous and sham controls43. In CNS injury there is infiltration of inflammatory/immune cells which secrete many proteinases, including matrix metalloproteinase (MMPs). Activities and expression of these enzymes increases in a number of CNS disorders, including multiple sclerosis (MS), Alzheimer’s disease, ischemia, and glioblastoma24,57-61. Since inflammation is common to CNS injury and MMPs are associated with inflammation, activities of these enzymes were examined in SCI and TBI. Activity of gelatinase B (MMP-9), an enzyme involved in the opening of the blood-brain barrier (BBB)62, was increased in the SCI lesion as well as in experimental brain trauma. Increased hippocampal expression of MMPs, including MMP-3, MMP-9, and gelatinase B are found in percussion TBI during functional recovery58,59. Upregulation in the expression of MMP genes has been reported in cerebral ischemia and intracerebral hemorrhage and stroke63,64. While MMPs are being increasingly implicated in the pathogenesis of several CNS diseases, MMPs also have been shown to facilitate recovery from CNS injury65. Readers can pursue the role of MMPs in different neurodegenerative disorders in the recent literature. As in the studies on cathepsins carried out in spinal cord injury, little has been reported on lysosomal proteinases in TBI. NFPs are known to be degraded by both calpain and cathepsins suggesting that the loss of these proteins in TBI may not be due to calpain alone. Other proteinases such as proteosome (multicatalytic proteinase complex) may also be involved.
CALPAIN HYPOTHESIS The calpain hypothesis of tissue destruction in CNS trauma was first developed as a result of studies conducted on spinal cord following injury. The findings of substantially greater neutral proteinase than acid proteinase activity in the lesion suggested a pivotal role for neutral proteinases (present in endogenous glial cells) in
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mediating tissue destruction in SCI before the infiltration of neutrophils/macrophages occurs41,42. The appearance of macrophages/inflammatory cells in the lesion occurs about 6 to 8 hours after injury, suggesting that cell death and tissue destruction occumng soon after injury is carried out by neutral proteinases while acid proteinases emanating from infiltrating lymphocytes contribute to this destructive process at later times. Taken together, the loss of cytoskeletal (NFPs) and myelin proteins (MBP), substantially greater neutral proteinase activity, increased calcium levels, granular degeneration of axon, vesiculation of myelin, and findings of similar changes in the CaC12-induced myelopathy model led investigators to implicate a role for calpain in tissue destruction associated with SCI33,39,42,44,66,67. This hypothesis derived from a host of studies was further supported by the demonstration that axonal (e.g., NFP, MAP2) and myelin proteins (MBP, MAG) were excellent calpain substrates. A direct role for calpain in tissue destruction was subsequently demonstrated by findings of increased calpain activity and translational expression by immunocytochemical technique in the lesion following trauma47,68-71. Our current studies further define a crucial role for calpain in cell injury/death not only in the lesion, but also in areas remote from the lesion epicenter. As in the SCI lesion, calpain activity and expression are progressively increased in the penumbra following trauma and the increase is greater caudal than rostral to the impact site72. Findings of increased intracellular calcium levels in regions of brain and spinal cord following trauma, axonal injury, and ischemia also implicated its role in calpain activation73-75. Subsequent studies demonstrated extensive loss of cytoskeletal proteins in TBI and ischemia3,6. These findings also implicated the involvement of a calpain-like protease in tissue damage related to brain trauma and ischemia5,76,77.
CHARACTERISTICS OF CALPAIN Calcium activated neutral proteinases (calpain), also known as cysteine endopeptidases (EC 3.4.22.17), are subclassified as ubiquitous and tissue specific. Ubiquitous calpain exists as microcalpain (µcalpain ) and millicalpain (mcalpain) isoforms requiring µM and mM calcium concentratiohs for activation, respectively. Both calpain isoforms consists of an 80kD catalytic and a 30kD regulatory subunit. At least 95% of calpain in the central nervous system (CNS) is present as the mcalpain isoform. µCalpain is largely associated with neurons while mcalpain is predominantly glial78,79. The mcalpain isoform in the CNS is present in cytosol as well as associated with myelin22,80,81 and the oligodendroglial cell body and processes82. Calpains are inactive in the cytosol and µcalpain is activated by autolysis of the 80kD catalytic subunit into the 76kD form and the 30kD regulatory subunit into the 19kD form in the presence of increased Ca2+ concentrations11,83. Both the µ and m calpain interact with membrane lipids (e.g., phospholipids, glycolipids) to increase the Ca2+ sensitivity for their activation84-89. Both µ and mcalpains are associated with the endogenous inhibitor, calpastatin, in the cytosol. Calpastatin is also degraded by activated calpain when the calpain:calpastatin ratio is increased. Calpain digests many proteins, including cytoskeletal (NFP), myelin (MBP, MAG), myofibrillar, enzymatic (phospholipase C, protein kinase C), histones, transcriptional factors (Fos, Jun), hormones, and others. In spite of its involvement in the degradation of many cellular proteins and processing of enzymes and hormones, the physiological function of calpain is unclear. Nevertheless, a large number of studies recently have implicated calpain as a primary mediator in the pathophysiology of many diseases. Its role is delineated in demyelinating diseases (e.g., MS) and degenerating diseases such as Parkinson’s disease, cerebral ischemia,
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Alzheimer's disease, and in rodent cataract formation 83,90-94. These findings also indicate an important role for calpain inhibitors as therapeutic agents for the treatment of a variety of diseases. To this end, the recent determination of the three dimensional structure of calpain will facilitate the development of specific inhibitors as agents for treatment95,96. In order to obtain a more detailed understanding of the biochemical and molecular pro erties of calpain, readers can consult several excellent reviews on related areas27,97,98.
ROLE OF CALCIUM IN SPINAL CORD INJURY One of the most important findings in SCI research was the demonstration of hydroxyapatite crystallites in both the axoplasm and mitochondria in the cord lesion as early as 15 to 30 minutes following injury. There was mitochondrial calcification and granular chan es in axonal filament in the SCI lesion at longer intervals following trauma8,38,99. The level of total calcium was determined in the lesion and found to be increased progressively following injury33,35,36. These findings suggested the involvement of increased calcium in tissue destruction in SCI. Subsequent studies provided strong support for a direct role for calcium in the mediation of tissue destruction in SCI with a CaC12-induced myelopathy model. This model, like SCI, showed granular degeneration of axons and vesiculation of the myelin sheath concomitant with the accumulation of calcium h droxyapatite crystallites66 and degradation of cytoskeletal and myelin proteins33,39,67. In contrast, other divalent or monovalent cations (e.g., Mg2+, K+, Na+, Cl-) did not induce any significant morphological or biochemical changes66. In both SCI and the CaC12-induced myelopathy models, the observed progressive granular degeneration of axon and vesiculation of myelin most likely resulted from the loss of structural proteins such as NFPs, MAPs, MBP, and PLP. Since these proteins are also calpain substrates, it is likely that increased calpain activation in SCI may be associated with higher levels of calcium in the lesion as well as in the penumbra to the lesion. Thus, elevated intracellular calcium levels have been implicated in cell death and axonal degeneration not only in SCI, but also in axonal injury in optic nerve and axotomyinduced axonal degeneration75,100-105. Morphological and biochemical changes similar to those in SCI were found in organotypic embryonic mouse spinal cord cultures as well as in spinal cord segments incubated with calcium106. The hypothesis that an increased intracellular calcium concentration causes cell death has been demonstrated in vivo in the CNS following injury and other systems, including ischemia57,107, muscular dystrophy108,109, toxic liver injury110, glutamate neurotoxicity, cataract formation111,112, and optic nerve degeneration113. Calcium-mediated cell injury/toxicity was partially inhibited by calcium channel blockers and calpain inhibitors114-117.
ROLE OF CALCIUM IN BRAIN INJURY Altered concentrations of ions in brain injury has been found to affect brain functions. Increased release of K+ may interfere with the membrane transport system, metabolism, and synaptic functions; disrupt energy homeostasis; deprive neurons of their oxygen supply; and lead to neuronal damage after TBI118-122. Decrease in the Mg2+ concentration following TBI not only may impair energy metabolism, glucose utilization and oxidative phosphorylation, but is also known to
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regulate transport and accumulation of Ca2+ in cells. Therefore, it is possible that alterations in Mg2+ concentrations in TBI may cause Ca2+-mediated neuronal loss in brain following injury 123. This is supported by reports of elevated intracellular Ca2+ levels in brain regions after experimental TBI and ischemia73,74,124-126 . In addition, the decrease in extracellular Ca2+ levels associated with profound functional disabilities following cortical compression/contusion injury in rats was not affected by pretreatment with glutamate receptor antagonists74,127. A recent study has suggested that excessive intracellular Ca2+ resulting from TBI in rats was adsorbed on mitochondrial membranes causing inhibition of the electron transport chain and energy metabolism128. It could therefore be concluded that in the pathophysiology of degenerative diseases, the influx and/or release of calcium from intracellular storage sites may cause cell death, involving many complex pathways, particularly in the secondary injury process following TBI and SCI. One of the pathways in which Ca2+ is involved is the activation of enzymes such as phospholipases and proteinases. An increased Ca2+ level also activates calpain, a Ca2+-dependent cysteine protease which mediates cytoskeletal protein degradation and neurodegeneration in human and experimental animal models of ischemia, SCI, and TBI3,5,,76,129-131. Diffuse axonal injury in white matter also has been implicated due to loss of cytoskeletal proteins and may be mediated by calpain132-134. In fact, the activation of calpain in the pathophysiology of CNS injury, ischemia, and other neurodegenerative diseases has now been firmly establised135-139.
CALPAIN ACTIVITY IN SPINAL CORD INJURY Since small samples of lesioned spinal cord are problematic, commonly used radioactive methods utilizing labeled substrates (e.g., casein) for assaying calpain activity are not reliable. These methods also require partial purification of the enzyme and removal of the endogenous inhibitor, calpastatin. In light of this problem, indirect methods for determination of calpain activity in limited amounts of tissue samples have been used39,47,70,76,77,140 . Several approaches have been taken to evaluate in vivo calpain activity in the lesion of spinal cord including (1) the degradation of endogenous proteins which are known calpain substrates; (2) the production of calpain-cleaved spectrin fragments; and (3) the formation or appearance of the active form (76kD) of µcalpain. Many endogenous proteins, including NFPs, MAP2, MBP, MAG, and spectrin, have now been identified as excellent substrates of calpain22,46,118,141-144. Some of these proteins are also degraded by other proteinases such as the cathepsins, matrix metalloproteinases, and caspase-343,145,146 whose cleavage sites and degradation products may be different as well as identical in some cases. For example, calpain and caspase-3 produce fragments of different sizes from spectrin. However, both in vivo and in vitro studies suggest a crucial role for calpain in neurofilament and spectrin degradation since the loss of 68kD NFP and spectrin is prevented (<90%) by the calpain specific inhibitors calpeptin, AK275, AK295, and others10,77,147,148. In recent years, the degradation of MAP2, NFPs, α -spectrin, and MBP in ischemia, CNS injury, multiple sclerosis (MS), Alzheimer's and Parkinson's disease has been exploited as an indirect measure of calpain activity in the neurodegenerative process5,47,70,72,76,93,149-152. Subsequently, the production of a calpain-cleaved 150kD D-spectrin fragment has been used as a direct measure of calpain activity in CNS injury, ischemia, cell death and neurodegenerative disorders by several laboratories 13,37,98,148,153.
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The extent of breakdown of cytoskeletal and myelin proteins (NFP, MBP, MAG), in the SCI lesion compared to control has been used as an indirect measure of calpain. One study on NFP revealed that as early as 30 minutes following injury, there was a 20% loss of both 68kD and 200kD NFPs while this loss was substantially greater (60%) at longer time (24 to 72 hours) after trauma70. The determination of the formation in the lesion of a 150kD calpain-cleaved fragment from the D subunit of fodrin, was shown to be greatly elevated (almost 130%) at 48 hours following injury and was taken as a measure of calpain activity37,71 (Figure 1). Similar progressive degradation of MAG and MAP2 was also observed in the SCI lesion
Figure 1. Calpain activity in SCI as measured by production of a 150 kD calpain-cleaved fragment of fodrin. Western blot (top) showing spinal cord samples at 4 and 48 hours after injury were quantified via densitometry and analyzed by one way ANOVA (+S.E.M.). Ray et al., 1999. With permission.
compared to sham operated controls154. The progressively decreased levels of 68kD NFP were also found in an experimental traumatic brain injury76. In the majority of neurodegenerative diseases, loss of NFP has been related to increased levels of calpain activity. However, it is important to point out that the NFPs are also substrates of cathepsins145,146. This finding suggests that these lysosomal proteinases have a crucial role in tissue destruction as well, particularly with the appearance of neutrophils and activated inflammatory cells at later (longer) times following injury. Two decades ago we correlated the progressive loss of cytoskeletal and myelin proteins in the lesioned cord at 30 minutes to 72 hours after injury with increased acid and neutral proteinase activity8,39. The activity of neutral proteinase was found to be greatly elevated in the lesion compared to acid proteinases and the neutral proteinase activity was increased early in injury. However, subsequent studies on the CaC12-induced myelopathy model revealed similar losses of NFP in the lesioned cord indicating that the neutral proteinase activity may be caused by a calpain-like enzyme39. There was no change in the neutral proteinase activity in the cord of other mono- or divalent cation (e.g., Mg2+, Na+)-induced myelopathy models. The demonstration of substantially increased calpain activity immediately after injury suggested that this was a major factor in the loss of myelin proteins, NFPs, and aspectrin37,39,47,70 . The finding of the calpain cleaved 150kD spectrin breakdown product in brain trauma, SCI, and Alzheimer’s disease also suggests a common but
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crucial role for increased calpain activity in these various conditions37,47,155. The participation of this important enzyme in tissue destruction was further confirmed by the observation of increased calpain expression (immunocytochemistry and Western blot analysis) and activity in the lesion soon after SCI37,68,71. This elevated calpain activity was not only confirmed in the lesion, but was also found to be upregulated in the penumbra (ie, areas adjacent to the lesion)72. Although the activity was highest in the lesion, it was greatly increased in the caudal area compared to the rostral segment. This finding suggests that at longer times following injury calpain is also involved in cell death and tissue destruction in the penumbra, particularly in the caudal area. Further, cellular protection can be obtained by preventing cytoskeletal protein degradation through early treatment of the spinal cord injured animals with calpain inhibitor(s)72.
CALPAIN EXPRESSION AND IDENTIFICATION OF CALPAIN-POSITIVE CELLS IN SPINAL CORD INJURY The finding of increased calpain activity in the SCI lesion and penumbra suggested that calpain expression may be elevated at both the translational and transcriptional levels in and around the lesion area. Western blot analysis revealed significantly increased calpain content in the lesion, approximately 22% at 30 minutes, 70% at 1 hour, and 90% by 4 hours following injury compared to sham. Although the calpain content then declined, the increased level is maintained over that of sham for up to 72 hours following injury70 (Figure 2). These results are further confirmed by subsequent independent studies47 as well as our own recent studies37,71. Since neutrophils and activated inflammatory cells appear later in SCI, the sudden and rapid increase in calpain translational expression at 30 minutes to 4 hours indicated this greater synthesis was primarily confined to endogenous cells, neurons and resident glial cells. Immunohistochemical studies revealed activated glial cells in the lesion at least 12 hours following injury. The relatively smaller increases in calpain content found in the lesion at longer time points may have been due to digestion of this enzyme by acid proteinases. Although increased calpain content in the lesion indicated elevated calpain synthesis in cells, it was not clear whether this increase was in endogenous cells or inflammatory cells. In order to identify which cells were responsible for increased calpain expression, immunocytochemical methods were used in the lesion of both impact and compression injury models. There was progressively increased calpain immunoperoxidase staining in the lesioned cords of both models compared to sham controls. Strongly stained neurons were swollen and necrotic. Using the astrocytic marker GFAP (glial fibrillary acidic protein) and microglial marker GSA (Griffonia Simplicifolia Agglutinin) antibodies to identify respective glial cells, the extent of increased calpain expression observed in astrocytes and microglia was shown to be dependent on time and severity of injury68,69.
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Figure 2. Increased calpain content following spinal cord injury. Using Western blot techniques, calpain bands (lesion and normal samples) were scanned and quantified via Quantity One software. Results are expressed as percent increase of calpain content compared to respective controls from 4-6 different experiments ± S.E.M. Banik et al., 1997. With permission.
Increased calpain immunoactivity was confirmed by double immunofluorescence labeling techniques using antibodies specific for calpain and GFAP (astrocytes) or calpain and OX-42 (microglia) in the lesion and penumbra (areas adjacent to the lesion). Spinal cord segments caudal and rostral to the lesion were investigated at different times after trauma. Like the immunoperoxidase staining, these studies also showed calpain immunostaining increased progressively in the lesion as well as in the adjacent areas and beyond37,71. Calpain immunoreactivity was increased in reactive astrocytes at 4 hours post injury and calpain immunofluorescence was evident in the astrocytic cell body and processes (Figure 3). Further increased immunostaining was observed at longer times following trauma in both caudal and rostral areas of SCI segments. Increased calpain expression was also present in activated microglia and macrophages which are known to secrete proteolytic enzymes capable of digesting various substrates in The calpain released from activated glial/inflammatory cells (as vitro156. demonstrated in vitro) may contribute to myelin/axonal protein degradation and lead ultimately to ultrastructural alterations or dissolution of the axon-myelin unit. Such correlation has been previously made in the lesioned cord following injury8,39.
CALPAIN ACTIVITY IN TBI The demonstration of increased calpain activity concomitant with alterations in the cytoskeletal and myelin proteins in SCI and Wallerian-type degeneration in optic nerve and spinal cord earlier prompted numerous investigators to examine whether similar changes may occur in TBI and/or any insult to brain due to ischemia or cerebrovascular injury. Such extensive degradation of cytoskeletal and other proteins was found in ischemia and indirectly implicated that this loss of proteins was due to calpain mediation. Degradation of these roteins, including MAP2 was observed in adult and neonatal ischemic conditions46, 157- 159. Loss of the NFP triplet proteins (extensively studied calpain substrate)149,160,161 and spectrin 11,91,150,162-166 in ischemia and cerebrovascular injury have also been attributed to calpain.
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Figure 3. Double immunofluorescent labeling of calpain and GFAP. Calpain positive astrocytes become apparent under a dual-pass optical filter. (a) Sham controls showed little calpain expression in astrocytes. (b) Spinal cords 4 hours after injury showed markedly increased calpain expression in astrocytes. (c) Astrocytic calpain expression remained upregulated 48 hours after injury. Ray et al.,72. With permission.
In recent years, an enormous number of investigations on calpain and related proteinases in experimental TBI and human TBI were initiated in a number of laboratories. Detailed and important studies on protein loss and related proteinase(s) associated with experimental TBI have emerged over the years3,6,137. As in SCI, ischemia and stroke, substantial degradation of cytoskeletal proteins was reported in TBI including observation of a significant degradation of dendritic MAP2 in tissue at 24 hours following TBI with loss amounting to several fold compared to control167169. Similar changes in the degradation of NFPs have also been reported in TBI76,77. In spite of the loss of NFPs, these investigators did not find any significant alterations in axons, at least at the morphological level, at 24 hours after trauma. Whether this is due to the very low grade of severity of injury or whether a longer time is needed to observe changes is not clear. Recent studies also indicated loss of NFPs in diffuse axonal injury in both gray and white matter following TBI131,170. Although degradation of neurofilament proteins in TBI and other CNS disorders has been attributed to calpain, the breakdown of these proteins are also known to be mediated by cathepsins145,146, as discussed earlier, indicating that loss of NFPs in SCI, TBI, and ischemia may not be entirely due to calpain. However, an elegant study by Posmantur et al.148 recently demonstrated that the possibly calpain-mediated 68kD NFP breakdown products of 53kD and 57kD in TBI are different from those mediated by cathepsins B and D148,171. For direct demonstration of calpain activity in CNS injury, investigators have recently used the extent of formation of calpain-specific spectrin degradation (SBDPs) products in tissue following trauma, ischemia, and other neurological disorders11,37,77,148,153. Accumulation of calpain-specific spectrin breakdown products (SBDPs) has been found not only at the contusion site in TBI, but also in adjacent areas, away from the lesion77,152. This finding further supported the immunohistochemical demonstration of calpain-specific SBDPs in dendrites following cerebral ischemia and TBI77,153. The implication of this direct role of increased calpain activity mediating axonal and dendritic degeneration in TBI was further confirmed by studies demonstrating prevention of cytoskeletal protein degradation by treatment of ischemic and/or TBI animals with calpain inhibitors107,155,169. Although there have been doubts expressed concerning the role of calpain in the mediation of axonal injury in the mild injury model172, significant changes in axons mediated by calpain in severe injury have been confirmed. The site
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manifesting little histological injury was found to lack calpain-mediated damage9,77. The indirect indications of calpain activation by trauma have now been confirmed by direct evidence for calpain activation using two parameters. One, by identification of the formation of active calpain and the other by determining calpain activity using casein zymography173. Activation of calpain by autolysis with the formation of an active form of calpain, a 76kD peptide produced from the inactive 80kD pcalpain isoform has been previously demonstrated in ischemia, Alzheimer's disease, MS, as well as in in vitro studies11,83,138,165,174. Similar activation of calpain with the production of the active form of µcalpain (76kD) has been found in TBI155. Further support for calpain activation was obtained from studies demonstrating increased activities of both µ and mcalpain at the injury site following TBI employing the casein zymography technique173. These investigators showed a progressive increase of calpain activity in TBI with time following injury, not only at the injury site, but also in the adjacent tissue. Even with the likelihood that other proteinases, including proteosome and cathepsins, participate in this process, substantial evidence now exists for a major role for calpain in tissue destruction following TBI. Although increased calpain activity concomitant with cytoskeletal protein degradation and its activation have been convincingly demonstrated in TBI, the overexpression of calpain in a particular cell type(s) whether endogenous or inflammatory at the site of injury or elsewhere has not been thoroughly studied. However, overactivation of calpain as shown by the immunohistochemical demonstration of increased spectrin breakdown products in dendrites in ischemia and TBI may indicate elevated calpain expression in neurons and their processes77,152,156. Since astrocytes and microglia are known to become reactive in TBI and there is infiltration of inflammatory cells, it is likely that calpain expression will also be increased in these cells following injury. In fact, overexpression of calpain in different cell types in ischemia and in the lesion as well as in the penumbra of spinal cord following injury has been recently demonstrated11,37,68. The degradation of cytoskeletal proteins and overactivation of calpain have been implicated in the degeneration of axons in TBI. Such changes may also lead to neuronal death following injury to brain and spinal cord. Thus, results from these various studies have suggested a role for calpain in ischemia, SCI, TBI and other neurodegenerative diseases in vivo and its role in cell death has been demonstrated in vitro11,47,77,91,117,148. CELL DEATH IN CNS TRAUMA Apoptosis or programmed cell death (PCD) is a natural phenomenon in the 177 developmental process of many tissues, including CNS175- . In CNS following injury and/or other diseases of various pathophysiology, cells may die of apoptosis and necrosis, and this death process is likely augmented or triggered by secondary injury factors. Occurrence of cell death by apoptotic and/or necrotic pathways has been demonstrated in a number of CNS disorders, including Alzheimer's disease178, amyotrophic lateral sclerosis179,180, Huntington's disease181,ischemia182,183, SCI184-186 and TBI187-191. Cysteine proteinases are among the main proteases involved in the mechanism of programmed cell death (PCD). Calpain and caspase-3 are two such proteases thought to participate in cell death in the injury site and penumbra of SCI and TBI. Such a role for calpain in the apoptosis of neurons and glial cells10,116,117 has been confirmed in vitro. Recently, calpain mediated PCD in thymocytes and lymphocytes has been demonstrated by preventing apoptosis through the use of the calpain inhibitors calpeptin, calpain inhibitor 1, and MDL28170192-194. Caspase-3
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when activated is known to degrade nuclear, cytosolic, and cytoskeletal proteins, including spectrin and can lead to cell death11,195,196. The participation of caspase-3 in neuronal apoptosis has recently been demonstrated11,148,191,197,198. Further studies are needed to define the role that each of these enzymes, calpain and caspase-3, plays in CNS injury.
