Molecular Basis of Mitochondrial Pathology 0121525171, 9780121525170

Citation preview

Current Topics in Bioenergetics Molecular Basis of Mitochondrial Pathology Volume 17

Advisory Board BRITTON CHANCE LARS ERNSTER YOUSSEF HATEFI DAVID W. KROGMANN GOTTFRIED SCHATZ

Current Topics in Bioenergetics Molecular Basis of Mitochondrial Pathology Edited by C.

P.

LEE

Department of Biochemistry School of Medicine Wayne State University Detroit, Michigan

VOLUME 17

ACADEMIC PRESS

San Diego New York Boston London Sydney Tokyo Toronto

This book is printed on acid-free paper.

©

Copyright © 1994 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

Academic Press, Inc.

A Division of Harcourt Brace & Company 525 Β Street, Suite 1900, San Diego, California 92101-4495 United Kingdom Edition published by Academic Press Limited 24-28 Oval Road, London NW1 7DX International Standard Serial Number: 0070-2129 International Standard Book Number: 0-12-152517-1 PRINTED IN THE UNITED STATES OF AMERICA 94 95 96 97 98 99 BB 9 8 7 6

5

4

3 2 1

Contents Contributors

ix

Preface

xi

Role of Mitochondria in Degenerative Diseases and Aging CHRISTOPH RICHTER I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII.

Introduction The Mitochondrial Respiratory Chain

1 2

Mitochondrial DNA Reactive Oxygen Species in Mitochondria

3 4

Oxidative Damage to DNA

5

Mutation Mechanisms Mitochondrial Diseases: The Introduction Changes of Mitochondrial DNA in Degenerative Diseases Changes of Mitochondrial DNA during Aging Aging Theories Based on Mitochondrial DNA Alterations

7 7 8 10 12

Prevention and Therapy of Mitochondrial Degenerative Diseases and Aging

12

Conclusions and Future Studies References

14 15

Mitochondrial Myopathies: Biochemical Aspects SARA SHANSKE AND SALVATORE DIMAURO I. II. III. IV.

Introduction Mitochondrial Metabolism Mitochondrial Disorders Concluding Remarks References

21 24 26 50 51 ν

CONTENTS

vi

Mitochondrial Myopathies: Genetic Aspects SCOTT W. BALLINGER, JOHN M . SHOFFNER, AND DOUGLAS C . WALLACE I.

Introduction

59

Human Mitochondrial Genetics

68

mtDNA Mutations Associated with Mitochondrial Myopathy

72

IV.

Contributing Factors

89

V.

Future Implications References

92 92

II. III.

Mitochondrial Disease: Noninvasive Approaches D . J. TAYLOR AND G . K. RADDA I. II.

Introduction

99

Techniques

101

III.

Primary Mitochondrial Disease

105

IV.

Secondary Mitochondrial Disease

120

Conclusions References

122 123

V.

Mitochondrial Antigens HAROLD BAUM I. II. III. IV.

Introduction The Spectrum of Antimitochondrial Antibodies Primary Biliary Cirrhosis (PBC) Concluding Remarks References

127 128 135 164 166

Mitochondrial Injury by Ischemia and Reperfusion JOE M . MCCORD AND JULIO F. TIJRRENS I. II. III. IV.

Introduction General Aspects of Ischemia-Reperfusion Responses of the Mitochondrion to Ischemia-Reperfusion Mitochondrial "Stunning" and Reperfusion Injury

173 174 177 189

CONTENTS V.

Conclusion References

vii 191 192

Mitochondrial Energy Metabolism in Chronic Alcoholism JAN B . HOEK I. II. III. IV. V.