Spinal Cord Injury Primary and/or secondary injury to the spinal cord has resulted in necrotic death of cells in the lesion as demonstrated by immunocytochemical studies, and it has also been thought that cells may die of delayed apoptotic death in the lesion and penumbra. Recently, cell death by both necrosis and delayed apoptosis in the SCI lesion and penumbra has been demonstrated72,184,185,199. Following SCI, damaged cells undergo either apoptosis or necrosis. With substantial severity of injury, cells in the lesion primarily die by necrosis, an irreversible process accompanied by an inflammatory reaction. In PCD there is no inflammatory reaction but a variety of other factors, including calcium, free radicals, cytokines, and proteases participate in the process. Increased calpain activity and expression in the lesioned cord suggest that calpain. may be one of the cysteine proteases responsible for necrotic and apoptotic cell death in the lesion and penumbra following SCI47,70. This hypothesis is supported by many in vitro studies employing various cell types, including lymphocytes, cardiomyocytes, neurons, and glial cells11,116,117,192,193. Subsequent studies have thoroughly investigated the mechanism of calpain-mediated apoptosis in SCI at the molecular level. Recent studies suggest that overactivation of calpain in SCI may be associated with altered expression of apoptosis related genes such as bc1-2, bc1-x, and bax. These studies also demonstrated intemucleosomal DNA fragmentation, a hallmark of apoptosis, in rat SCI lesions and penumbra72. Animals were sacrificed at 24 hours after injury and five spinal cord segments of equal length (centered on the lesion) were collected. Genomic DNA was isolated from these segments and resolved by agarose gel electrophoresis. No significant DNA damage was found in any of the five tissue segments in sham controls while segments from injured animals showed both internucleosomal and random fragmentation of DNA. The extent of DNA damage was less in the caudal and rostral segments at greater distances from the lesion site with somewhat more damage in the caudal segments than the rostral. The majority of cells in and immediately adjacent to the lesioned cord were found to be necrotic (random fragmentation) as well as apoptotic (intemucleosomal fragmentation)71,72. Cell death was also identified by TUNEL positive cells in these SCI segments. These changes correlate with an increased bax/bc1-2 ratio indicating predominance of the pro-apoptotic death gene. To elucidate the direct role that calpain may play in cell death in vivo, SCI animals were treated with intravenously administered calpain inhibitors (E64-d) for 24 hours after trauma. Spinal cord tissue segments were analyzed for different characteristics associated with cell death. These studies showed prevention of PCD, as determined by lack of DNA fragmentation, negative TUNEL assay, reversal of bc12/bax ratio, and reduction of calpain activity and expression in the lesion and penumbra in treated animals, compared to those untreated (vehicle)71,72 (Figure 4). Cell death and calpain activity were highest in the lesion and decreased at farther distances from the lesion. The extent of 68kD NFP loss was reduced in the lesion and penumbra of drug treated rats. These studies provided firm evidence of a role for calpain in apoptotic and necrotic cell death in SCI. A role for caspase-3 mediated cell death in SCI was also revealed185. These findings indicate that the use of calpain and caspase-3 specific
Pathophysiology of Central Nervous System Trauma
Figure 4. RT-PCR analysis for changes in the bax/bc1-2 ratio in spinal cord segments from different groups of rats. (a) RT-PCR products on the representative agarose gels showing mRNA expression of E-actin, bax and bc1-2 genes. (b) Densitometric quantification of changes in the bax/bc1-2 ratio. Results obtained from different groups of rats were compared by ANOVA with Fisher's PLSD post hoc tests. Significant differences between sham and vehicle plus SCI or vehicle plus SCI and E64-d are indicated by *, p<0.02; **, p<0.001. Ray et al., Brain Res. 867:80, 2000. With permission.
inhibitors as therapeutic agents may provide protection against cell death following SCI.
Traumatic Brain Injury The degradation of intracellular and membrane proteins at the injury site and penumbra following TBI may lead to loss of structural integrity and cell death by apoptosis and necrosis. Apoptotic cell death following TBI was demonstrated by determining different characteristics of apoptosis. Different laboratories have shown DNA laddering and nuclear chromatin condensation at the injury site following TBI188-190,200. These apoptotic cell death characteristics were also confirmed by demonstration of TUNEL positive cells at the same lesion site though the identity of the proteinase(s) responsible for this apoptotic mechanism was not determined. Subsequently, an elegant study by Yakovlev et al.191 correlated elevated caspase-3 activity with DNA laddering and TUNEL positive cells at the injury site and adjacent areas following TBI and suggested the involvement of caspase-3 in TBI neuronal death. This was further supported by demonstrating neuroprotection following treatment of TBI with caspase-3 inhibitors. Since then, a number of studies have provided evidence for a role of caspase-3 in apoptotic cell death in TBI198,201-203 .A role for calpain in apoptotic and necrotic cell death in TBI has been proposed in the past and timely reports on calpain's participation in this process have recently been These investigators exploited the degradation of spectrin and published11,204. determined the formation of calpain-cleaved spectrin breakdown products (145kD) and caspase-3 cleaved spectrin BDPs (120kD) in tissue following TBI. Their
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findings included accumulation of calpain-cleaved 145kD SBDP in cortical areas at 3 hours to 5 days following TBI while no significant amount of caspase-3 specific 120kD SBDP was found. In contrast, the 120kD SBDP was found to accumulate rapidly in the hippocampus while 145kD SBDP accumulation was delayed204. These investigators suggested that calpain is involved in necrotic cell death and caspase-3 mediates apoptosis in TBI. Although many other proteinases have been shown to participate in the cell death mechanism in vitro (including proteosome, matrix metalloproteinase, cathepsins)205,206, their role in cell death in CNS injury (SCI, TBI) has not been explored in detail. Since many proteases may be involved in tissue destruction in ischemia, TBI, and SCI as well as other neurological diseases, it is likely that these proteases may also be involved in the cell death mechanism in CNS injury.
CALPAIN REGULATION IN THE PATHOPHYSIOLOGY OF CNS DISORDERS A role for increased calpain activity and expression has been implicated in CNS injury, ischemia, Alzheimer's disease, and other neurodegenerative diseases by many investigators. In spite of its demonstrated pathophysiological role, the mechanism of activation and regulation of calpain in these conditions is poorly understood. One of the most critical regulators of calpain is calpastatin, an endogenous calpain-specific 207 inhibitor which exhibits no inhibitory effect on other proteinases . In normal physiological conditions, the calpastatin:calpain ratio is maintained for regulation of calpain activity. Calpastatin is also a suicide substrate of calpain when the regulatory ratio is changed208. Such a change in the calpain:calpastatin ratio has been demonstrated in muscular dystrophy models where calpastatin has been found to be de aded and the unregulated increased calpain activity is related to functional deficit209. Although not much is known about the role of calpastatin in CNS injury, changes in calpastatin expression and degradation in ischemia, SCI, TBI and other Such neurodegenerative diseases have been recently reported138,186,210,211. degradation of calpastatin will cause loss of its regulatory role and promote uncontrolled damage in cells and tissue following injury by the overactive calpain. In the pathophysiology of CNS injury and other diseases, intracellular calcium homeostasis is not properly maintained. Thus availability of excess calcium may activate calpain. The active calpain is further stimulated following interaction with lipids and cytokines released from inflammatory cells84-89,156. Recently calpain activation stimulated by free radicals has been found to cause thymocyte, C6 (glial) and PC12 (neuronal) apoptosis in vitro116,117,192. Thus, in CNS injury and other neurodegenerative diseases, calpain may be activated by free radicals generated by the arachidonic acid cascade and other pathways. Without calpastatin the overactivated calpain will remain unregulated in these pathological conditions37.
THERAPEUTIC APPROACHES WITH PROTEINASE INHIBITORS IN CNS TRAUMA Proteinase inhibitors are useful tools for studying the pathophysiology of the disease process. Since proteases have been implicated as one of the factors involved in secondary tissue destruction and cell death following CNS injury, it is logical to use protease inhibitors as therapeutic agents to provide neuroprotection. To this end, several studies have been reported employing calpain inhibitors in ischemia, SCI, and TBI 71,72,77,107,212 . The majority of the studies used calpain inhibitors (AK295,
Pathophysiology of Central Nervous System Trauma
E64-d, calpain I and II, calpeptin, leupeptin, MDL-28170) and examined prevention of protein degradation and attenuation of cell death. However, in order to obtain a more potent therapeutic effect in CNS injury, several important factors need to be considered. These include the window of opportunity for treatment following initial injury, time of drug delivery following injury, solubility of inhibitors (most of the calpain inhibitors are not very soluble), route of administration of drugs, and duration of treatment. Treatment of SCI Among the many agents which have been used to treat animals with SCI213, only a high dose administration of the glucocorticosteroid methylprednisolone has been found moderately beneficial to both animals and patients with spinal cord trauma 71,72,140,214-218 . However, because the degradation of axon and myelin proteins alters membrane structure and leads to cell death, the prevention of membrane protein loss is essential to the preservation of cells and for maintaining the axonmyelin structural unit. Because increased calpain activity is found in the lesion, calpain specific inhibitors were promising candidates as therapeutic agents in CNS trauma and disease219,220. Earlier studies with intravenous administration of calpeptin and/or E64-d for 24 hours to animals within 15 to 30 minutes of spinal cord injury showed reduced calpain activity and prevention of axon (NFP, MAP2) and myelin proteins (myelin associated glycoprotein, MAG) degradation221. The prevention of cytoskeletal protein degradation and attenuation of cell death was also shown following treatment of SCI animal with Riluzole47. These investigators also found reduced calpain activity in the lesion of treated animals compared to untreated. Since calpain activity may mediate irreversible cell death in the lesion and partial damage to cells in the penumbra, the possibility that the calpain inhibitor E64d may protect partially damaged cells in SCI was explored. The inhibitor was administered i.v. beginning 15 minutes after trauma and treatment continued for 24 hours. Five segments of the injured cord (near and distant rostral, lesion, near and distant caudal) were examined for calpain activity (68kD NFP degradation), transcriptional expression of µ and mcalpain and bax, bc12 genes, and occurrence of internucleosomal DNA fragmentation in the treated vs. untreated SCI animals72. The degradation of 68kD NFP, ratios of bax/bc12 and calpain/calpastatin, and DNA fragmentation were increased in the injured animals compared to control. All of these changes were maximally increased in the lesion followed by the adjacent areas of injured cord. In the penumbra, the increased calpain activity and expression was higher in the caudal areas than in the rostral. This finding was also correlated with the characteristics of cell death. These changes were largely undetectable in E64-d treated rats compared to controls. The results indicated that apoptotic cell death in rat SCI appears to be associated with increased calpain activity which is attenuated by the calpain inhibitor E64-d. Previous inhibitor studies have indicated the efficacy of calpain inhibitors199, AK295147, and riluzole47 for inhibition of calpain activity in SCI. Therapeutic use of calpain inhibitors may offer some advantages over ion channel blockers and glutamate antagonists, since calpain mediated proteolysis occurs at a later stage in apoptosis triggered by intracellular free Ca2+ levels and excitotoxicity. Thus, a wider time window provides greater opportunity for successful intervention by inhibition of increased calpain activity and apoptosis of neuronal cells. This delayed but effective antagonism of calpain ranging up to 3 hours after the injury with reduced neuronal death has been reported both in vivo107 and in vitro222. Apoptosis of both
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neuronal and glial cells could be responsible for neurological dysfunction following traumatic injury to the rat spinal cord223. Untreated SCI may cause onset of acute and chronic injury cascades leading to impairment of motor function because of myelin loss, axonal degeneration, and neural cell death. SCI and/or TBI are complex neuropathophysiological processes involving many neurochemical, cellular, and molecular cascades with unique timing. The interrelationships among diverse destructive mediators of the secondary injury enhance the neuronal damage and death with the progression of time. Therefore, it is important to make appropriate therapeutic intervention as early as possible. A therapeutic agent targeted to a single factor or pathway in the cascade of secondary injury may not provide enough neuroprotection if the therapy begins in the chronic phase of SCI. Since tissue destruction or cell death in trauma is multifactorial, a timely combination of rational therapeutic agents targeted to various pathways of secondary injury may be crucial for inhibition of progressive neurodegeneration in the chronic phase of SCI. Such therapeutic studies using a combination of calpain inhibitors and methylprednisolone, with its known efficacy in SCI, have been recently carried out in animals following SCI140,199. Methylprednisolone, as an inhibitor of free radical formation and antiinflammatory/immunosuppressive agent, may be ideal for this purpose since it also inhibits calpain activity 200,214,224. An added advantage of methylprednisolone is that it is beneficial for human patients with spinal cord injury215. Although calpainspecific inhibitors independently prevent 68kD NFP loss, our studies using these inhibitors (e.g., calpeptin) in combination with methylprednisolone, appear to minimize secondary tissue degeneration more than single treatment with either drug199,220. These studies concluded that calpain expression and activity were more reduced in the lesion with combination therapy than with either alone. The 68kD NFP degradation was also prevented more in the lesion. Apoptotic cell death was also reduced as determined by DNA fragmentation. A relevant similar study has shown that combination therapy usin riluzole and methylprednisolone together is more neuroprotective than either alone140.
Treatment of TBI The attenuation of neuronal damage by calpain inhibitors (AK295, E64-c, leupeptin) in ischemic brain injury has prompted examination of the efficacy of a variety of cell permeable calpain inhibitors in TBI101,107,150,162,163 . These studies provided evidence that inhibition of calpain activity prevented cytoskeletal protein degradation in vitro and ischemic injury in vivo. Detailed data on ischemic injury is found in the chapter by Emerich and Bartus. Recent studies on the effect of calpain inhibitors in TBI were focused on the prevention of cytoskeletal protein degradation, including NFP, D-spectrin, formation of the active form of calpain, and any improvement in cognitive function148,155,225. These studies successfully demonstrated downregulation of active calpain and prevention of cytoskeletal protein degradation. Treatment with the calpain inhibitor AK295 has been found not only to reduce infarct volume in brain ischemia107 but also to improve motor and cognitive function following TBI77. Subsequent studies with other calpain inhibitors (calpain II) also demonstrated reduction in the contusion volume as well as degradation of cytoskeletal proteins with preservation of axon and dendrite structure All of these studies, like those with SCI demonstrated the following TBI148. importance of targeting the calpain pathway for treatment of TBI with calpain inhibitors, and provided evidence that neuroprotection may be attained with proteinase inhibitors. Thus far, no significant studies have been carried out with a
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combination of drugs affecting other destructive pathways or with other protease inhibitors.
FUTURE STUDIES In order to understand the role of proteinases, particularly calpain, in CNS trauma, factors responsible for calpain activation in pathological conditions must be elucidated. Future studies should also investigate calpain activation via cytokines, free radicals, and quinolinic acid since the levels of these factors are increased in CNS injury. Since relatively little is known about the regulatory role of calpastatin, its status, localization, and activity must be determined not only in CNS injury, but also in the pathophysiology of other diseases. Further, the future goals should include the vigorous examination of combination therapies with calpain inhibitors and other agents and/or with other protease inhibitors (caspases, proteosome) which may be more effective in preserving cells and restoring motor and cognitive function following traumatic injury to the CNS.
Acknowledgements The work described in this review was supported over the years in part by grants from NIH-NINDS (NS-11066, NS-31622, NS-38146, and NS-41088), SCRF-1238 from the Paralyzed Veterans of America, RG-2 130B2 from the National Multiple Sclerosis Society, American Health Association Foundation and the MUSC Medical Student Training Program.
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LYSOSOMAL CYSTEINE PROTEASES AND THEIR PROTEIN INHIBITORS
Vito Turk1, Janko Kos1,2 Gregor Guncar1 and Boris Turk1 Jozef Stefan Institute Biochemistry and Molecular Biology Ljubljana SI-1000, Slovenia 2Krka, d.d. R&D Division Dept. of Biochemical Research and Drug Design Ljubljana SI-1000, Slovenia 1
INTRODUCTION Lysosomal cysteine proteinases, the cathepsins, have been viewed as mediators of terminal protein degradation. However, recent studies suggest more specific roles these enzymes play in processing and activating other proteins, in antigen presentation, bone resorption, apoptosis, etc. Alterations in their expression, processing and localization may lead to various pathological conditions. Alternatively, changes in synthesis and function of their endogenous inhibitors, resulting in uncontrolled proteolysis, may have similar consequences. This review focusses on the possible role of cathepsins and their inhibitors in some brain disfbnctions and neurological disorders, such as brain tumors, aging, neuronal death, Alzheimer disease, progressive myoclonus epilepsy (PME) and hereditary cystatin C amyloid angiopathy (HCCAA).
CYSTEINE PROTEASES The most abundant cysteine proteases are the papain-like enzymes (clan CA, family C l), which are widely distributed among living organisms1. The family comprises papain and related plant proteases, such as chymopapain, caricain, bromelain, actinidin, ficin, and aleurain, cruzipain and related parasite proteases, and the lysosomal Cathepsins. The sequences of 11 human cathepsins [B, H (I), L, S, C (J, dipeptidyl peptidase I), K (O2, O, X), O, F, V (L2), X (Y, Z, P) and W (lymphopain)] are currently known (Figure 1). They are relatively small proteins with Mr values in the range of 20 000-35 000 (see review2), with the exception of cathepsin C, which is an oligomeric enzyme with Mr ~200 0003. Most of the enzymes are widespread (cathepsins B, C, H, F, L, O, X), but some have more restricted localization (cathepsin K, osteoclasts and ovary; cathepsin S, spleen and antigen-presenting cells; cathepsin V, thymus and testis; cathepsin W, CD8+ Tlymphocytes), and have therefore been suggested to be involved in more specialized cellular processes. Cathepsin K has been shown to be essential for bone resorption and remodeling, cathepsin S to be responsible for MHC class II antigen presentation and
Role of Proteases in the Pathophysiology of Neurodegenerative Diseases, edited by Lajtha and Banik. Kluwer Academic/Plenum Publishers, New York, 2001.
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processing in most cell types and cathepsin C to be essential for processing a number of secretory serine proteases, including granzymes A and B. The precise physiological roles of other cathepsins, other than their role in protein degradation, are not well understood2,4.
Figure 1. Amino acid sequence alignment of the mature parts of all known human cathepsins. The sequences were taken from SWISS-PROT and GEN-BANK databases. The active site residues Cys and His are marked with the asterisk.
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STRUCTURE AND MECHANISM OF ACTION OF LYSOSOMAL CYSTEINE PROTEASES Lysosomal cathepsins are synthesized as preproenzymes, which undergo activation by limited proteolysis. The mature enzymes show strong sequence similarity (Fig. l), whereas the proregions show very little similarity and differ substantially in length. On the basis of sequence similarity, lysosomal cysteine peptidases have been divided into two families designated cathepsin L-like (L, V, K, S, W, F and H) and cathepsin B-like peptidases5,6. Cathepsins C and X are more distant, whereas cathepsin O is somewhat closer to the cathepsin L family. Within the cathepsin L family there are several subgroups: cathepsins L and V, cathepsins S and K and cathepsins F and W7. Cathepsin H, although being a member of the same family, is slightly more distant. Genes for cathepsins L and V are found on chromosome 9q21, for cathepsins S and K on chromosome 1q21 and for cathepsins F and W on chromosome 11q13. The cathepsin C gene maps at the same chromosome (11 q14). Four other cathepsins have different chromosomal localizations and map to 4q31-32 (cathepsin O), 15q24-25 (cathepsin H), 8p22-23 (cathepsin B) and 20q13 (cathepsin X)2. Papain-like proteases consist of two domains, referred to as L- (left) and R- (right), in accordance with the orientation used in the standard view (Fig. 2). The central helix, about 30 residues long, appears as the most prominent feature of the L-domain, whereas the fold of the R-domain is based on a beta-barrel motif. The two domains interact through a large planar interaction surface, reminiscent of a closed book with the spine in front. The two domain interface is open on the top, forming a 'V' shaped active site cleft. In the middle of the cleft the two active site residues, Cys25 and His159 (papain numbering is used throughout), are positioned with Cys25 at the N-terminus of the central helix from the L-domain, and His159 as part of the R-domain E barrel structure. In the active form of the enzyme both residues are charged, forming an ion pair8,9. The negatively charged Cys25 exhibits an unusually low pKa value in the range between 2.5 and 3.510. This negative charge, which appears to be stabilized by a strong positive electrostatic field originating presumably from the dipole of the central DBhelix, is responsible for the requirement of a reducing, slightly acidic environment for optimal activity of lysosomal cysteine proteases. A recent analysis of structural data has shown8 that the enzymes can accommodate relatively large substrates in an extended conformation in their active site clefts. However, only the substrate residues P2, P1 and P1' bind into well defined binding sites within the active site cleft. The S2 and S1' substrate binding sites are responsible for the diversity and selectivity of substrate and inhibitor binding. The primary specificity-determining site is the S2 subsite, where larger, hydrophobic residues (Phe) are preferred. An exception is cathepsin B, which can equally well accept positively charged residues in the P2 position8. Structure based sequence alignment has shown that the residues of the active site region (Cys25 and His1 59) and those interacting with the main chain of the bound substrate (Gln19, Gly64, Gly65, Trp177), together with Pro2 at the N-terminus and certain Cys residues, are completely conserved (Fig. 1). The core regions involved in the secondary structure elements are similar, whereas other regions and loops on the surface reveal little or no similarity. Most of the enzymes are endopeptidases, although cathepsin B also acts as a dipeptidyl carboxypeptidase and cathepsin H as an aminopeptidase11,2. The only true exopeptidases are cathepsin C, which acts only as dipeptidyl aminopeptidase , and cathepsin X, which acts as carboxy-monopeptidase12,13 or carboxy-dipeptidase13. Exopeptidases cathepsins H, B and X exhibit features that limit substrate access into their active sites (Fig. 2). Aminopeptidase cathepsin H utilizes the mini-chain, an eight residue long peptide originating from the propeptide region, which binds into the non-primed region of the active site cleft in a substrate-like orientation. With its negatively charged Cterminal carboxylic group it provides an anchor for the positively charged amino group of the P1 substrate residue. The C-terminal residue of the mini-chain binds into the place corresponding to the S2 binding site in related enzymes14. In the carboxydipeptidase cathepsin B, an 18 residue long insertion (Pro 107 - Asp 124), termed the occluding loop15 , blocks access into the active site in the primed binding regions and provides the two His residues, His119 and His111 (cathepsin B numbering) which bind the carboxyl group at the
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Figure 2. Ribbon diagrams of cathepsin L111 and cathepsin B15 structures. Standard view of two proteases shows mostly a-helical domain on the left side and E-barrel based right domain. Domain interface opens on the top into V-shaped active site cleft. Catalytic residue Cys is positioned on the top of the central α-helix and is shown in stick representation. Cathepsin L is an endopeptidase, whereas cathepsin B exhibits an exopeptidase activity that is defined by the occluding loop, which blocks a part of the active site cleft from the back.
C-terminus of the substrate. By analogy, the mini loop of another carboxypeptidase, cathepsin X which bears His23, serves the same function16.
BIOSYNTHESIS AND ACTIVATION Activation by limited proteolysis is a crucial step in controlling the proteolytic activity of cathepsins. Following synthesis as preproenzymes, the prepeptide is removed during passage to the endoplasmic reticulum, and procathepsins then undergo proteolytic processing to the active, mature enzyme in the acidic environment of late endosomes or lysosomes. The propeptide, which is partially or completely removed during activation, is responsible for proper targeting of the enzymes to the endosomal-lysosomal system via the mannose-6-phosphate receptor pathway and for the stability and proper folding of the proenzymes17. Propeptides are also able to specifically inhibit the activity of mature enzymes in vitro (see review18), although there is no evidence that they serve the same role in vivo after they have been proteolytically cleaved and removed from the body of their cognate enzyme. Proteolytic removal of the propeptide is autocatalytic for most of the enzymes, but can be also accomplished by the action of other peptidases, such as pepsin, cathepsin D and neutrophil elastase2. However, the true exopeptidases cathepsins X and C could only be activated by the action of another cysteine endopeptidase (cathepsin L, S)12,19. Based on the crystallographic studies of procathepsins B, L and K and on kinetic studies of procathepsin B processing, autocatalytic activation is an intermolecular process, although initial steps may be intramolecular20-24. The activation process is triggered by a pH drop that presumably substantially weakens the interactions between the propeptide and the catalytic part25. As a consequence, the proenzyme probably adopts a looser conformation, in which the propeptide is less tightly bound into the active site without the loss of secondary structure27,24. Such proenzyme molecules appear to exhibit a very small catalytic activity, which may be sufficient to initiate the chain reaction. Processing is then rapidly accomplished by the increasing number of catalytically active enzyme molecules28,24 . Propeptides, after serving their role to prevent inappropriate protease activity, dissociate from the protease, unfold and are proteolytically degraded24,29. Cathepsin C is an exception since a large portion of the proregion remains bound to the catalytic part also in the mature enzyme3.
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Glycosaminoglycans, which are found in lysosomes as a result of proteoglycan recycling, were suggested to be involved in in vivo processing of cysteine proteinases30 . However, in vivo processing appears to be more complicated and to involve some additional peptidases since, in a number of studies with recombinant enzymes, the processed forms had N-terminal extensions of several residues. A likely candidate for the trimming enzyme is cathepsin C, which has already been shown to remove the N-terminal extension of cathepsin B and does not cleave substrates with Pro in the P1 position2. Pro is a conserved residue in the +2 position of many lysosomal cysteine peptidases (Fig. 1).
REGULATION OF ACTIVITY There are several ways in which the enzyme activity of lysosomal cysteine proteases can be regulated (see review31), the most important for mature enzymes being inhibition by their endogenous protein inhibitors, the cystatins32,11 and thyropins (thyroglobulin type-1 domain inhibitors)33, and the general protease inhibitor D2macroglobulin34. In addition, lysosomal cysteine proteases are inhibited by the serpin squamous cell carninoma antigen 135 and by the cytotoxic T-lymphocyte antigen-2E which is homologous to the cathepsin L propeptide36.
The Cystatins On the basis of sequence similarity,the cystatins have been subdivided into three families, stefins, cystatins and kininogens37. Three residues, Glyl1, Gln55 and Gly59 (cystatin C numbering will be used throughout, if not otherwise specified), are strictly conserved in all the inhibitory cystatins and have been shown to have an important role in the inhibition32,11. The stefins are single-chain proteins of ~100 amino acid residues and lack carbohydrate and disulfide bonds. They are primarily intracellular proteins found in various cells and tissues in animals. Stefins have also been detected in extracellular fluids38. Originally, two types of stefins, A and B, were found in mammals, including rat, mouse, bovine and human11. Later two other stefins have been found, bovine stefin C39 and porcine stefin D40, suggesting that more family members exist. Members of the cystatin family are slightly larger than the stefins and contain ~115 amino acid residues. They are usually non-glycosylated, single chain proteins, having two intramolecular disulfide bridges and are found in many biological fluids at relatively high concentration32. The family consists of species variants of cystatin C, cystatin S variants, cystatin D41, E (M)42,43 and cystatin F38. Cystatins are widely distributed in nature and are also found in lower organisms44,11. Kininogens are large multifunctional glycoproteins circulating in mammalian blood plasma, and were first known as parent molecules for vasoactive peptides, the kinins. Three different types of kininogens have been identified, high molecular weight kininogen (HK), low molecular weight kininogen (LK) and T-kininogen (TK), the latter variant being found only in rats45,46. Both human HK and LK are products of the same gene, resulting from alternative mRNA splicing47. In addition to lysosomal cathepsins, kininogens are the only cystatins inhibiting calpain as well48. Furthermore, both HK and LK have been shown to simultaneously bind two molecules of target proteases with different affinities49,20.
Mechanism of Interaction of Cystatins and Cysteine Proteinases The cystatins are reversible, tight-binding competitive inhibitors of cysteine proteinases50. The interaction with their target proteases can be extremely tight, a Ki value of ~10 fM having been determined for the interaction between cystatin C and papain51. An important step in the elucidation of the molecular mechanism by which cystatins inhibit their target proteinases was the determination of the crystal structure of chicken cystatin52. The molecule consists of a five stranded antiparallel E-sheet, wrapped around a five turn Dhelix. On the basis of the chicken cystatin structure, it was suggested that there are three
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regions which provide contacts in the interaction with roteinases: the N-terminal segment and two hairpin loops that connect the strands of the E-sheet. This was later confirmed by the crystal structure of the complex between human stefin B and papain53. The interactions between stefin and papain are primarily non-specific and do not involve specific amino residues. The fold of stefin B in the complex is essentially the same as that of chicken cystatin (Fig. 3)53. Similar interactions have also been shown for stefin A by NMR54.
Figure 3. Stefin B structure53. Ribbon representation of the stefin B illustrates overall structure of the inhibitor. The five stranded E-sheet is wrapped around the D-helix. Interconnecting loops and both termini are important for stefin B inhibitory activity.
The mechanism of inhibition of the target proteinases has also been studied by other approaches. Glyl1 (Gly4 in stefins) is very important for inhibition. Any mutation of Gly11 or deletion of the N- terminal residues preceding Gly11 results in a substantial affinity drop55,56,11). However, residues immediately C-terminal of Gly11 have little influence on the interaction57. Similarly, mutations in the central Gln55-Gly59 (QVVAG) region and in the second hairpin loop (Trp106) largely reduce or even abolish the affinity for proteases (see review11). Although the interaction between cystatins and their target proteases is primarily nonspecific, cystatins are capable of discriminating between endo- and exo-peptidases. This is a consequence of the differences in the structures of the interacting regions of the enzymes. Endopeptidases (papain, cathepsins S and L) are tightly and rapidly inhibited by animal cystatins with Ki values in the pM to fM range, whereas exopeptidases (cathepsins B, H, C and X) are inhibited with substantially lower affinity in the nM range13,11. The active site cleft of endopeptidases is free to accommodate the inhibitors, whereas in the case of exopeptidases the active site cleft contains additional enzyme residues. In cathepsins B and X the occluding loop and the mini loop, respectively, partly occupy the active site cleft and need to be displaced in order to accept a cystatin molecule58,15,13. In cathepsin H sterical hindrance is provided by the mini chain14 .
PHYSIOLOGICAL ROLE OF LYSOSOMAL CYSTEINE PROTEASES With lysosomal concentrations as high as 1 mM, cathepsins L and B are, together with the aspartic peptidase cathepsin D, the most abundant lysosomal peptidases59. However, cathepsins B and D have been shown not to be very important for lysosomal
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protein degradation, indicating that cysteine peptidases other than cathepsin B do most of the work prior to their ubiquitin-mediated degradation or their denaturation and subsequent degradation by cathepsin D or some other peptidase (see review2). Lysosomal cysteine peptidases have been found to play important roles in processing and activating other proteins, such as thyroglobulin60, renin and a number of serine granule proteases8. Gene knock-out experiments have confirmed that cathepsin C is essential for the activation of granule serine peptidases from cytotoxic T-lymphocytes and natural killer cells (granzymes A and B)61. A number of other important cellular processes involve the action of lysosomal cysteine peptidases, including MHC class II antigen presentation with a major role of cathepsin S and, to some extent, cathepsin L, and bone resorption with the central role of cathepsin K (see review4). Any alterations in the normal balance of enzyme activity may lead to pathological conditions, and this has also been observed with lysosomal cysteine proteinases62 . Under normal conditions, small amounts of catalytically active proteases, released from lysosomes of infected or dying cells, are effectively blocked by their endogenous inhibitors. However, after massive lysosome leakage or failure in trafficking, the activity of the enzymes may escape inhibitor control. Alternatively, any changes from normal synthesis and function of endogenous inhibitors may have similar consequences. Uncontrolled proteolysis is thus often a result of an imbalance between catalytically active proteinases and their natural inhibitors. Lysosomal cysteine proteases play a role in a number of diseases such as cancer, rheumatoid arthritis and osteoarthritis, Alzheimer disease, multiple sclerosis, muscular dystrophy, pancreatitis, liver disorders, lung disorders, lysosomal disorders, inflammation, Batten's disease, diabetes, pycnodysostosis, periodontitis, myocardial disorders and many others. In many of these diseases lysosomal enzymes have been found in extracellular and extralysosomal environments in their proforms, which are substantially more stable than the mature enzymes and not susceptible to inhibition by endogenous protein inhibitors (see reviews63,2,64).
LYSOSOMAL CYSTEINE PROTEASES AND CYSTATINS DISFUNCTION AND NEUROLOGICAL DISORDERS
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BRAIN
Alzheimer's disease and aging There is a long line of evidence that lysosomal cysteine proteases are connected with various neurological disorders, although no precise role for them has been established63. Alzheimer's disease was one of the diseases where lysosomal cysteine proteases, in particular cathepsin B, are most often mentioned65-68. Cathepsin X, which has catalytic properties similar to cathepsin B, was even patented as one of the possible secretases69 . Although the recent discovery of a new aspartic protease BACE (beta amyloid converting enzyme) and its identification as E-secretase70 discounts the possibility that a lysosomal cysteine protease could serve this role there is still sufficient evidence that cathepsin activity is increased in Alzheimer brain71,72. Similarly, cathepsin B activity is largely increased in brain of patients with multiple sclerosis, although the reason is unknown73. There is also considerable evidence connecting lysosomal cysteine proteases with aging. Lysosomes in aged rat brain were found to be considerably less stable than from normal brain, with significant leakage of lysosomal enzymes being observed, either as mature enzymes or as inactive zymogens74,75 . The activity of cathepsin L was found to be greatly decreased in aged brain (~90%)76, whereas that of cathepsin B was not significantly affected77. Signs of aging were also observed when cultured hippocampal slices were treated with cysteine protease inhibitors resulting in an increased number of lysosomes with altered morphology. Lysosomes were translocated into the dendritic trees of pyramidal neurons where they fused, forming meganeurite-like structures78,79. In addition, lysosomal dysfunctions resulted in disrupted transport of hypothalamic releasing factors80 and reduced expression of brain-derived neurotrophic factor expression81. The latter is also characteristic of Alzheimer disease. One of the possible explanations for these changes is an increase in the pH of lysosomes, achieved experimentally by incubation of hippocampal
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slices with chloroquinone. This resemble the pattern observed in aging brain: greatly reduced cathepsin L activity and little effect on cathepsin B activity, followed by cathepsin D activation and subsequent tau protein degradation82. Lysosomal cathepsins are also involved in neuronal death either as initiators or direct agents65. Several studies have demonstrated an alteration in the distribution of cathepsins in CA 1 neurons in hippocampus after ischemic damage83. Intense cytoplasm labeling of cathepsin B was detected when neurons had become morphologically altered with obvious shrinkage of the cytoplasmic region84. When a specific inhibitor of cathepsin B CA-074 or CP inhibitor E-64c were administered immediately after the ischemic insult in monkeys, a significant proportion of CA1 neurons were saved from delayed neuronal death and the surviving neurons were associated with the decreased immunoreactivity for cathepsin B and cathepsin L83,85. These results indicate that CP inhibitors may provide a novel strategy for preventing ischemic delayed neuronal death. It is obvious that delayed death of CA1 neurons is an apoptotic and not necrotic process86,84,83. Additional evidence, although indirect, was provided by stefin B knockout experiments. Deficiency of stefin B, a lysosomal cysteine protease, but not caspase, inhibitor, resulted in cerebellar apoptosis in stefin B-null mice87.
Brain tumors The key biological feature of brain tumors is local invasion, due to early spread of tumor cells from the primary site into the surrounding brain88 . This process involves cell attachment to the extracellular matrix, motility and degradation of extracellular matrix proteins by proteolytic enzymes. The latter include endopeptidases of four classes (serine, metallo, aspartic and cysteine) acting either alone or in linked, proteolytic cascade reactions89. Cathepsin B was the first cysteine proteinase found to be related to malignant progression of human gliomas90,91. Its increased protein and transcript levels have been found in high grade glioblastoma and anaplastic astrocytoma compared with normal brain tissue and low grade gliomas92,93. As shown later, cathepsins L and H are also involved in the process of glioma progression94,95. Increased levels of cathepsin B have been found also in atypical and anaplastic meningiomas. Additionally, it has been shown that the level of cathepsin B in human glioma is significantly associated with Poor clinical symptoms and may serve as a prognostic factor for patients with brain tumors96,97. Cathepsin B is also expressed in endothelial cells, indicating its involvement in tumor-associated angiogenesis97. Stefins A and B appear to be down-regulated in tumors of neuroepiphelial tissues, suggesting that they are not able to balance the increased tumor-associated activity of cysteine proteinases97. Stefin B and EPM 1 Progressive myoclonus epilepsies (PME) are a group of inherited diseases characterized by myoclonic seizures, generalized epilepsy and progressive neurological deterioration, particularly dementia and ataxia. One of the five major types of PME is Unverricht-Lundborg disease (EPM l), a rare autosomal recessive disease with onset at the age of 6-1 5 years, which is more common in Finland and the Mediterranean. Patients have generalized, clonic or clonic-tonic seizures and marked progressive and incapacitating myoclonus. Seizures tend to diminish at age 25-30, but the patients generally develop ataxia and mild dementia late in the course of the disease98. Genetic analyses revealed that mutations in the gene for stefin B on chromosome 21q22.3 are res onsible for the disease99-101. Several mutations have been detected in the gene 99,101,102, one of them resulting in Gly4Arg substitution, which is characterized by a drastic drop in affinity for target proteases55. However, only in the minority of EPM 1 alleles mutations were found, while the majority contained large expansions of a dodecamer sequence (CCC CGC CCC GCG), located upstream of the 5' transcription start site of the stefin B gene100. The size of the expansion is 30-75 repeats, whereas only 2-3 repeats can be found in normal alleles103. As a result, stefin B mRNA is markedly reduced in the blood of patients but not in their cell lines. Replacement of the repeat expansion in the normal promoter with heterologous
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DNA fragments of similar size (700-1000 bp) showed similar reduced activity, suggesting that altered spacing of promoter elements due to the repeat expansion is among the causes of reduction of stefin B expression in EPM 1. In order to confirm the importance of stefin B for the development of the disease, stefin B-deficient mice have been generated87. Initially, the phenotype of young mutant mice did not differ from those of the wild-type mice, however, later during development (>6 months), stefin B-deficient mice developed ataxia and myoclonic seizures, similar to symptoms seen in the human disease. In addition, a large number of cerebellar granule cells are lost in the mutant mice by apoptosis, indicating that stefin B has a role in preventing cerebellar apoptosis by an as yet unknown mechanism.
Hereditary Cystatin C Amyloid Angiopathy (HCCAA) HCCAA is an autosomal dominant disorder characterized by deposition of amyloid primarily in the central nervous system, although amyloid deposits have also been detected in other organs. Clinically, this deposition results in brain haemorrhages in young individuals (25-35 years old), which are the major cause of death within couple of years from the first stroke104,105. The main component of the amyloid deposits was identified by protein sequencing as a variant of cystatin C. The amyloid cystatin C differs from normal cystatin C by an amino acid substitution of Leu68Gln resultin from a single T A mutation in the codon for Leu68 in exon 2 of the cystatin C gene106,107. As a consequence of this mutation the Alu I restriction site found in the normal gene is lost, resulting in an extra ~630 base pair Alu I fragment, which also enables diagnosis of the disease. Furthermore, the concentration of cystatin C in the cerebrospinal fluid of patients with HCCAA is ~3-fold decreased compared to healthy individuals108, suggesting that this could be another causative event. In vitro studies indicated that the Leu68Gln cystatin C variant is substantially less stable than the wild type protein and more prone to dimerization38,109. Moreover, increased accumulation was observed in NIH/3T3 cells expressing Leu68Gln cystatin C compared with same cells expressing wild-type cystatin C. The intracellularly accumulating mutant cystatin C was located mainly in the endoplasmic reticulum as insoluble protein suggesting that this is an important event in the molecular pathophysiology of HCCAA110.
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PROTEASES AND THEIR INHIBITORS IN GLIOMAS
Peter A. Forsyth1, Dylan R. Edwards2, Marc A. LaFleur2 and V. W. Yong1 Oncology & Clinical Neurosciences, University of Calgary and Department of Medicine, Tom Baker Cancer Centre Calgary, Alberta T2N 1N4, Canada 2 School of Biological Sciences, University of East Anglia Norwich, Norfolk NR4 7TJ, England 1
INTRODUCTION Extracellular proteases encompass several large families of molecules with a broad range of functions. These enzymes have been studied largely in the traditional context of their ability to degrade or cleave certain specific substrates, but it is becoming clear that they have important functions in diverse cellular activities such as angiogenesis, apoptosis and cell signaling that are not completely understood. Understanding the multifunctional nature of these molecules and their ability to affect a number of cellular control systems is very important for several reasons. First, knowledge of protease action will contribute to the unraveling of pathobiological mechanisms underlying a number of diseases. Second, these are important therapeutic targets and several clinical trials of protease inhibitors are already in progress. Third, since proteases are involved in a number of cellular processes, some beneficial and some deleterious, they must be clearly understood so that their manipulation is as selective as possible and the full therapeutic benefit maximized. In this chapter we outline the role of proteases and their inhibitors in gliomas. We highlight matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) along with the serine proteases and cathepsins since these are the most extensively studied proteases in gliomas. In addition, their potential as novel glioma treatments is the most advanced with MMPs; two clinical trials of synthetic MMP inhibitors in glioma patients are currently underway. Little is known about the large number of other proteases that have not been studied in gliomas.
GLIOMAS & THE BRAIN'S EXTRACELLULAR MATRIX Primary brain tumors are a heterogeneous group of tumors which arise from the tissues of the brain (glia, neurons, ependyma, choroid plexus etc.) or its coverings (the meninges). The two most common primary brain tumors are meningiomas and gliomas. The majority of meningiomas are cured by surgery and further treatment is generally not needed. In contrast, malignant gliomas are uniformly fatal in spite of aggressive surgical resection, radiotherapy and chemotherapy. Since gliomas are the major clinical challenge in neurooncology they are the focus of this review. Gliomas affect 25,000 Americans and Canadians a year and malignant gliomas (MGs) are the most common type. These are fatal tumors and the median survival of these MG patients is only about 12 months; only 2% of patients with the most malignant type Role of Proteases in the Pathophysiology of Neurodegenerative Diseases, edited by Lajtha and Banik. Kluwer Academic/Plenurn Publishers, New York, 2001.
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survive > 3 years1-3. Gliomas are important for three reasons: 1) they are the most common solid tumors in children, 2) they are becoming more common, and 3) their prognosis has not changed significantly in the past 20 years. Clearly, better treatments are desperately needed and inhibition of proteases may be an important novel glioma treatment when used in conjunction with conventional treatments. Proteases have been implicated in the two cardinal features of malignant gliomas: their marked invasiveness and vascularity. Gliomas extend tendrils of tumor cells into the surrounding brain. Isolated glioma cells migrate and invade several centimeters beyond the main tumor mass and render these surgically incurable. Similarly the most malignant of these tumors, glioblastoma multiforme (GBM) are among the most vascular tumors known. Both their invasiveness and vascularity remain major barriers to their effective treatment and are not targeted by our available treatments. Glioma invasion occurs predominantly along myelinated white matter tracts and blood vessels4. It occurs, with little or no destruction of the surrounding neuronal structures, at least when only a few cells are involved. As a consequence of its predilection for white matter tracts distant spread occurs along the optic radiations, corpus collosum or anterior commissure. Generally invasion is conceived of as a three step process: 1) receptor mediated adhesion of tumor cells to matrix proteins in the ECM, 2) degradation of the ECM by proteases creating a space and environment for the glioma cells to move into, and 3) active movement that requires receptor turnover, membrane synthesis and rearrangement of cytoskeletal elements. The second process is the focus of this review and can not be appreciated without some understanding of the brain's ECM4. There is very little ECM in the brain in contrast to other organs. The brain's ECM is mostly composed of proteoglycans and glycoproteins. The ECM's major components are collagen (particularly type IV), chondroitin sulphate, laminin, elastin, fibronectin, vitronectin, entactin, tenascin, heparan sulfate proteoglycan and hyaluronic acid. Classical ECM components (laminin, collagen type IV, fibronectin and vitronectin) are limited to vascular basement membranes (which glioma cells invade along but do not usually invade through and the glia limitans externa; the latter is another true basement membrane that covers the cortical surface. All of these aforementioned ECM components are considered as ligands that participate in glioma adhesion, ligand-receptor and signal transduction-messenger interactions that allow tumor invasion to occur. Many of these ECM components are also synthesized and deposited by glioma cells themselves creating a microenvironment that presumably facilitates their invasiveness, survival or proliferation. ECM macromolecules thought to be secreted by gliomas include tenascin, vitronectin, collagen types I, III, IV and VI, fibronectin, laminin, hyaluronan, chondroitin sulfate and heparin sulfate proteoglycans. Several families of cell surface receptors (e.g. integrins) have been identified that interact with specific domains of ECM proteins. These interactions trigger diverse cellular events such as cell attachment, adhesion, changes in cell morphology and activation of second messenger pathways. Several proteases have been implicated in the pathophysiology of gliomas, including the matrix metalloproteinases (MMPs), serine proteases (urokinase and tissue plasminogen activators; uPA and tPA), cysteine proteases (cathepsin B and S) and aspartic proteases (cathepsin D). In addition glycosidases are also important factors, but they will not be considered in detail here.