Introduction

197

Ethanol Metabolism and the Control of Mitochondrial Energy Supply

199

Membrane Actions of Ethanol in Relation to Mitochondrial Energy Conservation

222

Mitochondrial Protein Synthesis in Chronic Alcoholism

227

Conclusions References

232 236

Index

243

Contents of Previous Volumes

249

This page intentionally left blank

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

SCOTT W. BALLINGER (59), Center for Genetics and Molecular Medicine, Emory University School of Medicine, Atlanta, Georgia 30322

Faculty of Life Sciences, King's College University of London, London WC2R 2LS, United Kingdom

HAROLD BAUM ( 1 2 7 ) ,

London,

H. Houston Clinical Reseach Center for Muscular Dystrophy and Related Diseases, Department of Neurology, Columbia Presbyterian Medical Center, New York, New York 10032

SALVATORE DIMAURO ( 2 1 ) ,

Department of Pathology and Cell Biology, Jefferson University, Philadelphia, Pennsylvania 19107

JAN B . HOEK ( 1 9 7 ) ,

Thomas

Department of Medicine and Biochemistry, University of Colorado, Denver, Colorado 80262

JOE M . MCCORD ( 1 7 3 ) ,

MRC Magnetic Resonance Spectroscopy, University of Oxford, Headington, Oxford OX3 9DU, United Kingdom

GEORGE RADDA ( 9 3 ) ,

Laboratory of Biochemistry I, Swiss Federal Institute of Technology (ΕΤΗ), CH 8092 Zurich, Switzerland

CHRISTOPH RICHTER ( 1 ) ,

H. Houston Clinical Reseach Center for Muscular Dystrophy and Related Diseases, Department of Neurology, Columbia Presbyterian Medical Center, New York, New York 10032

SARA SHANSKE ( 2 1 ) ,

ix

CONTRIBUTORS

χ

JOHN M . SHOFFNER (59), Center for Genetics and Molecular Medicine, Emory University School of Medicine, Atlanta, Georgia 30322

MRC Magnetic Resonance Spectroscopy, University of Oxford, Headington, Oxford OX3 9DU, United Kingdom

DORIS TAYLOR ( 9 3 ) ,

Department of Medicine and Biochemistry, University of Colorado, Denver, Colorado 80262

JULIO F. TURRENS ( 1 7 3 ) ,

Center for Genetics and Molecular Medicine, Emory University School of Medicine, Atlanta, Georgia 30322

DOUGLAS WALLACE ( 5 9 ) ,

Preface Mitochondrial diseases occupy at present a highly active area of investigation in both molecular and cell biology, and in clinical physiology and medicine. By the end of 1992 the number of reports on mitochondrial diseases was approaching 1000 (Fig. 1). Research in this field has in recent years opened new insights, not only into the pathogenesis of such diseases, but also into the biological functions of mitochondria in general. In addition to the classical concept of mitochondria serving as the "powerhouse" of the cell, it is now evident that this organelle plays a crucial role in the genetic regulation of cellular energy metabolism, with important implications for 2+ various biological functions including electrolyte balance, C a homeostasis, cellular signal transduction, and antioxidant and immunologic defenses. There is growing evidence that a large number of pathological conditions, among them a number of myo- and neuropathies, certain degenerative and autoimmune diseases, and even the physiological aging and turnover of cells and tissues, may be related to mitochondrial functions. 1000

1960

1965

1970

1975

1980

1985

1990

Year FIG. 1. Data from 1959 to 1988 are from L. Ernster and C P . Lee (1990) in Bioenergetics: Biochemistry, Molecular Biology and Pathology (C. Kim and T. Ozawa, eds.), pp. 451-466. Plenum. xi

xii

PREFACE

Some of these developments are summarized in the chapters of this volume. I am indebted to the members of the Advisory Board who have provided invaluable advice and counsel, and to all the contributors for their cooperation in making the publication of this volume possible. Thanks are due to Dr. Lorraine Lica of Academic Press for her valuable editorial assistance during the preparation of this volume. C. P. Lee

CURRENT TOPICS IN BIOENERGETICS, VOLUME 17

Role of Mitochondrial DNA Modifications in Degenerative Diseases and Aging CHRISTOPH RICHTER

Laboratory of Biochemistry I Swiss Federal Institute of Technology CH-8092 Zürich, Switzerland I. II. III. IV. V. VI. VII. VIII.

IX. X. XI. XII.

(ΕΤΗ)

Introduction The Mitochondrial Respiratory Chain Mitochondrial DNA Reactive Oxygen Species in Mitochondria Oxidative Damage to DNA Mutation Mechanisms Mitochondrial Diseases: The Introduction Changes of Mitochondrial DNA in Degenerative Diseases A. syn- Mutations B. mit- Mutations C. p- Mutations D. Other Clinical Problems Related to Mitochondrial DNA Alterations Changes of Mitochondrial DNA during Aging Aging Theories Based on Mitochondrial DNA Alterations Prevention and Therapy of Mitochondrial Degenerative Diseases and Aging Conclusions and Future Studies References

I.