MATRIX METALLOPROTEINASES (MMPs) AND TISSUE INHIBITORS OF MMPs (TIMPs) Matrix Metalloproteinases (MMPs) The ability to breach tissue boundaries by active destruction of extracellular matrix (ECM) is the common denominator of tumor invasion, angiogenesis and metastasis. Tumor cells use a variety of degradative enzymes to destroy basement membranes and interstitial stroma5-8 endothelial cells may use the same machinery to form new blood vessels. The MMPs are the principal secreted proteinases required for ECM degradation in a variety of physiological and pathological tissue remodelling processes6-10. These are a family of zinc binding, calcium dependent endopeptidases. Twenty-two have been described11,12 (TABLE 1) and are subdivided principally by structure or by substrate preference into
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collagenases, gelatinases, stromelysins, and the membrane-type MMPs (MT-MMPs). There is a wealth of evidence for an association between deregulated production/activation of MMPs and aggressive behaviour in human cancers13-16; particularly for MMP-2 (gelatinaseA; 72 kDa) and MMP-9 (gelatinase-B; 92 kDa)5,6,17. A schema for the relationship between MMPs, tumor cells, the surrounding stroma, other proteases and the ECM is shown in FIGURE 1. Table 1: The family of vertebrate matrix metalloproteinases (MMPs) Group
Members
MMP numbers*
Collagenases
Interstitial collagenase (fibroblast-type)
MMP-I
Fibrillar collagens
Neutrophil collagenase Collagenase-3 Collagenase-4§
MMP-8 MMP-1 3 MMP-I 8
Fibrillar collagens Fibrillar collagens Unknown
Stromelysin-1
MMP-3
Stromelysin-2
MMP-IO
Laminin, non-fibrillar collagens, fibronectin Laminin, non-fibrillar collagens, fibronectin
Stromelysins
Main substrate**
Gelatinases
Gelatinase B MMP-2 (72 kDa type IV collagenase) MMP-9 Gelatinase B (92 kDa type IV collagenase)
Gelatin, Types IV and V collagens, fibronectin Gelatin, Types IV and V collagens, fibronectin
Membrane-type MMPs
MTl-MMP
MMP- 14
MT2-MMP
MMP- 15
MT3-MMP
MMP- 16
MT4-MMP MT5-MMP MT6-MMP
MMP- 17 MMP-24 MMP-25
Pro-MMP-2, collagens, gelatin Pro-MMP-2, collagens, gelatin Pro-MMP-2, collagens, gelatin Fibrinogen, pro-TNFa Pro-MMP-2 Pro-MMP-2
Matrilysin
MMP-7
Stromelysin-3
MMP-11
Metalloelastase No trivial name
MMP-12 MMP-1 9
Enamelysin Xenopus MMP CMMP Femalysin
MMP-20 MMP-2 1 MMP-21/22 MMP-23
Others
Laminin, non-fibrillar collagens, fibronectin D 1 proteinase inhibitor (serpin) Elastin Laminin, non-fibrillar collagens, fibronectin Not known Not known Not known
*MMP-4, -5 and -6 were found to be identical to other MMP family members and these designations are no longer in use. ** Although the principal substrates are listed, there is a great deal of substrate overlap. MMP = matrix metalloproteinase; MT-MMP = membrane-type matrix metalloproteinase; ? = numerical designation not yet assigned/clear. § Xenopus gene only at present.
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FIGURE 1. Representation of the relationship between tumor cells, the extracellular environment, various proteases and signalling molecules. The MMP/TIMP and uPA/uPAR systems are highlighted. The relationships are complex and the regulation of these proteases is interdependent. For example proteolysis of plasminogen (Pl’ogen) to plasmin by uPA when bound to uPAR on the cell surface also activates several MMPs which in turn contribute to proteolysis of the ECM. Furthermore, a number of membrane associated proteins, such as the MT-MMPs and uPAR activate proteases and serve to localize proteolysis to the invading edge of the tumor cell. The interaction of integrins, ECM molecules (e.g. vitronectin) with both MMPs and serine proteases is complex and further serves to localise regulation of proteolysis to the cell surface. Finally, once activated proteases influence tumor growth by liberating growth factors which in turn produce mitogenic signals through their receptors. Note that this figure applies to systemic cancers where, somewhat surprisingly, the surrounding stroma make most of the proteases. In gliomas, the tumor cells themselves may be the source of most of the MMPs. For example, IGFs are bound to IGFBP and not “free” to interact with their receptors but are liberated from IGFBP by MMPs. Abbreviations used: IGF = insulin-like growth factor; IGFBP = IGF binding protein; IGF1-R = IGF 1 receptor; M6P-R = nannose 6-phosphate receptor, also called IGF2-receptor; MMP = matrix metalloproteinase; MTMMP = membrane-type MMP; PAI-1 = plasminogen activator inhibitor type 1; Pl’ogen = plasminogen; TCF = T cell factor (also known as LeF), TIMP = tissue inhibitor of MMP; uPA = urokinase plasminogen activator; uPAR = uPA receptor.
All MMPs contain a signal peptide, a propeptide region, an n-terminal catalytic domain and a C-terminal hemopexin-like domain (with the exception of MMP-7 which lacks the C-terminus region). Intervening sequences further characterise certain MMPs such as fibronectin repeats in the gelatinases and the transmembrane region in the MT-MMPs. The propeptide is important for the control of MMP latency and activation. In the inactive zymogen, a cysteine residue within the Pro-Arg-Cys-Gly-X-Pro motif, the propeptide coordinates with the zinc ion in the active site disabling its proteolytic activity18. Activation of the MMPs requires proteolysis of the propeptide which exposes the active site. The catalytic centre consists of 3 conserved His residues and co-ordinate Zn2+ and is responsible for substrate and autolytic cleavage19. The C-terminal domain is similar in sequence to members of the hemopexin family. This domain appears to mediate substrate binding although both
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N-terminal and C-terminal domains are required for proper binding and cleavage. The Nterminal and C-terminal domains are linked by a flexible linker peptide20. Both MMP-2 and -9 have an additional fibronectin-like domain within the catalytic domain, consisting of 3 tandem repeats of a fibronectin type II-like module. This domain binds to denatured collagen type IV and V, elastin, and denatured and native type I collagen21,22. MMP-9 also has a collagen domain with sequence homology to the D2 chain of type IV collagen which is also thought to be involved in substrate binding23. The activities are tightly controlled at six levels: 1) gene transcription, 2) zymogen activation by proteolysis, 3) inhibition of active forms by the TIMPs, 4) mRNA stability, 5) translational control, and, 6) storage in secretory granules (as for MMP-8)6,23-26. The latter three of these are the least characterised mechanisms of MMP control. The MMPs are functionally related but differ in their expression and association with TIMPs. For example MMP-2 is widely expressed constitutively27, whereas MMP-9 has restricted expression and is inducible27-31. Gene transcription occurs in response to a variety of factors such as cytokines, angiogenic factors and hormones. MMP genes targeted for induction contain unique DNA sequences in their promoter regions which bind specific transcription factors and increase transcription. Several MMP genes (such as MMP-1, -3, -7, -9,-11,-13) contain an AP-1 (phorbol ester responsive element) site that can be induced by the fos/jun transcription factors in response to stimuli such as EGF. In contrast, the promoter region of MMP-2 lacks AP1 sites and contains GC-rich boxed more typical of housekeeping genes. However, the MMP-2 gene regulatory region contains AP2 and YB-1 sites that control it’s expression in a tissuespecific fashion in vivo32,33. Zymogen activation occurs in MMPs when there is a disruption of the interaction between the unpaired cysteine residue in the propeptide and the34 zinc atom in the active site (FIGURE 2). This has been called the “cysteine switch” . Once this interaction is disturbed a conformational change in the enzyme results in autocatalytic or proteolytic cleavage of the remainder of the propeptide giving rise to the mature catalytically competent enzyme. These proteolytic events are often the result of a complex proteinase cascade. Some MMPs, such as MT-MMPs and MMP-11 have a furin-like recognition sequence in their propeptide and thus can be activated intracellularly (in the trans-Golgi network) by the calcium dependent transmembrane serine proteinases of the subtilisin group (furin/PACE). Other MMPs, such as MMP-1, -3 and –9 can be cleaved in their propeptide via the serine proteases such as the uPA-plasmin system, elastase and trypsin35. Subsequently, some of these activated MMPs can also activate other proMMPs as is the case with MMP-3 (stromelysin- 1) which activates proMMP-1 and proMMP-97. MMP-9 is also activated by MMP-219 .
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FIGURE 2: All MMPs possess a pro-peptide which is responsible for maintaining the enzyme in a latent state. This is due to the interaction of a cysteine residue (Cys) in the pro-peptide with a zinc ion in the active site cleft. Once the pro-peptide has been cleaved N-terminally to the cysteine residue, the interaction between the cysteine and the zinc ion is destabilized, allowing further proteolysis to occur, possibly via inter/intramolecular proteolysis. The fully active zymogen is then generated once the pro-peptide has been completely cleaved.
The activation of pro-MMP-2 is unique in this family and thought to result from a cellsurface mediated mechanism involving associations between MMP-2, MT- 1, 2,3,5,6 MMP and TIMP-236. MT-1 MMP is present on the cell surface (as all other MT-MMPs) and can be inhibited by TIMP-2 which binds via its N-terminal domain to the active site of MT-MMP37. (FIGURE 3). This binary complex then acts as a receptor for pro-MMP-2 whose C-terminal domain binds to the TIMP-2 C-terminal domain. A second MT-MMP molecule in close proximity then cleaves the proMMP-2 and activates it. Activation of MMP-2 in this model is only possible if TIMP-2 concentrations are low. High levels of TIMP-2 will inhibit both MMP-2 and MT-MMP19 (FIGURE 3B) This mechanism localizes the proteolytic activity of MMPs to the cell surface where proteolysis and invasion occur. Some researchers have reported that MMP-2 activation occurs via the uPA-plasmin system though this is still controversial38.
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FIGURE 3: MT1-MMP is found on the cell surface of many cells in an active form and has been shown to activate pro-MMP-2. This activation mechanism is dependent on MT1-MMP-TIMP-2-pro-MMP-2 interactions on the cell surface. TIMP-2 can bind both pro-MMP-2 and MTI-MMP via its C-terminal domain and Nterminal domain respectively, forming a tri-molecular complex which localizes pro-MMP-2 to the cell surface. In the presence of low levels of TIMP-2 within the pericellular environment (A), other TIMP-2-free MT1-MMP molecules within close proximity to this tri-molecular complex can cleave the pro-peptide of the 72kDa proMMP-2, generating an intermediate 64kDa form. It is then thought that further autocatalytic cleavage occurs to generate the fully mature 62kDa form. However, if there are large amounts of TIMP-2 present within this system (B), all the MTI-MMP molecules will be bound and inhibited by TIMP-2 and would therefore not be able to cleave pro-MMP-2.
Tissue Inhibitors of MMPs (TIMPs) TIMPs block the deleterious effects of elevated productiod/activation of all MMPs in vitro and in vivo 6,39,40. The four TIMP family members (TABLE 2)41-44 have distinct properties and functions23,45. Common features include the characteristic 6 loop structure, resulting from 12 conserved cysteine residues forming intrachain disulphide bonds. TIMPs also possess two domains; a highly conserved N-terminus that is critical for binding to, and inhibiting, MMP activity and a C-terminus, which governs TIMP-pro-MMP interactions46. Structural differences among TIMP proteins include the presence of N-linked glycosylation sites on TIMP-1 and -3, but not TIMP-2 or –4. Both TIMP-1 & -2 cDNA encode for 21 kDa proteins but TIMP-1 can be either singly (24 kDa) or doubly (28 kDa) glycosylated26; the functional significance of glycosylation is unclear. There are important differences between TIMPs in diffuseability, tissue distribution, transcriptional regulation, and specific association with latent gelatinases. TIMP-1, 2 & 4 are freely diffuseable but TIMP-3 is ECM-associated47. Differences in tissue distribution are outlined in TABLE 2. TIMP-1 might be actively transported into the nuclei of gingival fibroblasts48 but the significance is unclear. TIMP- 1 transcription is regulated by a number of cytokines, hormones and growth factors (eg. TGFE IL-1, IL-6 TNFD and retinoic acid)49,50 . TIMP-3 gene transcription is induced by TPA and TGFE147. Gene silencing by methylation has been described only for TIMP-351. Both TIMP-2 and - 4 are constitutively expressed and their promoter regions have distinctive features and lack the AP1 sites that confer inducibility on TIMP-1 and – 352-54. The binding
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of TIMP to the activated MMPs catalytic site leads to inhibition55. In addition, binding of specific TIMPs to the hemopexin-like domain of some MMPs regulates activation of their proforms. TIMP-1 binds to and slows activation of the latent proMMP-9 while TIMP-2 binds and regulates activation of pro-MMP-255-58. TIMPs can be inactivated by a variety of proteinases such as neutrophil elastase and trypsin59. Table 2: Properties of tissue inhibitors of metalloproteinases (TIMPs) TIMP-1
TIMP-2
TIMP-3
Chromosome gene location (human)
Xp11.23-11.4
17q2.3-2.5
22q 12.1-1 3.2
Protein (kDa)
28*
21
24,27*
22
Major sites
Ovary, bone, uterus
Lung, brain, testes
Kidney, decidua, brain
Brain, heart
Expression
Inducible
Largely constitutive
Inducible
Constitutive
Predominant form of expressed molecule
Secreted
Secreted
ECM-associated
Secreted
Pro-MMP complex
MMP-9
MMP-2
MMP-2
?
lnhibition of MT-MMP
No
Yes
Yes
?
Inhibition of gelatinases
Yes
Yes
Yes
Yes
Matrix bound
No
No
Yes
No
-/+1
+
?
No
No
Yes
No
TNFRI
No
Yes
?
?
TNFRI
?
?
Yes
?
TNFRII
No
Yes
?
?
L-selectin
No
No
Yes
?
HER2/ncu
Yes
No
?
?
IL6R
No
No
Yes
?
Apoptosis Inhibition of protein ectodomain shedding: proTNFD
TIMP-4 3p25
TIMP = tissue inhibitor of metalloproteinase; MMP = matrix metalloproteinase; MT-MMP = membrane-type matrix metalloproteinase; ECM = extracellular matrix; TACE = tumor necrosis factor alpha converting enzyme; - = inhibits apoptosis in some tissues; + = promotes apoptosis in some tissues; ? = unknown Apoptosis effects are likely tissue and context dependent 1 inhibits apoptosis in melanoma cells (168) but promotes it in lymphocytes * size of glycosylated protein
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As expected, manipulation of TIMPs affects invasive/metastatic behavior. TIMP-1 inversely correlates with metastatic potential60,61 and exogenous TIMP-1/ TIMP-2 proteins can inhibit invasion in vitro and invasion and metastases in vivo62-67. Antisense-mediated suppression of TIMP-1 induces malignant behavior in Swiss 3T3 cells68 and forced overexpression of TIMP- 1/TIMP-2 reduces the metastatic ability of melanoma cells65,66,69. Adenoviral delivery systems which over-express TIMP-370,71 find inhibition of invasion, increased apoptosis and reduced attachment. Little is known about TIMP-4 other than it will inhibit invasion, metastasis, tumor growth and angiogensis when transfected into a breast cancercell line72 .
The multifunctional role of MMPs and TIMPs It is critical to appreciate that MMPs/TIMPs do a great deal more than simply interact with each other and mediate the invasive process. They have dramatic and potent effects on a broad range of cellular functions such as growth and proliferation, apoptosis, and angiogenesis45,23,73. The effects of MMPs/TIMPs on these processes are poorly understood and this has important implications for developing novel glioma therapies. In terms of growth and proliferation the multifunctional nature of MMPs has been highlighted by intravital video microscopy which shows that extravasation occurs independently of MMP or TIMP expression74. Instead MMPs may be more important in creating and maintaining a favourable growth environment once extravasation has occured23,45 rather than in extravasation per se. The mechanisms are unknown but MMPs may indirectly stimulate growth in vivo by influencing growth factor bioavailability75-77 in the ECM or through G-protein-mediated proteolytic growth factor processing at the cell surface78. The inhibition of growth factors, such as heparin-binding EGF-like growth factor, has recently been shown to be a metalloproteinase-dependent process which can be inhibited with a synthetic MMP inhibitor (BP-94). Inhibiting the “shedding” of growth factors, such as EGF, which are critical to the signalling of tumors like gliomas is a novel mechanism by which MMPs affect tumor growth and proliferation. Other potential mechanisms through which MMPs/TIMPs may influence growth factor bioavailability have been described. One involves the liberation of IGF from the soluble binding proteins (IGFBPs). MMP-1, -2, and -3 can degrade IGFBP-3 and thus release active IGF75 and TIMP-1 can inhibit tumor growth by reducing IGF bioavailability. In this scenario TIMP-1 inhibits the MMP mediated degradation of IGFBP-3 resulting in elevated IGFBP-3 protein levels which in turn results in less “free” unbound IGF-II being available to stimulate tumor growth79. Also MMP-3 can cleave the membrane-anchored precursor form of heparin-binding epidermal growth factor that can act on cells in a paracrine or autocrine fashion76. MMPs can also negate mechanisms designed to moderate cytokine signals, as occurs in cleavage and release of the inactive type II cell surface “decoy” receptor for interleukin 177. Any or all of these types of events may be occurring in the tumor microenvironment but particularly at the tumor-stromal interface. These indirect mechanisms of influencing tumor growth have not yet been explored in gliomas. TIMPs also have unexpected effects that are potentially important. TIMPs may stimulate cell growth independently of MMP inhibition. For example TIMP levels are not always negatively correlated with tumor grade (expected if decreased TIMPs led to a more malignant and invasive phenotype80). Furthermore, experimental manipulation of TIMPs can inhibit tumor growth in addition to invasion and metastasis. TIMP-2 overexpression produces reduced melanoma growth but not metastases69 and transfection of TIMP-2 into transformed fibroblasts reduced tumor growth as well as metastases81. Finally, TIMP-1 &-2 are being recognized as potentially important cell signaling molecules; TIMP- 1 stimulates proliferation in erythroid precursors41,82 (possibly independent of its MMP inhibitory activity77) and other cell types in vitro in the absence of serum83,84. TIMP-1 also activates steroidogenesis in testis85 and has been described as accumulating in the nucleus in gingival fibroblasts48. Similarly TIMP-2 has growth-promoting activity in vitro at picomolar concentrations86 but inhibits bFGF-induced endothelial proliferation87 Whether TIMPs will be growth stimulators or inhibitors may be tissue specific, or (as appears to be the case with TIMP-2) co-mitogens that depend for their actions on other factors such as insulin or the IGF-1 receptor84. TIMPs may have dramatic effects on apoptosis but the mechanism is unresolved. Furthermore, presumably by regulating several levels of cellular control, individual TIMPs have dramatically different effects on apoptosis. The ECM may act as a “survival factor” and suppress apoptosis since proteolytic modification of the ECM or disruption of cell-matrix
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contacts produces apoptosis88,89. Proteolytic processing of TNF-D and FAS ligand (FasL) from the surface of lymphoid cells by MMP-like activity also alters apoptosis 90,91. These effects on apoptosis may underlie some of the reported effects of TIMPs on cell growth84,85 and the apparent paradox (paradoxical if one expects high levels of TIMP-1 would inhibit invasion and metastases and produce a better prognosis) between high TIMP-1 levels in tumor specimens and the patient's poorer prognosis80,92. To outline what is known about TIMPs and apoptosis briefly. TIMP-1 protects against apoptosis in Burkitt's lymphoma cells and normal tonsillar B cells in a manner that is independent of its MMP-inhibitory activity93. This was not found with TIMP-2 or a synthetic MMP inhibitor, was reversed with TIMP-1 antibodies, and was associated with up-regulation of the anti-apoptotic protein Bc1-XL (but not Bcl-2). Since TIMP-1 is secreted, binds to the cell surface, and the effect is blocked by TIMP-1 antibodies, it may act through a receptormediated autocrine loop though a receptor has not been isolated. TIMP-1 has also been associated with a mature, activated phenotype in Burkitt's lymphoma cell lines93. Another mechanism though which TIMP-1 may affect cell signaling and hence apoptosis directly or indirectly is by inhibiting the shedding of the extracellular domain of growth factor receptors as has been found for the HER2 ectodomain in breast cancer cells94 (TABLE 2). The data for TIMP-2 are less clear and the effects of TIMPs on apoptosis are likely tissue-specific, dependent on the stage of differentiation and/or the cellular milieu. For example TIMP-2 inhibited mitomycin-induced apoptosis in melanoma cell lines95 but induces apoptosis in simulated human T lymphocytes (Dr. M. Lim personal comm.) possibly by inhibiting shedding of FasL or TNF-D The best-characterized TIMP in apoptosis is TIMP-3 which has been reported to protect TNF-α receptors from proteolysis by metalloproteinses. TIMP-3 transfectants in colon carcinoma lines inhibited tumor formation in nude mice96. TIMP-3 transfectants had fewer mitotic figures, were delayed in G1, died after serum starvation and their conditioned media caused cell death that was inhibited by anti-TNF-D antibodies. Transfectant cell lysate contained p55 TNF-α receptor while controls had p55 TNF-α receptor and p46 soluble TNFα inhibitor. Control conditioned media also had p46 while no soluble receptor was found in the transfectant condition media96. Others have found that adenoviral expression of TIMP-3 induced apoptosis in melanoma cells71, vascular smooth muscle cells and HeLa cells70. There are no published studies regarding the effects of TIMP-4 on apoptosis. TIMPs are involved in regulation of the shedding of a variety of protein ectodomains in addition to the TNF Receptors (TABLE 2). These effects may be connected with growth and apoptosis through modulation of adhesive signals (e.g. L-selectin) or signaling receptors or ligands (e.g Her2/neu94,97). Angiogenesis allows solid tumors to grow beyond a certain critical size (2-3mm3)98. The acquisition of angiogenic capabilities (referred to as the angiogenic switch) is a key event in tumour progression which is controlled by the balance between angiogenic factors and inhibitory molecules99-102. Extracellular matrix (ECM) remodeling is an important aspect of angiogenesis because of the structural barriers encountered by invading endothelial cells (ECs). The ECs must first dissolve their underlying BM as well as degrade ECM components as they invade into the surrounding perivascular stroma, forming new capillary sprouts. Angiogenesis is dependent, at least in part, on the actions of MMPs since both TIMPs and synthetic MMP inhibitors such as BB-94 (Batimastat) and AG3340 (Prinomastat) are anti-angiogenic103-106. In vivo, tumors arising from B16F10 melanoma cells overexpressing TIMP-2 show reduced angiogenesis95 . Moreover, endothelial tube formation induced by bFGF and VEGF is inhibited by TIMP-2 and TIMP-3, but not TIMP-1103. Several MMPs may be involved in the angiogenic process, but prime candidates include MMP-2 (gelatinaseA), cell surface MT-MMPs, and MMP-9 (gelatinase-B). All of these can degrade a wide range of substrates including interstitial collagens, gelatin, laminin, and fibronectin107-110. Several observations indicate a link between MMP-2, MT-MMPs and angiogenesis. First, during in vitro EC capillary-like structure formation, an increase in activated MMP-2 is observed compared to a monoculture of EC111. Second, pro-MMP-2 activation may involve an association with the D v E integrin, itself an essential function for EC adhesion to BM112,113. Third, PEX, a fragment of MMP-2 comprising of the C-terminal hemopexin-like domain blocks pro-MMP-2 activation on the chick chorioallantoic membrane where it disrupts angiogenesis and tumor growth 114. Fourth, in MMP-2 null mice, tumors generated by malignant cell lines displayed reduced tumor volumes and decreased levels of angiogenesis115. Fifth, endothelial sprout formation from a muscle explant embedded within
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a fibrin gel was found to be MT1-MMP dependent116. Finally, MTl-MMP-null mice show defects in vascular invasion of cartilage and fail to produce new blood vessels in response to the bFGF in a mouse corneal angiogenesis assay117,118. The role of MMP-9 in tumor associated angiogenesis is also important though less extensively studied than that of MMP-2; furthermore it is likely that both enzymes are important in tumor associated angiogenesis. Degradation of the type IV collagen in the basement membrane by endothelial cells can be mediated by both MMP-2 and -9. Type IV collagenase activity is very important in the early steps of endothelial cell morphogenesis and capillar formation. In addition MMP-9-null mice demonstrate very abnormal growth plate vascularization and ossification problems73. The precise mechanism by which MMP-9 regulates growth plate angiogenesis is unknown. Presumably it functions to release angiogenic factors or to inactivate angiogenic inhibitors. As yet unpublished data suggest the progression to the angiogenic switch and malignancy are markedly reduced in MMP-9-null mice. It is possible, for example, that MMP-9 releases sequestered VEGF or other angiogenic factors and contributes to the induction of angiogenesis in these tumors. MMPs/TIMPs have a complex role in angiogenesis that is not yet clear. The simple notion that excess activity of all MMPs leads to increased angiogenesis is not accurate. Rather these may have both angiogenic and angiostatic effects and the net effect may be MMP and tissue specific and depend on spatial-temporal expression. For instance, MMP-7, MMP-9 and, MMP- 12 have all been linked with the generation of angiostatin from plasminogen119 . Processing of endostatin from collagen XVIII also involves the actions of cathepsins and MMPs120. Clearly the contribution of MMPs/TIMPs to tumor associated angiogenesis needs to be better understood so these can be effectively manipulated therapeutically.
Synthetic Inhibitors of MMPs A number of low molecular weight synthetic MMP inhibitors are under various stages of development by the pharmaceutical industry (TABLE 3). In general, these have a peptide backbone similar to the cleavage site on collagen that binds the MMP, and they contain a hydroxamate group that coordinates the catalytic zinc ion in the active site form121. Several promising studies have been published that find antitumor activity of synthetic MMP inhibitors in a variety of in vivo tumor models. The most widely studied of these is batimastat (also called BB-94: British Biotech Ltd., Oxford United Kingdom), which produced prolonged survival in an ovarian tumor xenograft122; inhibited metastasis of melanoma123 , breast124 and colon cancer125 cell lines. The BB-94 also inhibited growth of a colon cancer xenograft126, a breast cancer cell line127 and a hemangioma105. Other synthetic MMP inhibitors (Celltech Therapeutics, Ltd., Slough, United Kingdom; Agouron Pharmaceuticals, San Diego, U.S.A.) inhibit the tumor growth in a variety of tumor models in vivo 128-130. Table 3: Metalloproteinase Inhibitors in Clinical Trials Company
Compound
Indication
Status
Agouron Bayer British Biotech British Biotech Chiroscience Chiroscience Chiroscience Roche Roche Biosciences
AG3340 Bay 12-9566 Marimastat BB-2516 BB-3644 D2163 D1927 D5410 Ro 32-3555 RS 130830
Cancer and Gliomas Cancer Cancer and Gliomas Multiple Sclerosis Cancer Cancer Inflamm. Bowel Arthritis Osteoarthritis
Phase II/III Suspended Phase II/III Phase I Phase I Preclinical Phase II Phase I Phase I
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MMPs In Gliomas The evidence that MMPs are important in the pathophysiology of gliomas is based on three observations: 1) A number of MMPs (mostly MMP-2 and -9)/ TIMPs are found in gliomas or their tumor vasculature, 2) Over-expression or dysregulation of some MMPs is associated with a poorer clinical outcome131-135. Synthetic MMP inhibitor inhibit glioma proliferation, tumor growth and vascularity in a glioma model in vivo136,106. The latter two observations are the strongest evidence that MMPs are a critical requirement for glioma growth and not simply associated with it. Various genetic manipulations in vivo, such as glioma formation in MMP-knock out mice or inducible overexpression systems in vivo have not been published in gliomas. MMP-1, -2, -3, -7, –9 131-146, TIMPs 1-4 135,140,143,147-150 and MT 1-6 MMPs144,151-153 are found in glioma cell lines and surgical specimens. MMP-2 and -9 are the most extensively studied and it seems likely both are involved in invasion and angiogenesis. MMP-2 may become important in glioma invasion and MMP-9 in glioma angiogenesis. Both MMP-2 and -9 are present in normal brain and are expressed in neurons and to a lesser extent in the glia and vasculature using both in situ hybridization (IS) and immunohistochemistry (IH)132,146; MMP-2 is also found in fetal astrocytes154. Levels of expression (using RT-PCR or Northerns), amount of protein and activity are higher for all gliomas, irrespective of grade, than in normal brain131,132 for both MMP-2 and -9. However, expression levels of MMP-2 remain relatively constant with increases in glioma grade whereas MMP-9 expression increases dramatically. This is probably due to the different tissue sources of MMPs in gliomas; the more malignant gliomas are very vascular tumors and MMP-9 is more closely associated with the vasculature in gliomas. MMP-2 is mostly expressed in glioma cells (using IS and IH) and to a much lesser extent in the vasculature and surrounding glia; MT1 -MP localization follows a similar pattern 138,144-146 . In contrast, MMP-9 is predoqinantly expressed in the vasculature with variable expression in glioma cells and little, if any staining in surrounding glia132,134,135,146 . In some patients MMP-9 expression is almost exclusively confined to the vasculature132 but in others high levels are also seen in the glioma tumor cells suggesting expression may be variable among patients. In the vasculature the MMP-9 expression seems to originate from the endothelial cells and perivascular cells. Two other comments regarding the localization of MMP-2 and -9 in gliomas should be made. First, we have never observed MMP expression in isolated tumor cells which were distant from the main tumor mass as one might expect if the early expression of MMPs facilitated invasion of isolated tumor cells. However the methods of IS and IH may be too insensitive to detect very small levels of expression. The failure to observe the expression does not mean it does not occur. In situ zymography may detect MMP activity in these cells but it has not been reported yet. Alternatively, our underlying hypothesis, that MMPs are critical factors in glioma invasion, may be incorrect and these function to maintain a favorable environment for glioma growth. Second, in contrast to systemic cancer where MMP expression predominantly localized to surrounding stromal cells and not found in the tumor cells, MMP-2 and -9 expression were found mostly in the glioma cells and vasculature and not in the surrounding glia or stroma. The reasons for this difference from systemic cancer are unknown. It could be related to the specialized "stroma" of the brain which largely lacks the tough basement membranes and collagen-rich tissue planes that are major barriers to the spread of tumors outside the CNS. The CNS ECM may pose less of a barrier to glioma invasion or the brain's ECM may regulate proteinase expression/activity in glioma cells. There is considerable evidence that MMP-2 plays a major role in glioma invasiveness. In vitro studies usually find that the best correlations are found between invasion and MMP-2 expression/activity8,144,155,156. In addition, there is a close correlation between MT1-MMP expression and activation of MMP-2 during the malignant progression of gliomas144. While MMP-2 is present in normal brain, it is both overexpressed and activated in malignant gliomas. One interpretation of these data from tumor specimens is that MT 1 -MMP is overexpressed in malignant gliomas where there is an accompanying activation of MMP-2; small amounts of MTI-MMP are found in glia surrounding gliomas but not in normal brain144 . So the idea is that the presence of both MT1-MMP and MMP-2 on glioma tumor cells allows for activation of MMP-2 at the glioma/stroma interface and this process facilitates glioma invasion. An alternative explanation is provided by in vitro
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experiments with the rat C6 glioma line in which the critical proteolytic enzyme was MT1MMP and not MMP-2157. These investigators found that the ability of C6 glioma cells to overcome the inhibitory properties of myelin and allow invasion along myelin occurred with MT1 -MMP expression; this invasive phenotype could be mimicked when fibroblasts were transfected with MT1-MMP. This is independent of MMP-2 activity since invasion was not inhibited with TIMP-1, a potent inhibitor of MMP-2 but not MT1-MMP. However, as inhibition was also not observed with BB-94, an effective inhibitor of MT- 1 -MMP158 there may be contributions from other metalloproteinases. Whether these results apply to humans, or in vivo, remains to be determined. The six MT-MMPs have all been found in gliomas144,152,153 . In addition to activation of MMP-2 they may also activate other MMPs such as MMP-13 (collagenase-3). Information on the MT-MMPs is at present very preliminary but there are clear differences in their expression in normal brain and gliomas. MT1-, 2- and 6-MMPs are not expressed in normal brain whereas MT3-, 4-, 5-MMP transcripts are151-153. The expression in gliomas is somewhat inconsistent but MT5-MMP may be the most commonlyoverexpressed; MT1-, 2-, 3-, 4- and 6- are over expressed in a smaller number of samples 151-153 . The cellular origin of these has been described for MT1-, 2-, and 3-MMP but not yet for MT4-, 5-, 6-MMP. MTland 2-MMP are found in malignant glioma cells and, to a lesser extent, in the endothelial cells. Little or none is found in normal brain. MT3-MMP was found in normal brain but the cellular origin was not clear151 . Clearly the importance of these enzymes which may be critical to glioma invasion needs to be better understood.
Integrins and Protease Activity Integrins are cell surface receptors that mediate the physical and functional interactions between a cell and its ECM. These are being increasingly studied in glioma migration and invasion159 . Cell surface integrins may physically "grip" the ECM proteins and simultaneously interact with cytoskeletal elements within the cell to regulate cell adhesion, shape and motility. In addition, the interaction of ECM components with integrins can affect signalling pathways and regulation of protease activity. For example, ligation of the DvE3 integrin provides a survival signal for endothelial cells in vitro and in vivo160 and disruption of this receptor inhibits angiogenesis by inducing endothelial cell apoptosis112. Similarly, vitronectin (ligates DvE 3 and DvE 5 integrins) may protect glioma cells from apoptosis161 . αvβ3 integrin may also influence glioma migration and invasion by regulating the localization and activation of tumor-derived proteases at plasma membrane of glioma cells in two ways. First the αvβ3 integrin directly binds activated MMP-2, concentrates its proteolysis at the tumor cell surface113. Second, vitronectin (the ligand of DvE3) binds, depending on its confirmation, either the protease plasminogen, its inhibitor plasminogen activator inhibitor type 1 (PAI-1), or the urokinase receptor162. These mechanisms may serve to focus both serine proteinase and metalloproteinse activities in the vicinity of the cell membrane. Clearly the complexity of αvβ3, vitronectin and plasminogen, PAI-1 or the urokinase receptor may permit integrins to regulate serine protease-mediated proteolysis. In this scenario plasminogen binding to av b3 and vitronectin would produce enhanced proteolysis and invasion whereas binding of PAI-1 may reduce proteolysis but allow a better "grip"for tumor cell locomotion162 . PAI-1 over expression, somewhat paradoxically, is correlated with increased tumorginecity in gliomas163. This tumor enhancing role of PAI-1 is endorsed by studies of PAI- 1 -/- mice, which have shown that transplanted malignant keratinocytes fail to invade and establish vascularized tumors in these animals, but angiogenesis and tumor growth could be restored upon adenoviral deliver of PAI-164.
TIMPs In Gliomas Although all TIMPs seem to block glioma invasion their effects on a variety of other cellular processes such as proliferation apoptosis or angiogenesis may be quite distinct from each other. Little is known regarding TIMPs in gliomas and inconsistencies exist. Some investigators find a reduced expression of TIMP-1 &-2165 with increasing glioma grade. This suggests a lack of TIMP contributed to the increased aggressiveness of malignant gliomas. However, we 149 and others 135,143,150 find an upregulation of TIMPs -1 &-2 with increasing grades of gliomas. Interestingly, over-expression of TIMP- 1 in a glioma cell line166produced
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the expected reduction in invasion in vitro but also decreased proliferation. This observation was not otherwise explained but is potentially important. TIMP-3 in gliomas has been reported by only three groups149,150; one51 found reduced expression by gene silencing via methylation. No study has reported TIMP-4 in gliomas. We evaluated the expression of TIMPs 1-4 in gliomas149 and found that TIMP-1 was positively, whereas TIMP-4 was negatively, correlated with glioma grade. TIMP-4 levels are initially high in low and midgrade tumors but in GBMs return to levels seen in normal brain tissue. TIMP-2 & -3 expression levels did not vary with tumor grade and are expressed at low levels. The patterns of TIMP localization (using IS and IH) were also different . TIMP-1 was expressed in the tumor vasculature > tumor cells, whereas TIMP-2 was diffusely expressed. TIMP-4 was expressed only in glioma tumor cells though we cannot rule out very low levels of expression in the tumor vasculature using these techniques. Since TIMP- 1 & -4 have dissimilar localizations and expression patterns this suggests their functions in gliomas may be very different. This is certainly not consistent with the simple notion that a loss of TIMP expression allows the malignant progression of gliomas. The increase in TIMP- 1 expression with glioma grade may stimulate glioma proliferation (e.g. via VEGF upregulation167 ), act as a growth factor or be a compensatory increase in expression. In contrast, the reduction of TIMP-4 expression in higher grade tumors may reflect enhanced tumor growth, angiogenesis or invasion or alternatively, enhanced expression of TIMP-4 may confer growth advantages early in the progression of gliomas which are less important once transformation to the highly malignant GBM has occurred. Breast cancer lines which over-express TIMP-4 have produced decreased proliferation, invasion and microvascular density168. Like TIMP-2, TIMP-4 can associate with the Cterminal hemopexin domain of pro-MMP-2169 and this raises the possibility that the TIMP-2 and -4 may have different abilities to regulate MT-MMP-mediated pro-MMP-2 activation.
Synthetic Inhibitors of MMPs In Gliomas There are only four studies of synthetic inhibitors of MMPs (SynMMPIs) in gliomas to our knowledge 106,136,170 (Penny Costello unpublished observations). In terms of in vitro studies Tonn et al. 1999170 reported that both BB-94 and BB-2516 had the expected effects on invasion using the Matrigel assay or spheroid confrontation assay. Neither compound was found to be cytotoxic, however marked inhibition of proliferation, which in some cases inhibited proliferation completely, was found using the U251 cell line. This contrasts to our own data136 using a different MMP inhibitor called AG3340. This did not inhibit proliferation or viability at concentrations 100µM. The mechanism by which BB-94 or BB2516 might affect proliferation in vitro was not explained. It should be noted that the concentration of 50µM of BB-2516 used to inhibit proliferation is approximately 1000 times higher than IC50 for collagenase, MMP-2 or MMP-9. The observation, therefore, is intriguing but unexplained. One possibility is that the BB-94 and BB-2516 (but not AG3340) inhibit a sheddase which liberates EGF from membrane-bound heparin-binding EGF and subsequently allows this liberated EGF to interact with its receptor. Alternatively BB-94 or BB-2516 may inhibit the liberation of other growth factors from the matrix or their binding proteins in serum. Further studies are needed to clarify the potential growth modifying actions of these inhibitors in vitro and in vivo. All in vivo studies are done using the U87 malignant glioma cell line implanted subcutaneously in SCID-NOD or nude mice106,136 (Costello unpublished papers). These all find a markedly reduced rate of glioma growth, proliferation, angiogenesis and invasion in vivo using either BB-94 (Penny Costello unpublished observations) or AG3340106,136 (FIGURE 4). Since AG3340 does not affect glioma viability or proliferation in vitro (at least in our hands at pharmacologically relevant concentrations) we speculated that AG3340’s effects in vivo are mediated by inhibiting angiogenesis and/or by affecting growth factor bioavailability. The effect of AG3340 on glioma vascularity is particularly striking 106. All of these SynMMPIs only slowed tumor growth in vivo and neither produced tumor “cure” or caused tumor regression. Indeed they were not designed or expected to act as cytotoxic agents. Finally, it is still unknown if SynMMPIs are effective in intracerebral glioma models. The mechanism by which synthetic MMP inhibitors reduce angiogenesis in gliomas is unknown but probably complex. As mentioned previously the simple notion that excess MMP activity leads inevitably to increased angiogenesis may not be accurate. MMPs may be involved in the generation of the endogenous angiogenesis inhibitors angiostatin and
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FIGURE 4: Effects of AG3340 on s.c.. growth of the malignant glioma cell line U87. Effects of vehicle control or AG3340 treatment (begun on day 0) on tumor length (length X width); treatment began when tumors were easily measurable and clearly growing. A, in experiment lA, a significant difference in tumor size appeared by day 21 (P < 0.01, Wilocxon test) and remained until day 31 (P<0.01, Wilcoxon test) when the control group (n = 8) needed to be sacrificed. On the same day (day 31) several animals from the AG3340 group were sacrificed, and the others (n = 4) were allowed to grow until sacrifice was indicated on day 70. The survival of the AG3340 group was 2.3 times longer than the control group. B, the same experiment is repeated with similar results. C and D, the animals were sacrificed earlier in both groups, whereas the tumors were smaller and the mice were unaffected. A-C, n = 8 in both group; D, n = 9 in AG3340 and 5 in control groups, respectively;–, AG3340-treated group; ' vehicle control-treated groups, Bars, SD. (Source-Clinical Cancer Research 5:847- with permission)
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endostatin from their precursors.119,120,171 . However, if the only effects of inhibiting MMPs were on these angiogenesis-inhibitor processing pathways one would expect that blocking MMP activity would increase tumor angiogenesis by inhibiting the bioavailability of angiostatin and endostatin (i.e. the opposite of what we observe in vivo) . Clearly MMPs and TIMPs play a complex role in the control of glioma-associated angiogenesis and each pathway needs to be appropriately targeted to produce the maximal effect on angiogenesis. Since SynMMPIs only slow glioma growth (though very significantly) these have been tested in combination with radiotherapy (RT). RT remains the most effective glioma therapy clinically. Using the same subcutaneous U87 human malignant glioma model described above we found106 that the effectiveness of AG3340 was dramatically enhanced when combined with RT. To mimic the clinical scenario we selected a dose of RT which significantly slowed tumor growth but not produce “cure”. Qualitatively similar results were obtained with other angiogenesis inhibitors when combined with RT172 These studies, and other preclinical cancer models evaluating SynMMPIs, prompted the initiation of two trials of SynMMPIs in gliomas. Both are still underway, one uses Marimastat (BB-25 16) and the other Prinomastat (AG3340). Both study GBM patients following RT and patients are randomized to the inhibitor or a placebo. In the Prinomastat trial all patients (ie. in both the control and AG3340 groups) also receive temazolamide as adjuvant chemotherapy. The Marimastat trial has finished accrual but is still following patients. The Prinomastat trial has just begun and in neither case are preliminary results available. The toxicities of these are expected to be relatively mild and “non-overlapping” with chemotherapy or radiotherapy in that these have no myelosuppression. Instead joint pains in the shoulders or hands are most commonly seen and only occasionally necessitate dose reduction for a significant period of time.
SERINE PROTEASES Extracellular serine proteases play central roles in physiological functions like coagulation (thrombin), digestion (trypsin), fibrinolysis, cell migration, and embryogenesis (uPA and tPA), and mast cell function (cathepsin G, tryptase, chymase). The protease thrombin exerts a wide range of cellular responses in a variety of different cell types via PAR-1, - 4 . The thrombin receptor is a G protein coupled receptor. Since little is known about thrombin in brain tumors it is not reviewed here.
Plasminogen Activation System This is nicely reviewed elsewhere173. Plasmin is a proteolytic enzyme that is generated from the proenzyme plasminogen by the action of urokinase-type plasminogen activator (uPA) and tPA. uPA is synthesized and secreted by tumor cells and normal cells and interacts with a specific cell surface associated receptor (uPAR) which localizes the enzymatic activity of plasmin at the cell surface. The activity of uPA is controlled by the plasminogen activator inhibitors type-1 and type-2 (PAI-1 and PAI-2). Many tumor cells secrete uPA and have uPAR and these activate the proenzyme plasminogen into the active trypsin-like protease plasmin. Plasmin in turn degrades fibrin and other components of the ECM and basement membranes. uPA also digests ECM proteins such as fibronectin, independent of plasmin, and activates other proteases such as MMP-9. Binding of uPA to uPAR dramatically increases the catalytic conversion of plasminogen to plasmin. Bound uPA to uPAR is still susceptible to inhibition by PAI-1 PAI-1 is also synthesized by tumor cells and is required for efficient cell invasiveness174 . The ternary complex of uPA, uPAR and PAI- 1 are rapidly internalized175. uPAR is recycled to the leading edge of invading tumor cells and ensures proteolysis is localized to the cell membrane that leads the invasive front176. uPA is produced as the proenzyme pro-uPA by normal and cancer cells and it has little or no intrinsic enzymatic activity173 . Pro-uPA is activated to uPA by plasmin, kallikrein, cathepsins B and L and NGF- J177-180. uPAR lacks a transmembrane domain and is attached to the membrane by a glycosyl phosphitidyl inositol moiety181 . A strong association between dysregulation of various components of the plasminogen activating system and prognosis in a variety of malignancies has been consistently found including brain tumors173 , For example uPA, uPAR and/or PAI-1 or -2
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are significant prognostic factors173 in patients with breast cancer. It seems paradoxical that elevated levels of an inhibitor (e.g. PAI-1) correlate with a poorer prognosis in cancer patients. Perhaps local down-regulation of protease activity results in the formation of new tumor stroma and blood vessels. Also both PAI-1 and uPAR associate with vitronectin182 so that by competing with uPAR binding to vitronectin PAI-1 might interfere with cell attachment and migration183-185 .
Multifunctional role of the Plasminogen Activator System in Tumor Biology Much like the scenario for MMPs/TIMPs above it is becoming clear that the plasminogen system mediates a number of biological effects (mitogenic, chemotactic, migratory, and adhesive) in normal and tumor cells that are not related to its fibrinolytic function. For example treatment of ovarian cancer cells with uPA produces induction of cfos independent of its enzymatic activity186 . Since uPAR is a GPI-liked protein and lacks a transmembrane domain it would seem counterintuitive that it could affect intracellular signaling directly and the search is on for transmembrane adaptors linking the uPA/uPAR system with signal transduction.
Serine Proteases in Gliomas In addition to the MMPs gliomas also use uPA and uPAR in the proteolysis required for glioma invasion. tPA has a high affinity for fibrin and works mainly in vascular fibrinolysis, while uPA is mainly involved in degradation of the ECM. tPA is the less extensively studied in gliomas than uPA. Benign brain tumors have more tPA than malignant tumors187 but tPA immunoreactivity was more prominent in endothelial cells of high grade than low grade gliomas188. The significance of tPA in brain tumors is unknown but it is produced by racine and human gliomas189,190. Like systemic cancers uPA, uPAR and PAI-1 and –2 are critical proteolytic enzymes used by gliomas to invade the surrounding stroma. uPA and its inhibitors have been found in brain tumors in situ and in vitro191-195. Both uPA and uPAR are expressed in normal cells and overexpressed in gliomas. uPAR expression is higher in high grade than low grade gliomas163 . One of the strongest indications of the importance of uPAR in glioma invasion is provided by an adenovirus delivery of the antisense of uPAR. This inhibited glioma invasion in vitro and glioma growth in vivo in a subcutaneous glioma model in nude mice196. Furthermore, transfecting a low grade glial tumor, which normally expresses low levels of uPAR, with uPAR produces a marked increase in invasion compared to the parent line147. Interestingly there is in vitro evidence that uPA/uPAR induces glioma migration independently of uPA-mediated proteolysis192 . In this case an amino terminal fragment which binds to, and activates uPAR but has no catalytic activity was used192. Similar effects on migration were found with melanoma cells197 and keratinocytes198. The mechanism whereby activation of uPAR increases glioma invasion independent of proteolytic activity is unknown. Therefore, activation of the uPA/uPAR system seems to promote glioma invasion both by increasing proteolysis and by increasing glioma migration independent of its proteolytic actions; the mechanism of this latter effect is unknown. UPA, tPA, PAI-1 and VEGF may all cooperate in malignant gliomas. In vivo, these are all concentrated at the invasive tumor border when the gliomas are >1mm3 190. PAI-1 is produced by endothelial cells, vascular smooth muscle cells and a number of tumor cells. Human and rat GBM cell lines produce PAI-1 in vitro191,194. Expression of PAI1 is higher in GBMs than in low grade gliomas and IS and IH analysis shows the message and protein is confined entirely to blood vessels and necrotic areas and is not found in glioma or normal cells199,200 . The expression was higher not just because GBMs are more vascular tumors but also because expression was higher in individual vessels. It may act here to limit plasminogen activation to areas of invasive endothelial cell growth and limit indiscriminate ECM degradation. The association of PAI-1 with areas of tumor necrosis suggests that its overexpression may be responsible for necrosis by interfering with microvascular patency.