Introduction

During the last decade the importance of mitochondria as a major contributor to various types of diseases and to natural aging has become apparent. These diseases include myopathies and encephalomyopathies, heart diseases, late-onset diabetes, and other degenerative diseases that come with age, such as Parkinson's, Huntington's, and Alzheimer's diseases. Since 1988, point mutations, deletions, and large-scale rearrangements in l Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.

CHRISTOPH RICHTER

2

mitochondrial DNA (mtDNA) have been explicitly related to aging and several of the diseases, but genetic analyses show that nuclear factors can also contribute to them. The etiology of disease- and aging-related mitochondrial gene modifications is presently not clear. Reactive oxygen species (ROS) can damage DNA. Mitochondria are the foremost intracellular source of ROS, and mtDNA bases are heavily damaged by them. This is indicated by the high steady-state level of 8-hydroxydeoxyguanosine (80HdG), one of many oxidatively modified bases. 8 0 H d G is mutagenic, since it causes base mispairing and point mutations. mtDNA is also oxidatively fragmented to some extent. Conceivably, such fragmentation is the basis of deletions found in mtDNA. Future studies in this new frontier in acquired and inborn gene defects will address the relationship between ROS and mitochondrial dysfunction, the mechanism(s) leading to mutations in the mitochondrial genome, the role of nuclear gene products in the decline of mitochondrial functioning, the possibile application of gene therapy in mitochondria, and the improval of diagnosis at the molecular level.

II.

The Mitochondrial Respiratory Chain

In nucleated cells, mitochondria provide most of the ATP, the remainder being formed during glycolysis. Mitochondrial ATP is generated by oxidative phosphorylation in a process which uses molecular oxygen as the final electron acceptor. Oxidative phosphorylation provides most of the energy in brain, heart and skeletal muscle, liver, kidney, and pancreatic islets. It engages five protein complexes situated in the inner mitochondrial membrane. Four of them (complexes I through IV) catalyze the transport of electrons to molecular oxygen and thereby create an electrochemical proton motive force, the fifth (complex V) uses this force to form ATP from ADP and inorganic phosphate. Complex I (NADH dehydrogenase, consisting of over 30 polypeptides) oxidizes NADH. Complex II (succinate dehydrogenase, built from four polypeptides) receives electrons from succinate and subsequently donates them to ubiquinone to form ubiquinol. This small, lipid soluble, mobile compound then reduces complex III (ubiquinol:cytochrome c oxidoreductase, consisting of 10 polypeptides), from where the electrons flow via cytochrome c to complex IV (cytochrome oxidase, comprising 13 polypeptides). There, most of the molecular oxygen consumed by mitochondria during respiration is reduced with four electrons to water without the liberation of partially reduced oxygen species. The protons which are expelled during electron transport over com-

mtDNA MODIFICATIONS IN DISEASE AND AGING

3

plexes I through IV from the mitochondrial matrix into the intermembrane space flow back into the matrix via complex V (ATP synthase, formed by 12 polypeptides). The continuous operation of oxidative phosphorylation also requires a supply of ADP for complex V via an active adenine nucleotide translocator, which exchanges intramitochondrial ATP with extramitochondrial ADP.

III.