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CYSTEINE PROTEASES CATHEPSINS B, H, L Cathepsins The six major families of cysteine proteases are classified into the papain, calpain, clostipain, streptococcal, viral and caspace cysteine proteases. The cathepsins (Cat) B, H L, S and several newer members (K, O/2, F, W and U) are all members of the papain family201. Their structure is similar to papain and subtle differences account for their distinct substrate specificity, regulation and inhibition by protein and synthetic inhibitors. In general these are synthesized as 30-50 kDa precursor proteins and are glycosylated and phosphorylated in the Golgi apparatus. They localize to endosomal vesicles and finally to lysosomes where they are present in their fully active and mature forms. Their optimal activity occurs at slightly acidic pHs. These proteases are involved in protein degradation in the lysosome and at least some Cats may also degrade the ECM extracellularly, either at the plasma membrane or in the ECM as secreted forms. Their intracellular protease functions are well recognized. Partially degraded ECM components may be endocytosed and digested intracellularly in acidic vacuoles and mediated by Cat D202,203 or Cat B203. In some cases Cat B may be present in two pools, one an intracellular lysosomal pool and the other plasma membrane associated; the latter of these would directly facilitate tumor invasion. The mechanism of Cat secretion or attachment to the plasma cell membrane is not understood. Plasma membrane proteins that bind pro-Cat B, D, and L have been found204-207 to be involved in tumor invasion208. Translocation of lysosomal vesicles to the plasma cell membrane and release of Cats may also mediate cell surface Cat activity. Cat B has been found on the outer surface of breast, brain, and melanoma cell lines 209. In terms of extracellular proteolytic activity the mechanism of secretion of lysosomal Cats is not fully understood. Cats are secreted in normal and tumor cells mainly as precursor forms for Cat B, D, and L while many types of tumor cells also secrete mature, active Cat B210-212. .It is unknown whether the pro-Cats are activated at the cell membrane or in the extracellular environment.
Cathepsins in Gliomas The most widely studied Cat in gliomas is Cat B. Overexpression of Cat B transcripts is found in glioma cell lines and tissue specimens and increases with glioma grade213,214. As expected, the expression of several cysteine protease inhibitors, which inhibit a broad range of cysteine proteases including Cat B, is reduced in human glioma and meningioma specimens215. Increased secretion of pro-Cat B is found in glioma cell lines216. The cellular distribution of Cat B in glioma cell lines is also unusual and is found in both cytoplasmic processes and perinuclear regions. In normal cells it is found only in perinuclear regions217. Inhibition with cysteine protease inhibitors reduces glioma invasion in vitro in Matrigel and into normal brain217 . Furthermore, active Cat B localized to the cytoplasm and in cell processes and was also present on the surface of glioma cells217. Taken together these results suggest that Cat B is important in glioma invasion though this has not yet been demonstrated in vivo. Whether it is important in glioma invasion or some other process in gliomas its expression, as measured by immunohistochemistry in glioma and endothelial cells, was recently found to be a prognostic factor for survival218 inglioma patients. Cat D, L and H are also upregulated in the malignant progression of gliomas219-221. All of the above work focuses on Cats as mediators of the invasive process but they may also be important in tumor angiogenesis. In one angiogenesis model Cat B inactivated the anti-angiogenic effects of TIMP-1 and —2. This suggests that in addition to its contribution to tissue remodeling necessary for neovascularization Cat B may facilitate angiogenesis by inactivating two angiogenesis inhibitors222.
CONCLUSION We have highlighted here only the very small number of proteases which are known to be involved in gliomas. The astonishing large number of proteases which have not so far been characterized in brain tumors suggest these may also be critical in the pathophysiology
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of brain tumors. Most of the work herein described is largely descriptive and there are a small number of studies that use either genetic manipulation or specific inhibitors of protease function to clarify their contributions. A great deal of work needs to be done to clarify their roles but preliminary approaches are therapeutically promising and already some protease inhibitors are in clinical trials. Therapeutic approaches which use protease manipulation as potential treatments need to take into account interplay between the various proteases here which may lead to several unintended effects. Finally it is likely that none of these approaches to protease manipulation alone would be entirely effective as treatments in brain tumors and these would need to be combined with other conventional agents approaches such as surgery, radiation and chemotherapy. In spite of these cautionary comments this manipulation of proteases offer a very exciting new avenue of treatment to a group of patients who are desperately in need of better therapies.
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PROTEOLYSIS OF MUTANT GENE PRODUCTS ARE KEY MECHANISMS IN NEURODEGENERATIVE DISEASES
Vivian Y.H. Hook Departments of Medicine and Neurosciences University of California, San Diego La Jolla, California 92093-0822
INTRODUCTION Proteolytic events are primary mechanisms in the development of major neurodegenerative diseases that include Alzheimer's Disease (AD), Huntington's disease (HD), and Parkinson' Disease (PD). This chapter will illustrate the theme that proteolytic mechanisms are primary contributors to the pathogenesis of AD, HD, and PD (Figure 1). In each of these diseases, genetic mutations that are linked to each of these neurodegenerative diseases result in expression of protein precursors that undergo limited proteolysis to result in the formation of neurotoxic peptides. Of paramount importance is the deposition of each of these toxic peptide fragments as protein aggregates in the brain, which are manifested as specific neuropathologies. Moreover, these gene mutations and resultant peptide fragments contribute to the behavioral abnormalities that are characteristic for each of these neurodegenerative diseases – AD, HD, and PD (Table 1). In Alzheimer's Disease (AD), genetic mutations in the amyloid precursor protein (APP) gene and the presenilin 1 and 21-3 , result in enhanced conversion of APP into the smaller, neurotoxic ß-peptide (Aß) in the brain. Aß becomes deposited in extracellular brain amyloid plaques, the hallmark of AD neuropathology. Evidence from studies of transgenic mice expressing these mutant genes suggests that elevated Aß leads to cognitive deficits in1-3 . In Huntington's Disease (HD), the IT15 gene undergoes expansion in CAG trinucleotide repeats, resulting in an expanded polyglutamine domain in the huntingtin protein4,5. Of particular interest is the finding that an NH2-terminal fragment(s) of huntingtin becomes deposited in nuclear inclusions of HD brains6 and transgenic mice7,8. These neuropathologic inclusions are implicated in the pathogenesis of HD. With respect to Parkinson's Disease (PD), genetic mutations in the D-synuclein gene have recently been found to be linked to PD9,10 The D-synuclein gene product is represented by the NCAP protein (non-Aß component precursor11-13 that is proteolytically cleaved to form the NAC peptide that possesses amyloidogenic properties14-16. Moreover, D-synuclein is present within Lewy bodies that are characteristic of PD brain neuropathology. It is clear that proteases are important in AD, HD, and PD neurodegenerative disease mechanisms. However, the specific brain proteases responsible for these proteolytic events have not been identified. In this chapter, the proteolytic processing of APP, the huntingtin protein, and D-synuclein will be discussed as a means towards understanding the pathogenic mechanisms that contribute to these neurodegenerative diseases.
Role of Proteases in the Pathophysiology of Neurodegenerative Diseases, edited by Lajtha and Banik. Kluwer Academic/Plenurn Publishers, New York, 2001.
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Figure 1. Pathogenesis of mutant gene products by proteolysis in neurodegenerative disease. A common feature of mechanisms involved in AD, HD, and PD neurodegenerative diseases involve proteolysis of the mutant protein gene product. Proteolysis results in peptide fragment(s) derived from the protein precursor. These neurotoxic peptide fragments become incorporated into protein aggregrates that are involved in the pathogenesis of neurodegenerative diseases.
Table 1. Neurodegenerative disease gene products in pathological brain deposits. Neurodegenerative Disease gene
Pathogenic peptide generated by proteolysis product of gene
Neuropathological deposits
Amyloid Precursor Protein (APP) in Alzheimer’s Disease
Aß peptides
amyloid plaques
Huntingtin Protein in Huntington’s Disease
NH2-terminal Huntingtin Protein fragments.
nuclear inclusions
Alpha-synuclein in Parkinson’s Disease
NAC (non-amyloid component)
Lewy bodies
ALZHEIMER’S DISEASE Clinical Features Alzheimer’s Disease is an age-related cognitive disorder that generally appears in the 6th or 7th decade of life, and results in a gradual degeneration of memory and cognitive processes. The cognitive deficits incapacitate the AD patients to the point that they are totally dependent on supplemental health care for survival. While several drug therapies are available for treating AD, some are palliative, but none are effective for any length of time17. Ten percent of people over 65 years of age are afflicted with AD. With the gradual
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aging of the American population, it is predicted that this disease will affect a larger fraction of the population.
Elevated Aß Peptides in Amyloid Plaques of AD Brains AD brains are characteristically diagnosed postmortem by typical neuropathology illustrated by the presence of amyloid plaques, especially in hippocampal and cortical brain regions1-3. The primary component of amyloid plaques is the ß-peptide (Aß peptide) that consists of three peptide forms that differ in their COOH-termini. These ß-peptides are comprised of the Aß1-40 Aß1-42, and Aß1-43 forms that possess 40, 42, or 43 residues of the same primary sequence except for their COOH-termini. The Aß1-42 form contains two additional amino acids at the COOH-terminus of the Aß1-40 peptide form, and the Aß1-43 form contains one additional amino acid at its COOH-terminus compared to the Aß1-42 form. The three forms of Aß are first synthesized as the larger amyloid precursor proteins (APP)18-22. Clearly, proteolytic processing of APP is required to generate the smaller Aß peptides. All forms of Aß accumulate in AD brains and have been shown in numerous studies to be neurotoxic23-25. Moreover, there appears to be a preferential elevation of the Aß1-42 and Aß1-43 peptide forms in AD. Genetic mutations in the APP gene, and in the presenilin 1 and 2 genes, have been characterized in transgenic mice and in AD patients1-3; these studies show a strong relationship between APP and presenilin gene mutations with the observed increases in levels of Aß1-42 and Aß1-43 amyloid plaque formation, and cognitive deficits. These data support the hypothesis of a causal relationship between enhanced Aß production and amyloid plaques in AD brains associated with the cognitive deficits that are characteristic of AD.
Production of Aß Peptide by Proteolysis of the Amyloid Precursor Protein (APP) All forms of Aß peptides are derived from a larger precursor protein, the amyloid precursor protein (APP) (Figure 2). There are three major forms of APP consisting of 695, 751, and 771 amino acids, which result from alternative splicing of the APP gene product. Each form of APP contains the Aß peptides18-22. The APP-751 and APP-771 forms include a kunitz protease inhibitor domain. Proteases, known as 'secretases,' produce Aß peptides by cleaving APP at specific peptide bonds at or near the NH2- and COOH-termini of the Aß peptide sequences within APP1,2,18-22. The secretases are categorized according to their specific cleavage sites within APP, which are related to the production of Aß peptides. The secretase that cleaves at the NH2-terminal end of Aß is known as the ß-secretase. The ß-secretase is predicted to cleave between Met- Asp to generate the NH2-terminus of Aß. The protease(s) that cleaves at the COOH-termini of Aß are known as J-secretase(s), which determines whether Aß1-40 Aß1 -42, or AB1 -43 is produced. Production of Aß1-40 would require J-secretase cleavage between Val- Ile. Aß1 -42 and Aß1-43 production would require J-secretase cleavages between Ala- Thr and Thr- Val, respectively. It is not known whether different Jsecretases produce the three different forms of Aß peptides. However, because specific increases in Aß1-42 and Aß1-43 occur in AD, compared to lesser changes in Aß1-40 it is likely that several J-secretases exist to generate the COOH-termini of the different Aß peptides. In addition to ß- and J-secretases, normal cleavage within the Aß sequence occurs between Lys- Leu by D-secretase1-2. Alpha-secretase cleavage of APP, thus, precludes formation of Aß peptides. Genetic studies point to the critical role of the ß- and γ-secretasesto increase the production of Aß peptides in AD, especially the extended Aß1-42 and Aß1-43 peptide forms. Mutations in the APP gene, which are located near secretase processing sites within APP, are genetically linked to familial AD in certain families 1-3,26-29. Transgenic mice that overexpress mutant APPs develop brain amyloid plaques, show elevated Aß peptide levels in brain, and display deficits in cognition and memory1-3,30-32. Moreover, numerous AD-linked genetic mutations in the presenilin 1 and 2 genes enhance the production of Aß to favor the elevation of Aß1-42 and Aß1-43 over Aß1-40 in transgenic mice33,34 and tranfected cell lines34-36. The selective increase in Aß1-42 and Aß1-43 by
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mutant presenilins suggests that different J-secretases may be responsible for producing the Aß peptide forms.
Figure 2. Amyloid precursor protein (APP), Aß peptides, and secretases. The structure of the amyoid precursor protein (APP) is schmatically illustrated. APP contains a signal peptide (SP) sequence, a cysteinerich region (C-rich), a KPI (kunitz protease inhibitor) domain for APP-751 and APP-770 forms (APP-695 lacks the KPI domain), a transmembrane domain, and a cytoplasmic COOH-terminal domain. Most importantly, Aß peptides are present within APP near the transmembrane domain. The APP precursor undergoes proteolysis by secretases to generate the Aß peptides that are known to be neurotoxic, and accumulate in amyloid plaques in AD brains. Proteolytic processing of APP generates three forms of Aß peptides of 40, 42, and 43 amino acids in length. These peptide forms possess the same NH2terminus beginning with Asp; they differ in their COOH-termini, as illustrated. Proteolysis at the ß-secretase site generates the NH2-terminus of Aß peptides. Proteolysis at the COOH-termini of Aß peptides occurs at Jsecretase sites; it is noted that there are three different J-secretase sites. Proteolysis may also occur within the Aß peptide at the a-secretase site, which precludes formation of Aß peptides. Although the APP processing sites have been named as the ß-, J- and D-secretases, the protease responsible for cleavage at these sites have not yet been definitively identified.
APP Trafficking and Processing in the Secretory Pathway Neuronal peptides destined for secretion are typically routed to the secretory pathway to allow release of these peptides to the extracellular environment. Studies of the cellular trafficking of APP and its processing are important to define the possible locations of secretases within the cell. Thus, although the secretases themselves have not been found, numerous studies have established that APP subcellular trafficking and processing occur in the secretory pathway1-3. The deduced primary sequence of the human APP cDNA indicates that it possesses an NH2-terminal signal sequence, which serves as a mechanism to route translated proteins to the secretory pathway. The secretion of peptides routed to the secretory pathway is typically stimulated by neuronal receptor activation; indeed, muscarinic receptor stimulation of hippocampal neurons releases Aß peptides37. In vivo, APP undergoes axonal transport to nerve terminal38,39 which is consistent with trafficking of vesicles to axon terminals for secretion. In brain, receptor-mediated stimulated secretion of APP and its products demonstrates functional localization of APP products within secretory vesicles
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at the nerve terminal that undergo exocytosis and secretion. Thus, in vivo studies have provided ample evidence for the trafficking and processing of APP in the secretory pathway. The in vivo studies of APP have been extended into in vitro cellular systems, that is, APP transfected into cell lines, to provide further evidence for APP trafficking and processing in the secretory pathway. Investigations of Aß peptides, detected by sensitive sandwich ELISAs, show trafficking of APP in the secretory pathway, where Aß peptide production occurs. Evidence suggests that APP processing occurs in the early secretory pathway, including the RER (rough endoplasmic reticulum) and Golgi apparatus, and in post-Golgi vesicles1,2,40,41. In addition, a high proportion of APP exists in a membranebound form, and becomes incorporated into the cell membrane1-3. The APP protein can be internalized from the cell surface to endosomes, where some APP processing may also occur. These findings predict that APP processing into Aß peptides may occur at several locations within the secretory pathway. It is, therefore, logical that the corresponding secretases are present with APP in the secretory pathway.
Criteria for Identifying Authentic Secretases and Recent Studies of Candidate ßsecretase Activities Knowledge of the cell biology of APP trafficking and processing is important for consideration of candidate secretases. Identification of proteolytic enzymes as secretase processing enzymes for APP are expected to meet three stringent criteria that are expected of authentic neuroproteases responsible for production of Aß. Firstly, the protease(s) must represent the primary secretase activity at an in vivo site of Aß production from APP, corresponding to the subcellular organelle site of APP processing from an in vivo tissue source that naturally produces Aß. Based on studies of APP trafficking and processing in the secretory pathway, the secretase enzymes are predicted to be localized to secretory vesicles, or other organelles within the secretory pathway. Secondly, the protease(s) must possess the appropriate cleavage specificity for processing at the designated secretase cleavage site. Thirdly, the protease(s) should demonstrate optimum activity at an acidic pH to illustrate that it would be active within the acidic pH environment of secretory vesicles (or the secretory pathway) where APP processing and Aß peptide production occur. These criteria have been effective in the identification of authentic proneuropeptide processing proteases42-44, including the carboxypeptidase E/H45-47 and subtilisin-like prohormone convertases44,48. Thus far, reported candidate secretase enzymes have not yet been demonstrated to meet all three criteria. However, ongoing studies in our laboratory demonstrate novel proteases that meet all criteria. These studies will soon be reported and the details will be discussed at that time. Therefore, this section will discuss candidate ß-secretase enzymes that have reported at this time. Recent in vitro studies in transfected cells have suggested that caspases may cleave APP to Aß, and, thus, caspases have been proposed as candidate secretases49. However, caspases are present in the cytosol of cells, whereas APP is inaccessible to cytosolic components, since APP is contained within the subcellular organelles of the secretory pathway. Therefore, caspases are not considered to be candidate50. Several recent studies reported the identification of a candidate aspartyl protease known as BACE (beta-site APP-cleaving enzyme) or Asp251-54, for ß-secretase processing of APP. This novel aspartyl protease cDNA clone was obtained by expression cloning of a human embryonic kidney cell cDNA library expressed in HEK293 cells51, purification and cloning of the human aspartyl protease52, and bioinformatic approaches to identify aspartyl proteases based on their predicted conserved active site residues53,54. BACE, or Asp2, increases Aß formation when cotransfected with APP in cell lines. Recombinant BACE, or Asp2, has been shown to cleave at the ß-secretase site. This enzyme is expressed in brain, with highest expression in pancreas, as well as in kidney and other tissues. Studies have not yet tested for colocalization of the aspartyl protease with APP, APP-derived intermediates, and Aß within the identical cell type and subcellular compartment in vivo. Moreover, it will be important to test these candidate ß-secretase enzymes in knockout mice to assess their likelihood as proteases involved in Aß formation.
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It will be most exciting when authentic secretases are established, which is now an area of intense investigation. Knowledge of the secretases is essential for understanding the proteolytic mechanisms underlying the development of Alzheimer’s Disease.
HUNTINGTON’S DISEASE Clinical Features Huntington’s Disease (HD) is a neurodegenerative disorder of adult onset, characterized by dementia, neurological signs of chorea, and personality change5,55. The onset of the disease occurs in mid-life in approximately 90% of HD patients. In some cases, HD occurs in juveniles; sporadic cases of HD also occur56,57. HD is characterized by neuronal loss, especially of striatal neurons. Such neuronal loss results in modified activity of the nigrostriatal dopamine pathway, and explains the clinical observation of chorea that accompanies the disease58. Degeneration of the striatum in HD brains occurs in a gradient, with degeneration beginning dorsomedially and extending ventrolaterally. The severity of the disease is graded 0 to 4. In grade 1, 50% of neurons in the caudate nucleus are lost, and the putamen and ventral striatum are intact. However, in grade 4, almost all neurons in the dorsal striatum have been destroyed, and ventral neurons are spared; grade 4 represents the end-stage of the disease59. Moreover, affected neurons contain nuclear inclusions of protein aggregates, a typical neuropathological manifestation in HD. The accumulation of nuclear inclusions has been implicated in the pathogenesis of HD, since formation of neuronal inclusions precedes onset of disease symptoms. Proteolysis of the Trinucleotide Repeat Expanded IT15 Gene Product: Deposition of NH2-terminal Fragments of the Huntingtin Protein in Nuclear Inclusions Huntington’s Disease is inherited in an autosomal dominant fashion. The HD gene, the IT15 gene, maps to chromosome 4, and encodes the huntingtin protein of 3144 amino acids4. Importantly, the IT15 gene in HD contains an expanded CAG trinucleotide repeat region, that is part of the first exon. The CAG repeats results in a polyglutamine domain near the NH2-terminus of the huntingtin protein. The repeat is polymorphic in normal brain with 8 to 39 repeats. In HD patients, expansions of 36 to 121 repeats have been reported60. The length of the repeated polyglutamine expansion is inversely correlated with the age of onset of the disease. In addition, the primary sequence of the huntingtin protein is unique4, and possesses no significant homology with known proteins, except for a single leucine zipper motif61. Importantly, NH2-terminal fragments of the huntingtin protein are contained within nuclear inclusions of HD brains6. Using an antibody generated against 17 residues of the NH2-terminus of the huntingtin protein, anti- 1-17 immunoreactivity was detected in nuclear inclusions by immunocytochemistry and western blots. However, nuclear inclusions were not recognized by antibodies specific for the COOH-terminal regions of the huntingtin protein. These data indicate cleavage of the intact 350 kDa (approximately) huntingtin protein, such that the NH2-terminal fragment(s) of the huntingtin protein, containing the polyglutamine expansion, becomes deposited as protein aggregates within nuclear inclusions. Nuclear inclusions also stain positively for ubiquitin, suggesting possible ubiquitin-mediated proteosome degradation of the huntingtin protein; however, this possibility has not yet been definitively determined. The intact huntingtin protein is normally located in the cytoplasm of cells. Thus, proteolysis of the huntingtin protein is presumably followed by nuclear translocation of NH2-terminal fragments containing the polyglutamine region, and incorporation of these peptide fragments within nuclear inclusions (Figure 3). In our studies, several NH2terminal fragments are detected by the anti-1-17 serum (Hook et al., manuscript in preparation) that are generated in a tissue-specific manner that differs in striatum compared to cortex. Moreover, selected NH2-terminal fragments contain ubiquitin, suggesting a role for the ubiquitin-proteosome system in proteolysis of the huntingtin protein. Several distinct ‘protease susceptible’ domains have been mapped within huntingtin However, the precise cleavage sites of the huntingtin protein have not yet been determined. Knowledge of the proteolytic cleavage sites that result in the huntingtin protein NH2-terminal
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fragments, that become deposited in nuclear inclusions, will be essential for future identification of proteases that process the huntingtin protein.
Figure 3. Proteolysis of huntington to generate NH2-terminal fragments. The huntingtin protein represents the gene product of the IT15 gene that is genetically linked to Huntington's disease. The mutant gene product contains expansion of a poly glutamine region near the NH2-terminus of huntingtin. Proteolysis generates NH2-terminal fragment(s) that become incoporated into nuclear inclusions of affected brain neurons. The precise proteolytic cleavage sites of huntingtin have not been determined, as indicated by the ragged peptide fragments. It will be important to define the cleavage sites that will allow identification of proteases involved in huntingtin protein processing.
Expression of mutant NH2-terminal fragments in transgenic mice has confirmed the role of these peptide fragments in the pathogenesis of HD. Mice expressing huntingtin protein fragments with 115-156 repeats developed brain nuclear inclusions, and mice showed behavioral symptoms of the disease7,8. These nuclear inclusions in mice were also stained by ubiquitin antibodies, suggesting involvement of a ubiquitin/proteosome system. In Drosophila, expression of a huntingtin protein NH2-terminal fragment with 120 repeats resulted in rapid degeneration of photoreceptor cells. Lower degrees of degeneration were produced when a fragment containing a fewer number of 75 repeats was expressed in Drosophila62. Moreover, suppression of polyglutamine toxicit in Drosophila has been found to involve genes related to human heat shock protein63, that may be involved in reducing aggregation of huntingtin and formation of nuclear inclusions related to HD. These results support the hypothesis that huntingtin-derived peptide fragments, containing expanded polyglutamine repeats, are involved in neurotoxicity and neurodegeneration in HD.
Cell Biology of Normal Huntingtin Protein Huntingtin is a cytoplasmic protein expressed in many tissues, yet the mutation of the protein only affects neuronal cells. Huntingtin protein expression is high in medium sized spinal neurons that contain GABA and enkephalin, or GABA and substance P58. The normal protein is thought to function in vesicle trafficking64-66. The protein was localized by immunoelectron microscopy to microtubules and vesicle membranes, and western blots detected the huntingtin protein in synaptosomal fractions67. In subcellular fractionations of fibroblasts, huntingtin co-localized to clathrin-coated vesicles and with plasma membranes66. Clathrin coated vesicles are part of the trans-Golgi network and secretory system68.
Proteolytic Mechanisms in Other Trinucleotide Repeat Neurodegenerative Diseases HD represents one of several neurodegenerative diseases that are due to expansions of trinucleotide repeats in the disease gene. The spinocerebellar ataxias (SCA) types 1, 2, 3, 6, and 7 involve CAG repeats in affected genes69-73 SCA 3 is also known as Machado
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Joseph disease71. Expansions of trinucleotide repeats in affected genes are involved in spinal and bulbar muscular atrophy (SBMA, or Kennedy disease)74, and dentatorubralpallidoluysian atrophy (DRPLA)75. Among these diseases, expansions of CAG trinucleotide repeats are present in unrelated genes on different chromosomes. In each case, the expanded polyglutamine region produces toxic effects on vulnerable neurons. Notably, huntington7,8 and SCA376 transgenic mouse models expressing polyglutamine expanded regions of the respective mutant genes show the formation of nuclear inclusions. These triplet repeat neurodegenerative diseases involve CAG repeats in the coding region of the mutant disease gene, resulting in a polyglutamine expansion in the mutant gene product. In contrast, several triplet repeat diseases contain expansions in noncoding sequences of the gene, which includes fragile X, myotonic dystrophy, and Friedrich's ataxia77; the roles of these gene mutations in non-coding regions in disease pathogenesis is unknown. Clearly, proteolysis of proteins encoded by mutant genes containing expansions of trinucleotide repeats may represent similar molecular mechanisms responsible for neurodegeneration involving mutant genes containing expansions of trinucleotide repeats.
PARKINSON'S DISEASE Clinical Features Parkinson's Disease (PD) is the second most common neurodegenerative disease, following Alzheimer's Disease. PD is a movement disorder in which affected individuals display resting tremor, rigidity, and bradykinesia (slowness in initiating movements), and is sometimes associated with difficulty in maintaining posture. The prevalence of PD increases with aging to approximately 3.4% among those above 75 years old78-80. PD involves neuronal degeneration which results in loss of dopaminergic cells, and deficiency of dopamine in the substantia nigra of PD brains. The degree of dopamine depletion in the caudate nucleus and putamen correlates with loss of cells in the substantia nigra. Other dopaminergic systems in the brain are also affected, but to a lesser degree than nigrostriatal projections. Neuropathologic characterization of PD brains indicates the presence of intracytoplasmic inclusion bodies, known as Lewy bodies (LB) that represent a significant marker for PD. LBs are found in several brain regions including substantia nigra, locus coeruleus, hypothalamus, cerebral cortex, and other regions79-81. LBs contain accumulate neurofilaments and ubiquitin, as well as other protein components. LBs in brain are also a characteristic of certain dementias. Molecular mechanisms involved in development of PD are thought to be multifactorial, with environmental factors influencing genetically predisposed individuals as they age. Moreover, PD most likely involves polygenic inheritance.
Genetic Mutations in the D-synuclein and parkin Genes in PD Identification of mutant genes involved in PD have only recently been discovered. Two mutations in the D-synuclein gene have been identified in PD (autosomal dominant), consisting of missense mutations resulting in an Ala to Thr substitution at position 53 (Ala53Thr)9, and an Ala to Pro substitution at position 30 (Ala30Pro)10. Importantly, Dsynuclein has been identified as a major component of LBs in PD80,81, as well as in LBs in dementia and AD11,14-16. Alpha-synuclein belongs to a family of related proteins, which includes ß- and J-synuclein82-83; however, ß- and J-synuclein are not found in LBs80. Possible relationships between LB pathology and neuronal loss are not clear at the present time.
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Alpha-synuclein was first isolated form vesicles from the electric organ of Torpedo californica; in addition, rat homologues have been described82,84. Human α-synuclein is homologous to zebra finch synelfin85. Phosphoneuroprotein 14 (PNP 14) represents another member of the synuclein family, and is homologous to ß-synuclein13. ß-synuclein lacks the NAC (non-amyloid component peptide) peptide sequence present within Dsynuclein. The J-synuclein represents a third member of the synuclein family, which is expressed in numerous breast tumours82, suggesting a role in cancer. Because genetic mutations of the D-synuclein gene involved in PD have only recently been discovered, transgenic mice expressing mutant α-synucleins have not yet been reported. Expression of the human D-synuclein Ala30Pro substitution could be expressed in transgenic mice, since the mouse α-synuclein gene sequence is identical to human at position 30. However, expression of the human Ala53Thr mutation in transgenic mice is not predicted to produce phenotypic effects, since the normal mouse α -synuclein gene possesses Thr at position 30, which represents the human mutation in PD. With these prediction of mouse phenotypes upon expression of mutant α-synuclein, it is of notable interest that recent expression of the wild-type human D-synuclein gene in transgenic mice resulted in do aminergic loss and inclusions body formation that resemble in vivo neuropathology in PD86. Future evaluation of mice expression normal and mutant a-synuclein genes will be important for understanding their role in the development of PD. Even more recently, mutations in the parkin gene87,88 have been identified in juvenile parkinsonism87. The newly identified parkin protein consists of 465 amino acids, with a segment possessing some homology to ubiquitin. Resemblance to ubiquitin suggests a role for yet another possible proteolytic component, parkin, in PD. Characterization of the parkin protein in brain and its possible presence in LBs has not yet been determined. Thus, it is not currently known whether the parkin protein undergoes proteolysis.
Proteolytic processing of the α-synuclein gene product Prior to the discovery of the genetic mutations in the D-synuclein gene in PD, a proteolytic fragment of a-synuclein was originally isolated and identified from amyloid plaques of Alzheimer’s Disease (AD). This peptide fragment was known as ‘non-amyloid component’ (NAC) of AD amyloid plaques11,12 . NAC is a 35-amino acid peptide that is acidic, and has been demonstrated to be highly amyloidogenic14,15. Molecular cloning of NAC revealed that it is first synthesized as a precursor protein, NACP, of 19 kDa11. NACP and D-synuclein are the identical protein; this protein is currently more commonly referred to as D-synuclein. LBs contain D-synuclein, but its identity as the D-synuclein precursor protein, or the 35-residue NAC peptide has not yet been established. Clearly, D-synuclein (NACP) undergoes proteolytic processing to generate the smaller amyloidogenic peptide NAC (Figure 4). It is of interest to note that NAC is flanked by monobasic or dibasic lysines at its NH2- and COOH-termini within the Dsynuclein precursor. These basic residues suggest proteolytic processing at these sites to generate the NAC peptide. Specific proteases cleaving at these basic lysine residue processing sites within D-synuclein would be essential in the production and deposition of NAC in amyloid plaques of AD. It will also be important to assess whether the NAC peptide is also a component of LBs in PD. Alpha-synuclein (NACP) is co-localized with synaptic vesicles, assessed by colocalization with subcellular organelles containing the vesicle protein synaptophysin12,89. Comparisons of the subcellular localization of D-synuclein (NACP) and its proteolytic peptide product NAC have not yet been determined. Such knowledge will be helpful in predicting the location of D-synuclein cleaving protease(s).
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Figure 4. Proteolysis of D-synuclein to generate the NAC peptide. The structure of the a-synuclein precursor contains the NAC peptide. Alpha-synuclein also contains seven repetitive degenerate sequences of the consensus sequence KTKEGV (indicated by the numbered regions 1-7). Proteolysis of a-synuclein (also known as NACP, for NAC precursor) is required to generate the NAC peptide product. Proteolytic processing of the precursor at single and paired lysine residues that flank NAC at its NH2- and COOHtermini is required to generate NAC. Proteases responsible for generating NAC have not been identified.
FUTURE POTENTIAL OF PROTEASE INHIBITORS FOR THERAPEUTIC TREATMENT OF NEURODEGENERATIVE DISEASES It is clear from the discussions presented in this chapter that proteolytic processing of mutant gene products into potentially neurotoxic peptide fragments, which are deposited in neuropathological inclusions and aggregates, represent significant mechanisms responsible for the development of AD, HD, and PD neurodegenerative diseases. Identification of the specific proteases that mediate proteolytic processing of APP in AD, the huntingtin protein in HD, and D-synuclein in PD and AD are critical to future discovery of drug inhibitors that block proteolysis of these mutant gene products, thereby preventing the neurotoxic effects of neurodegenerative disease peptides. It will, therefore, be important to find the authentic brain proteases that are responsible for the development of these devastating neurodegenerative diseases. These protease enzymes will provide logical drug targets for inhibition by chemical molecules as therapeutic agents for the treatment of these neurodegenerative diseases.
ACKNOWLEDGEMENTS This work was supported by grants from the Hereditary Disease Foundation and the National Institutes of Health.
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MAMMALIAN PROTEINASE GENES
Hahn-Jun Lee1, Koichi Suzuki2, and Takaomi C Saido1 Laboratory for Proteolytic Neuroscience RIKEN Brain Science Institute Saitama, 351-0198, Japan 2Tokyo Metropolitan Institute of Gerontology Tokyo, 173-0015, Japan 1
INTRODUCTION Proteinases play key roles in many essential physiological processes, and are of great importance in maintaining various cellular functions. With the advent of molecular biology in the 20th Century, remarkable progress has been made in understanding the molecular structure and function, catalytic mechanism, and evolution of proteinases. Further, it has become evident that proteinases can be classified into families, with the members of each family having similar structures and catalytic mechanisms1. In parallel with this, we also have witnessed dramatic advances in understanding the term proteolysis, which is one of the most important enzymatic protein modifications. Careful control of the levels of structural proteins, enzymes, and regulatory proteins is essential to maintain the proper cellular functioning. Generally, structural proteins or proteins that are constitutively required are longlived, whereas regulatory proteins are otten rapidly degraded. Furthermore, their degradation must be precisely timed and regulated, because unregulated degradation would be quite hazardous to cells. Ultimately, the balance between the rate of synthesis and the breakdown of cellular proteins determines the fate of cells in vivo. Thus, the highly complex and regulated mechanism, termed proteolysis, has evolved to accomplish this purpose. As indicated in Table 1, the widely used term protease, which is synonymous to peptidase, can be applied equally to both exopeptidases and endopeptidases. In contrast, the term proteinase is applied only to endopeptidases, and four mechanistic classes of proteinases are recognized by the IUBMB as described below1. This chapter will mainly describe mammalian proteinases possessing endopeptidase activity. However, to describe all mammalian proteinases is beyond the scope of this chapter. It is the aim of this chapter to summarize mammalian proteinases briefly and to explain the structural and evolutionary characteristics of their genes on the basis of recent information. Role of Proteases in the Pathophysiology of Neurodegenerative Diseases, edited by Lajtha and Banik. Kluwer Academic/Plenum Publishers, New York, 2001.
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Table 1. Classification of proteinases. PEPTIDASES (PROTEASES) Endopeptidases (Proteinases) Serine proteinases Cysteine proteinases Aspartic proteinases Metalloproteinases Unclassified proteinases
Exopeptidases Aminopeptidases Dipeptidyl peptidases Tripeptidyl peptidases Carboxypeptidases Dipeptidases Dipeptidases Tripeptidases Omegapeptidases
CLASSIFICATION OF MAMMALIAN PROTEINASES Generally, mammalian proteinases can be subdivided into serine, cysteine, aspartic, and metalloproteinases on the basis of their catalytic mechanisms as shown in Table 1. This classification has been recommended by IUBMB according to the evolutionary relationships, and is also available in the public data base1. Besides these four classes, proteinases whose catalytic mechanisms have not yet been identified also exist. Thus, it is possible that novel types of as yet unidentified proteinases exist in living organisms1.
CHARACTERISTICS OF MAMMALIAN PROTEINASES Serine Proteinases General Features. Serine proteinases are one of the best studied of mammalian proteinases both at the nucleic acid and protein levels. Serine proteinases comprise two distinct subfamilies, the mammalian chymotrypsin family and the bacterial subtilisin family2,3. Mammalian serine proteinases include many regulatory enzymes involved in digestive function, coagulation, fibrinolysis, complement systems found in blood plasma, and various other polypeptide processing enzymes4,5. The processing of precursor trypsinogen to active trypsin by enterokinase, as well as the blood clotting cascade, is one of the best examples of a regulatory system involving serine proteinases6,7. Generally, serine proteinases have different substrate specificities, and these result from amino acid substitutions at various enzyme subsites interacting with substrates8,9. It is well known that three residues, Asp 102, His 57, and Ser 195 (chymotrypsin numbering), in the active site are essential for catalytic action10. Further, catalysis proceeds via a tetrahedral transition state intermediate during both the acylation and deacylation steps of catalysis11. Characteristics of Serine Proteinase Genes. The number and distribution of introns differ among genes coding for trypsin, chymotrypsin, and elastase, which are representative serine proteinases 12. Although exon shuffling and intron insertion are not consensus events in all mammalian serine proteinases, they are well-known mechanisms in the 5’-halves of serine proteinase gene structures13. Further, since the exon-intron junctions of mammalian serine proteinase genes map to the protein surface, the overall conformation of the protein is not affected by simple insertion and deletion of coding segments at the splice junctions10,13. Taken together, serine proteinases have preserved their catalytic triad successfully under evolutionary pressure.
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Trypsin. Trypsin (EC3.4.21.4) is selectively synthesized as an inactive precursor, a zymogen, at high levels in the exocrine pancreas of mammals. The role of trypsin is considered to be in the digestion of foods by itself and by activation of other zymogens. In addition, a homologue of pancreatic trypsin, termed TESP4, has been found to be involved in the penetration of the egg zona pellucida by sperm like acrosin14. Trypsin has been reported to comprise a multigene family on mammalian genomes. Although the reason for the presence of these multiple forms is not known, it is certain that they are products of separate genes judging from their distinct sequence varieties15,16. Among the 8 human trypsinogen genes, 5 are transcribed, 2 are pseudogenes, and one is a relic gene17. These genes are intercalated in two pieces within the human ß-T cell locus on chromosome 7q3517,18. Three nonfunctional trypsinogen genes are located at the 5'-end of the locus, whereas known trypsinogen isozymes are located near the opposite end of the locus. These corresponding genes have also been found in the same loci in rat and chicken19,20. Kallikrein. Most kallikrein (EC3.4.21.35) and kallikrein-like genes that have been identified so far are composed of 5 exons, and the tertiary structures of the encoded enzymes show remarkable similarity. For mouse and rat, these genes are highly conserved, tightly linked, and tandemly arranged. In contrast, in humans, the family is less well defined and seems to be much smaller than in mouse and rat. The human kallikrein gene subfamily comprises three genes, for tissue kallikrein, human kallikrein HK2, and semenogelase21. Although the exact physiological function remains to be elucidated for many kallikrein gene family members, they are considered to play important roles in processing biologically important peptide precursors22 and maintaining low blood pressure20,23,24. Chymase. Chymases (EC3.4.2 1.39) are chymotrypsin-like serine proteinases secreted by mast cells, and are thought to play a role in the formation of angiotensin II, which is responsible for the pathogenesis of cardiovascular diseases. Alpha- and beta-chymases differ in their structures, functions, and mast cell subset and species-specific expressions25. The chymase genes examined so far are identical in the number, phase, and placement of introns. High similarity has been found in the intronic and 5'-flanking regions of the dog, human, and mouse alpha-chymase genes, but little similarity is found in the corresponding beta-chymase genes. Repetitive elements probably derived from retroposons are unique features of the 5'flanking region in dog25. The regulation of chymase gene expression is very complex, and the products of many chymase genes in different mast cells are tightly controlled by various normal and pathologic stimuli. Interestingly, the chymase genes in both humans and mice are reported to cluster with other granule-associated serine protease genes in lymphocytes and myelomonocytes. This clustering may facilitate the expression of hematopoietic protease genes26,27. Plasminogen Activators. The fibrinolytic system comprises an inactive proenzyme, plasminogen, that is converted by plasminogen activators to the active enzyme, plasmin (EC3.4.2 1.7). Plasmin degrades a number of ECM proteins including fibrin, fibronectin, laminin and proteoglycans, and further may activate other proteases such as MMP-9. Two physiological plasminogen activators have been identified: tissue-type plasminogen activator (t-PA) (EC3.4.21.68) and urokinase-type plasminogen activator (u-PA) (EC3.4.21.73)28. uPA is secreted as an inactive single-chain 55 kDa proenzyme. Binding to its receptor (uPAR) results in accelerated conversion to the double-chain, enzymatically active protein that generates plasmin29. The human urokinase-type plasminogen activator (uPA) gene is 6.4 kb in size, comprising 11 exons and 10 introns, located on chromosome 1030. Comparison of the murine uPA gene with the porcine and human uPA genes reveals a higher sequence similarity even in the introns and flanking sequences. The murine gene is also organized into 11 exons comprising 34.7% of the 6710 bp region spanning the interval between the presumed transcription initiation and polyadenylation sites. The transcription initiation site is flanked by common RNA polymerase II promoter elements, including a TATA box and a potential transcription factor Spl binding site30. On the other hand, the human tissue-type plasminogen activator (t-PA) gene, located on chromosome 8, consists of 14 exons. Its exon-intron organization strongly suggests exonshuffling, whereby the distinct structural domains are encoded by a single exon or by adjacent exons31. The proximal promoter sequences in the human t-PA gene contain typical TATA and
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CAAT boxes and potential recognition sequences for transcription factors (e.g. AP1, NF1, SP1, It is likely that consensus sequences for a CAMP-responsive element and an AP-2 AP2)32,33. binding site found in the flanking region have a cooperative effect on constitutive t-PA gene expression34. Furthermore, allelic dismorphism has been observed as a result of an Alu insertion/deletion that occurred early in evolution35,
C1r and C1s. Clr (EC3.4.21.41) and Cls (EC3.4.21.42), responsible for the activation and proteolytic activity of the C1 complex of complement, share similar overall structural organizations featuring five nonenzymic protein modules followed by a serine protease domain. Both human C1r and C1s genes are located in region 12q1336, and have been shown to be arranged in a tail-to-tail orientation37. Both proteinases have interaction properties associated with their N-terminal regions, including the ability to bind Ca2+ ions with high affinity, to associate with each other in a Ca2+-dependent C1s-C1r-C1r-C1s tetramer, and to interact with C1q upon C1 assembly38. It has been also reported that partial or complete genetic deficiencies in Clr correlate with lupus erythematosus and renal disease39. Cysteine Proteinases General Features. Cysteine proteinases are widely distributed among living organisms, and play an essential role in a variety of cellular functions. Based on the most recent classifications1,40, this family comprises plant proteinases such as papain (EC3.4.22.2), caricain (EC3.4.22.30), stem bromelain (EC3.4.22.32), and actinidain (EC3.4.22.14), several mammalian cathepsins and calpain (EC34.22.17), as well as several parasitic proteases. The papain-like cysteine proteinases are one of the most abundant species in this family, and share similar sequences and structures41-43. Cathepsins B (EC3.4.22.1), C (EC3.4.14.1), H (EC3.4.22.16), and L (EC3.4.22.15) are ubiquitous in the lysosomes of animals, whereas cathepsin S (EC3.4.22.27) has a somewhat restricted localization44,45. Besides the enzymes described above, interleukin- 1-beta converting enzyme (EC3.4.22.36), as well as a related proteinase, termed caspase, has been shown to represent a novel type of cysteine proteinase46,47. The higher sequence similarity of ICE (caspase-1) to the Caenorhabditis elegans death gene CED-3 provides the hints to link caspases to apoptosis. For now, caspase constitutes a new superfamily of cysteine proteinases targeting Asp groups. In the case of cysteine proteinases, the major catalytic amino acid residue is cysteine 25, acting like serine 195 in chymotrypsin10. It has been revealed that catalysis proceeds through the formation of a covalent intermediate and involves a cysteine and histidine residue10. Characteristics of Cysteine Proteinase Genes. Judging from phylogenetic analyses, gene duplications are often found on cysteine proteinase gene structures, and several of them are thought to have occurred very early in evolution48. Calpain. Among mammalian cysteine proteinases, the gene structures of calpain are especially well characterized. Calpain (EC3.4.22.17) plays a key role as a biomodulator in various cellular functions in response to Ca2+-mediated signaling49-53. Recent advances in molecular biological approaches have helped to clarify the fact that calpain constitutes a superfamily consisting of ubiquitous and tissue-specific homologues. Mammalian calpains that have been identified so far are summarized in Table 2. The ubiquitous forms, CAPNl and CAPN2 (µ- and m-calpain, respectively), are the best-characterized mammalian isozymes. They are heterodimers consisting of a unique large subunit and a common small subunit (CAPN4) (also called 30K). The mammalian tissue-specific calpains include the muscle-specific CAPN3 (also called p94, nCL- 1)54, the stomach-specific CAPN8 (nCL-2)55, the digestive tract-specific CAPN9 (nCL-4)56, and two atypical family members, CAPN5 and CAPN657. In addition, the recently identified CAPNl1 may be the human orthologue of µ/m calpain58. Among mammalian calpain genes, CAPN3 (p94, nCL-1) is well characterized in terms of its gene structure (Figure 1). When the sequences of mouse, rat, and human p94 genes are compared, even the 5'-flanking region is highly conserved (Figure 1)59. Quite recently, the calpain 3 gene has been reported as one of the causes of limb-girdle muscular dystrophy type 2A (LGMD2A). Various mutations such as missense point mutations, non-sense mutations,
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frameshift mutations, and splice site mutations have so far been identified in the calpain 3 gene6062. Taken together, the calpain 3 gene may be a useful tool for use in the diagnosis of 15q-linked familial cases of LGMD as well as sporadic cases62.
Figure 1. Schematic structure of the p94 gene. Top, gene structure of mouse p94. Closed numbered boxes represent exons. Bottom, comparison of the 5’-flanking region of the p94 gene among mammals. Numbers at the top are nucleotide residue numbers of mouse genomic DNA. Open and hatched boxes indicate the 5’-noncoding and coding regions, respectively. Numbers on the arrows are the nucleotide identity of the segment between mouse and rat or human.
The chromosomal localizations of mammalian calpains are also summarized in Table 2 on the basis of the gene cards database and references63,64. It is also reported that gene duplications may have occurred in calpain gene families as in other multigene families in vertebrates during evolution64. Moreover, it has been suggested that the conserved chromosomal regions containing calpain genes may also be regulated in a coordinated manner64.
Cathepsins (B, H, L, and S). Cathepsins, which are involved in intracellular protein degradation, can be subdivided into two groups, the cathepsin L-like or ERW/FNIN proteinases and B-like proteinases on the basis of a highly conserved interspersed amino acid motif (ERW/FIN) in the proregion65,65a. Human cathepsin B is encoded by a single-copy gene located on chromosome 8p22. It consists of 13 exons, and the gene encompasses approximately 27 kb66. Cathepsin B expression has been found to be a prognostic indicator of colon carcinoma67. Multiple cathepsin B mRNA species resulting from alternative splicing68 may be related to tissue- and tumor-specific expression69. In contrast, the human cathepsin S gene localizes on chromosome 1q2170 and consists of six exons and introns. Analysis of the 5’-flanking region reveals that this gene lacks canonical TATA and CAAT boxes but contains an AP1 site and CA microsatellites, suggesting specific regulation70. The mouse genes for cathepsins B, H, L, and S have been mapped to chromosomes 14,9, 13, and 3, respectively, suggesting these four cysteine proteinases were dispersed over different chromosomes before separation of mice and humans during evolution71.
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Table 2. Localization of calpain genes on human chromosomes. GBD Name
Chromosomal Localization
Gene Product
CAPN 1
1lq12-q13.1
µ-calpain large subunit (µCL, µ80K)
CAPN2
lq41
m-calpain large subunit (mCL, m80K)
CAPN3
15q 15.1 -q21.1
p94 (nCL-1)
CAPN4
19q13
µ-, m-calpain small subunit (30K)
CAPN5
1lq14
hTRA-3 (calpain 5, nCL-3)
CAPN6
Xq23
Calpamodulin (CANPX)
CAPN7
3p24
PalBH
CAPN8
lq41
nCL-2,2'
CAPN9
lq42.1-q43
nCL-4
CAPN11
6p12
calpain 11
CAPN12
16p13.3
SOLH
Aspartic Proteinases General Features. Most mammalian aspartic proteinases belong to the pepsin family, including digestive enzymes such as pepsin (EC3.4.23.1) and chymosin (EC3.4.23.4), intracellular cathepsins D (EC3.4.23.5) and E (EC3.4.23.34), processing enzymes such as renin (EC3.4.23.13, and some fungal proteases10. A second family also comprises viral proteinases such as retropepsin (EC3.4.23.16) in HIV virus. The characteristic active site residues are aspartic acids 33 and 213 (penicillopepsin numbering), which are in close proximity to each other72. The great majority are most active at acidic pH, but a few are also active at neutral pH, for example, renin. Characteristics of Aspartic Proteinase Genes Pepsin. Pepsin is one of the most important acidic proteinases found in stomach. Pepsin is synthesized in gastric mucosa and secreted into the stomach as a zymogen, pepsinogen. Pepsinogen, an inactive precursor of pepsin, is synthesized in the chief cells of gastric glands. There are two major groups of pepsinogen, namely pepsinogen A (PGA) and pepsinogen C (PGC) (or progastricsin), and each also has isozymogens. Each gene is organized into nine exons and eight introns of various length, as in the case of other aspartic proteinase zymogens73. The human pepsinogen gene, containing nine exons in 9.4 kb, strongly suggests the duplication from a common ancestral gene during evolution73,74. The 5'-flanking region of the human PGA gene is different from those of human and rat PGC genes, while the human and rat PGC genes are similar to each other. This implies that the expressions of the PGA and PGC genes are regulated somewhat differently73. Moreover, it has been reported that PGA gene expression is controlled in a species-specific manner75. Cathepsin D. Cathepsin D is a major aspartyl lysosomal proteinase expressed in most animal tissues, but it can also be regulated by estradiol, calcitriol76 and retinoic acid77. The organization of the cathepsin D gene is almost same as that of other aspartic proteinases. Although
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its exact promoter structure remains confusing as no classical estrogen response element has been identified78,79, cathepsin D gene transcription is increased by estrogen and growth factors in estrogen-receptor-positive breast cancer cells and by an unknown mechanism in estrogen-receptornegative cells. No classical TATA or CAAT boxes have been identified, but SP1 and AP2-binding sites exist in the 5'-flanking region80.
Renin. Although the gene organization of human renin includes 10 exons separated by nine introns81, the structures of mouse and rat renin contain nine exons separated by eight introns82,83. Based on the gene organizations of renin as well as other aspartic proteinases, it is possible that the two exons containing the two active site Asp residues arose from a duplication of a common ancestral gene, eventually resulting in the bilobal aspartic proteinases74,83. Metalloproteinases General Features. Metalloproteinases are found in variety of living organisms from humans to bacteria. They differ in sequence and structure, but the great majority contain a divalent metal cation such as zinc, but sometimes the zinc may be replaced by cobalt or nickel without loss of activity. Further, metalloproteinases can be divided into two groups according to the number of metal ions required for catalysis, those for which only one zinc is required and those needing two metal ions for catalysis84,85. The great majority contain the characteristic HEXXH motif, and this motif occurs in a nine residue consensus sequence, bXHEbbHbc, in which b is an uncharged residue, c is hydrophobic, and X can be any amino acid1. Characteristics of Metalloproteinase Genes. Among metalloproteinases, cell surface proteinases participate mainly in post-secretory processing and the metabolism of neuropeptides and peptide hormones. Neutral endopeptidase-24.11(NEP) is the prototype of a family of zinc metalloproteinases that also includes the endothelin-converting enzymes (ECE), the erythrocyte cell-surface antigen, KELL, and the putative product of the PEX gene, which has been associated with X-linked hypophosphatemic rickets86. These metalloproteinases share close sequence similarity and are all type II integral membrane proteins, featuring a large luminal domain and a shorter cytoplasmic tail. Neutral Endopeptidase-24.11 (NEP). A wide range of biologically active peptides, such as enkephalin and atrial natriuretic peptide, have been reported as substrates for NEP (EC3.4.24.11). The human NEP gene is located on chromosome 3q21-q2787, showing that it spans more than 80 kb, and is composed of 24 exons88. The pentapeptide sequence (His-Glu-IleThr-His) related to metalloproteinase zinc binding and substrate catalysis is encoded within exon 19. As shown in Fig. 2, three types of NEP cDNA resulting from alternative splicing of exons 1, 2a, and 2b to the common exon 3 have been identified so far in human88. On the other hand, 4 types of NEP cDNA have been identified in rat89. Interestingly, in the case of human, exons 2a and 2b share the same 5' sequence but differ from each other in having two distinct donor splice sites 171 bp apart in the gene88. Further, a comparison of the nucleotide sequences of human NEP and rat NEP reveals a high degree of sequence similarity within noncoding exons 1 and 289. Interestingly, in the rat gene, a downstream noncoding exon 3 can also be detected as indicated in Figure 2. Two transcripts derived from exon 2 by alternative splicing, termed type-2a and type-2b, were demonstrated to present in human kidney and lung, whereas only the type-2b transcript was present in these same tissues in rat89. Although the type-1 transcript was detected in human kidney, lung, and brain, this transcript appears to be the major species in brain. Therefore, the expression of these neutral endopeptidase mRNAs can be regulated in a tissue-specific and/or developmentally regulated manner89. NEP KO mice appear to be developmentally normal except for some small differences in lymphoid development. However, the NEP KO mice show a hypersensitivity to endotoxic shock compared to their control litter mates90.
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Figure 2. Schematic representation of NEP 5’ genomic regions and their spliced mRNA products. (A) For human, 3 types of mRNAs are generated by ligation with 3 different noncoding exons to the common coding exon 3. On the other hand, for rat, 4 types of mRNAs are generated by ligation with 4 different noncoding exons to the common coding exon 4 (B). Common coding exons are indicated by hatched boxes.
Endothelin-Converting Enzymes. Endothelin-converting enzyme catalyzes the final step in the biosynthesis of the vasoconstrictor peptide, endothelin. Complementary DNAs encoding two ECE forms, termed ECE-1 (EC3.4.24.71) and ECE-2, have been isolated91-95. Recently, a novel ECE specific for big endothelin-3 was purified from bovine iris microsomes96. ECE- 1 and ECE-2 are also type II integral membrane-bound proteinases similar to neutral endopeptidase-24.1 1 (NEP) and kell blood group protein91. Four isoforms of human ECE-1 have been identified to date: ECE-la and ECE-1b91, ECE-1c93, and, more recently, ECE-1d97. These isoforms are encoded by the same gene with four distinct promoters92,94,97. However, ECE-2 is encoded by a distinct gene95. The isoforms differ in their N-terminal cytosolic regions, and have distinct tissue distributions and intracellular localizations. PEX. The PEX gene is associated with hypophosphatemic rickets, a disorder characterized by impaired renal tubular phosphate reabsorption using a positional cloning approach98. Matrix metalloproteinases (MMPs). Besides the metalloproteinases described above, matrix metalloproteinases (MMPs), formerly called matrixins, are an important group of zinc enzymes that are thought to be involved in the degradation of extracellular matrix components such as collagens, proteoglycans, elastin, laminin, fibronectin, and other glycoproteins99-101. The MMP family includes collagenases (MMP- 1, MMP-8, and MMP- 13), gelatinases (MMP-2 and MMP-9), stromelysins (MMP-3 and MMP-10), stomelysin 3 (MMP-1 1), membranetype metalloproteinases (MT1-5MMPs), matrilysin (MMP-7), and metalloelastase (MMP- 12). The cDNAs of these proteinases have been cloned and are all well characterized. However, information about the roles of various metalloproteinases in vivo, as well as regulatory patterns of their genes, is very limited. Generally, these proteinases are either secreted or membrane-bound and are synthesized as pre-proenzymes. They have a well conserved Zn2+-binding catalytic site and can be inhibited by specific tissue inhibitors known as TIMPs (tissue inhibitors of metalloproteinases). Moreover, they are clearly distinguished from other metalloproteinases by a unique sequence motif, PRCG[V/N]PD, in the propeptide, and the zinc-binding motif, HEXGHX[L/M]G[L/M]XH, in the catalytic domain in which three histidines bind to the catalytic zinc atom. Most MMPs possess at
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least a C-ter domain similar to hemopexin and vitronectin that may help to bind the enzyme to the extracellular matrix101. The gene structures of matrix metalloproteinases have well studied and characterized, and it has been shown that a single exon encodes the region of the protein bearing the zinc ligand. The gene loci of human matrix metalloproteinases are summarized in Table 3. However, no association between MMPs and degenerative genetic diseases have so far been identified. On the other hand, the gene regulation as well as the 5'-flanking regions of some MMPs have been identified. For example, the promoter region of MMP-1 contains a TATA box, an activator protein-1 (AP-1) site, and a polyoma virus enhancer A3 (PEA3)-like element. The APsite in the collagenase promoter plays a crucial role in the transcriptional control of the gene. This site is essential for basal transcription, and contributes to the induction by phorbol esters, although other sites in the proximal promoter are also essential102.
Table 3. Localization of MMP genes on human chromosomes. Gene Name
Collagenases
Gelatinases
MMP No.
Chromosomal Localization
Interstitial collagenase (EC.3.4.24.7)
MMP- 1
11q21-q22
Neutrophil collagenase (EC.3.4.24.34)
MMP-8
11q21-q22
Collagenase 3
MMP-13
11q21-q22
Gelatinase A (EC. 3.4.24.24)
MMP-2
16q2 1
Gelatinase B (EC.3.4.24.35)
MMP-9
20q11.2q13.1
Stromelysin 1 (EC.3.4.24.17)
MMP-3
11q21-q22
Stromelysin 2 (EC.3.4.24.22)
MMP-10
11q21-q22
MT1-MMP
MMP-14
14q12.2
MT2-MMP
MMP-15
16q12.2
MT3-MMP
MMP-16
8q2 1
MT4-MMP
MMP-17
12P
MT5-MMP
MMP-24
20q11.2
Stromelysin 3
MMP-I 1
22q11.2
Metalloelastase
MMP-12
11q21-q22
Matrilysin
MMP-7
11q21-22
Stromelysins
Membrane-type MMPS
Others
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Studies of rat MMP-1 have also revealed that interleukin-1 does not induce APl-binding activity strongly, but increases MMP- 1 gene transcription through sequences in the distal promoter. It is clear that the regulation of collagenase gene expression by pro-inflammatory cytokines such as IL-1 requires both transcriptional and post transcriptional mechanisms103.
CONCLUDING REMARKS In recent years, the rapid expansion of nucleotide sequence data available in public data bases has facilitated the search for homology and the evolutionary relationships among the huge proteinase family, and this information has contributed greatly to our understanding of proteinases. Especially, the expressed sequence tags data base (dbest) has proven very useful for identifying novel proteinase genes. In fact, the in silico method has led to the discovery of several new members of the proteinase family. In addition, recent dramatic advances in functional genomics by genome projects may help to clarify proteinase structure and function. In order to study the physiological function of proteinases, precise information about the gene structures is essential. Although the physiological function of many proteinases remains to be elucidated, the study of proteinases by molecular biological approaches will provide clear insight into their basic biological mechanisms in vivo, and lead to further medical applications to the fight against degenerative diseases.
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AUTHOR INDEX Abraham, C.R., 101 Azuma, M., 85 Banik, N.L., 25, 199 Bartus, R.T., 75 Berg, M.J., 155 Bromme, D., 47 Chan, S.L., 117 Cong, J., 63 Cuzner, M.L., 5 Edwards, D.R., 241 Emench, D.F., 75 Eng, L.F., 199 Figueiredo-Pereira, M.E., 137 Forsyth, P.A., 241 Fukiage, C., 85 Goll, D.E., 63
Guncar, G., 227 Hogan, E.L., 199 Hook, V.Y.H., 269 Kos, J., 227 LaFleur, M.A., 241 Lampi, K.J., 85 Lee, H.-J., 283 Li, H., 63 Ma, H., 85 Marks, N., 155 Mattson, M.P., 117 Matzelle, D.C., 199 Moskowitz, M.A., 179 Petanceska, S., 47 Ray, S.K., 199 Rockwell, P., 137
Saido, T.C., 283 Schulz, J.B., 179 Shearer, T.R., 85 Shields, D.C., 25 Shih, M., 85 Slot, F., 101 Smith, M., 1 Suzuki, K., 283 Thompson, V.F., 63 Turk, B., 227 Turk, V., 227 Wang, K.K.W., 189 Wilford, G., 199 Yong, V.W., 241
SUBJECT INDEX Aging, cysteine proteases, 233 Alzheimer’s disease $E peptide production, 271 amyloid and presenilin proteases, 155 caspases, 122 clinical features, 270 cysteine proteases, 233 metalloendopeptidases, 101 proteases, 54 ubiquitin/proteasome pathway, 142 Amyelotrophic lateral sclerosis, 143 Amyloid precursor protein proteolysis, 271 secretory, pathway, 272 Amyloid proteases in Alzheimer’s disease, 155 Apoptosis cascade, 181 caspases, 122, 180 in spinal cord injury, 209 ubiquitin/proteasome pathway, 142 Calpains in Alzheimer’s samples, 35,37 in cataract formation, 92 cellular distribution, 64 in cerebral ischemia, 75,78 characterization, 204 in demyelinating diseases, 25, 38 cDNA sequences, 86-88 in EAE, 27 expression, 29, 50 expression, in spinal cord injury, 208 general properties, 76 hypothesis, of trauma, 202 inhibitors with cathepsin, 52 isoforms in eye, 85 lens specific, 95,97 in multiple sclerosis, 34 in muscular dystrophy, 67, 69 in neurodegeneration, 124 in Parkinson’s disease, 35, 37 protein sequences, 90, 91 regulation, 124 regulation, in CNS disorder, 214 role in neuromuscular disease, 63 in spinal cord injury, 206
substrate specificity, 96 substrates, 121 in synaptic plasticity, 125 system, in brain, 65 system, properties, 64 in traumatic brain injury, 213 Calpastatin in Alzheimer’s, multiple sclerosis, and Parkinson’s samples, 37 calpain complex, 77 properties, 64 Caspa ses activation, 120 in Alzheimer’s disease, 123 in apoptosis, 122 characteristics, 117 development of inhibitors, 184 in Huntington’s disease, 183 inhibition, and inflammation, 183 inhibition, in therapy, 183 in multiple sclerosis, 183 in neurodegeneration, 122 in Parkinson’s disease, 180 PS processing, 168 regulation, 117 in stroke, 182 substrates, 121 in synaptic plasticity, 127 trauma, 182,213 Cathepsins activation, 230 amino acid sequence, 228 biosynthesis, 230 expression, 50 families, 258 genes, 49 in ischemia, 56 in neurological disorders, 53, 233 phylogenesis, 50 synthetic inhibitors, 51 in tumors, 258 Chymase, 285. Cystatin in aging, 233 amyloid angiopathy, 235 families, 231 299
300 interaction with synthetic proteins, 231 in neurological disorders, 233 Cysteine proteases catalytic mechanism, 48 characteristics of genes, 286 distribution, 227 interaction with cystatins, 231 lysosomal, 227,232 mechanism of action, 229 in neurodegenerative diseases, 47, 117 properties, 272 protein inhibitors, 227 structure and localization, 48, 229 in synaptic degeneration, 117 Demyelination in animal models, 27 calcium-activated neutral protease, 25 calpain in, 25 cell-mediated, 14 cellular source of proteases, 15 mechanisms, 13 in peripheral nerve, 16 proteases in, 5 proteinase inhibitors, 17 and proteolytic enzymes, 1 in vitro, by proteases, 7 Encephalopathies, spongioform, 55 Experimental allergic encephalomyelitis calpain activity, 28 calpain expression, 29 Extracellular gliomas, 241 Eye, calpain isoforms, 85 Gene transcription, metalloproteases, 245 Genes aspartic protease, 288 calpain, 286 cathepsin, 287 cathepsin D, 288 endopeptidase, 289 endothelin-converting enzymes, 290 of mammalian proteases, 261 matrix metalloproteinases, 290 metalloproteinases, 289 mutant product, proteolysis in neurodegeneration, 269 neutral endopeptidase, 289 pepsin, 288 renin, 289 Gliomas extracellular matrix, 241 metalloprotease inhibitors, 25 1 metalloproteases in, 252 proteases and their inhibitors, 241 serine proteases in, 257
Subject Index Huntington’s disease caspases in, 183 clinical features, 274 Huntington fragments, 274 protein, cell biology, 269 Inflammation caspases in, 183 metalloendopeptidases, 111 ubiquitin/proteasome pathway, 141 Inhibitors of metalloproteases, 251 proteases, 191, 192 of proteases, for therapy, 212 synthetic, of metalloproteases, 237 therapy, for neurodegeneration, 278 Injury, proteases involved in, 200 Ischemia calpains, 75,78 cathepsins, 52 focal, 78 global, 80 Kallikrein, 285 Lysosomes, cysteine proteases, 227, 232 Metalloendopeptidases Alzheimer’s disease, 107 gene structure, 106 history, 101 hormonal, implication, 110 inflammation, 111 inhibitor, profile, 104 molecular weight, 103 in neurodegeneration, 101 pain and analgesia, 110 properties, 104 substrate specificity, 105 tissue distribution, 107 Metalloproteases family, 243 gene transcription, 245 inhibition, in gliomas, 234,240 inhibitors, 247 matrix, 242 multifunctional role, 249 synthetic inhibitors, 251 tissue inhibitors of, 247 zymogen activation, 249 Multiple sclerosis calpain activity, 25 caspase in, 183 Myelin basic proteins, 8 damage, 14 glycoproteins, 9 proteases, processing, 5, 10, 12 protein composition, 6
Subject Index Neurodegeneration calpains, 124 caspases, 122, 179 and cysteine proteases, 47, 117 metalloendopeptidase in, 101 protease classes, 179 protease inhibitor therapy, 189 proteolytic mechanisms, 275 Neurological diseases cathepsins, 53 cysteine protease inhibitors, 56 Neurological disorders protease inhibitor therapy, 189 ubiquitin proteasome pathway, 137 Neuromuscular diseases, role of calpain, 63 Oxidative stress, ubiquitidproteasome pathway, 140 Papain-like cysteine proteases, 47 Parkinsons’s disease caspases in, 183 clinical features, 276 genetic mutations, 276 ubiquitin/proteasome pathway, 145 Plasminogen activation syndrome, 256 activators, 285 in tumor biology, 257 Presenilin proteases in Alzheimer’s disease, 155 Prion diseases, ubiquitidproteasome pathway, 138 Processing enzymes, 156 Protease inhibitors in demyelination, 17 in gliomas, 254 of lysosomal proteases, 227 Proteases characteristics, 284 classes, in neurological diseases, 189 classification, 284 nhibitors, 190, 191 inhibitors, in therapy for neurological diseases, 189 Proteasomes degradation, 138 substrate recognition, 139 ubiquitin pathway in neurological disorders, 137 Proteolipid proteins, 9 Proteolytic enzymes in autoimmune diseases, 1 in demyelination, 5 Secretases, identification, 259
301 Serine characteristics, 270 genes, 270 in gliomas, 243 proteases, 284 Signal transduction notch signalling, 157 pathways, 120 Spinal cord injury apoptosis, 198 biochemical changes, 201 calpain activity, 209 morphological changes, 20 1 proteases, 121,201 protein loss in EAE, 30 role of calcium, 205 Stefin and epilepsy, 234 structure, 232 Stroke apoptosis in, 182 and caspase activity, 182 Synapse degeneration, and cysteine proteases, 117 Synuclein gene product, proteolytic processing, 277 mutations in Parkinson’s, 276 Therapy for CNS trauma, 199 by protease inhibitors, 189 Trauma caspase activity, 182 cell death in, 208 proteases in, 202 proteolytic mechanisms, 199 treatment of injury, 213 Trypsin, 285 Tumors and cathepsins, 53,234, 258 plasminogen activator system, 257 and proteases, 244 serine proteases, 257 Ubiquitin cleavage, 161 -like proteins, 132 pathway in ALS, 143 presenilinase, 164 presinilins, 164 proteasome pathway in neurological disorders, 137 protein inclusions, 138 secretases, 1 5 8 Ubiquitination, 137
302 Ubiquitin/proteasome pathway Alzheimer’s disease, 142 amyotrophic lateral sclerosis, 143 Angelman syndrome, 144 apoptosis, 142 aspartyl proteases, 152 inflammation, 141 in oxidative stress, 140 Parkinson’s disease, 145 prion diseases, 146 role in hereditary disorders, 142 SAG polyglutamine expansion diseases, 144 Wilson’s disease, 145 yeast Asp proteases, 160 Wilson’s disease ubiquitin/proteasome pathway, 146 Wolfgram protein, 9
Subject Index