Mitochondrial DNA

There is convincing evidence that mitochondria originate from symbiotic bacteria (Margolis, 1981). This explains not only why these cell organelles contain their own DNA but also some of the many peculiar properties of mtDNA, such as cell cycle-independent replication or the sensitivity to inhibitors of prokaryotic DNA metabolism. During evolution, genes were transfered from mitochondria to the nucleus, and the size and coding capacity of mtDNA were thereby reduced (Gray, 1989; De Giorgi and Saccone, 1989). Furthermore, gene transfer from mitochondria to the nucleus during the lifetime of some organisms has been detected (Osiewacz, 1990; Thorsness and Fox, 1990). Mammalian mtDNA has very little redundancy and a high information density. The 16,596 base-pair human mtDNA codes for two ribosomal RNAs, 22 transfer RNAs, and 13 peptides which are part of the five multisubunit enzymes of the respiratory chain and the oxidative phosphorylation machinery in the inner mitochondrial membrane (Capaldi, 1988). The peptides coded for are seven subunits of complex I (coded for by genes ND1, ND2, ND3, ND4L, ND4, ND5, and ND6), one subunit of complex III (coded for by gene cytochrome b), three subunits of complex IV (coded for by genes COI, COII, and COIII), and two subunits of complex V (coded for by genes ATPase 6 and 8). The remaining several hundred mitochondrial polypeptides are coded for in the nucleus and must be imported into the organelle from the cytosol (Attardi and Schatz, 1988). The normal genetic code is used in unaltered form in mitochondria of green plants, but nonplant mitochondria have a code which includes changes from the universal code (Jukes and Osawa, 1990). Mammalian mitochondria are largely, but not entirely, maternally inherited: In mice, paternally inherited mtDNA shows up at a frequency of 4 10~ , relative to the maternal contribution (Gyllensten et al, 1991). The number of mitochondria present in a cell depends principally on the cell type, but also on the energy demand of a given cell. Between one and several hundred mitochondria are present in nucleated cells, with each mitochondrion containing several DNA molecules.

CHRISTOPH RICHTER

4

The organelles proliferate independent of the cell cycle by bacteria-like growth and division. In adult rats, their DNA has a half-life between several days and a month, depending on the organ (Gross et ai, 1969). mtDNA is a closed-circular molecule, formed by two complementary strands, heavy (H) and light (L). The two strands contain separate origins of replication (Clayton, 1982). A special region is the displacement loop (D-loop), which is about 1,000 base-pairs long and contains the origin of replication of the Η-strand and the promoters for H- and L-strand transcription. It is also a region of intracellular intergenomic communication, where nuclear-encoded proteins and riboproteins interact with mtDNA and regulate its replication and transcription (Clayton, 1991). In mammals, mtDNA mutates 5 to 10 times faster than nuclear DNA (Brown et al., 1979). In sharks, which have a metabolic rate about 5 to 10 times lower than mammals of similar body weight, the rate of mtDNA evolution is about 7 to 8 times lower than in primates or ungulates (Martin et ai, 1992). During cell division, mitochondria are randomly distributed to the daughter cells. This stochastic separation is called replicative segregation. A cell can contain a uniform population of wild-type or mutated mtDNA (homoplasmy), or a mixture of various mtDNA types (heteroplasmy). As cells divide, the relative proportions of heteroplasmic mtDNAs change randomly ("drift") and may thereby even produce homoplasmic cells. The accumulation of mutated DNA, together with segregation during cell division, can result in tissue bioenergy mosaics. Phenotypic expression of variant mtDNA depends on the extent of segregation of heteroplasmic mtDNA. In a heteroplasmic cell the wild-type mtDNA can apparently make up for some of the weaknesses confered by the mutants, because the cell is compromised only when a certain threshold level of mutations is reached. IV.

Reactive Oxygen Species in Mitochondria

Reactive oxygen species (ROS) such as superoxide radical, hydrogen peroxide, hydroxy 1 radical, and singlet oxygen are products of normal metabolism (Chance et al., 1979; Cadenas, 1989). Mitochondria consume about 90% of the body's oxygen and are a particularly rich source of ROS, since about 1-2% of oxygen metabolized by mitochondria is converted to superoxide by several constitutive sites in the respiratory chain and matrix (Chance et al., 1979). Calculations show that one rat liver mitochondrion 7 produces during normal metabolism about 3 x 10 superoxide radicals per day (Richter, 1988). The steady-state concentrations of mitochondrial su-

mtDNA MODIFICATIONS IN DISEASE AND AGING

5

peroxide and hydrogen peroxide, the predominant precursors of the highly reactive hydroxyl radical, are estimated to be in the picomolar and nanomolar ranges, respectively (Forman and Boveris, 1982). In addition to normal ROS production in mitochondria, reactive oxygen is formed in large amounts in the presence of certain compounds (e.g., so-called "redox cyclers" such as alloxan) and during some pathological states. Thus, alloxan (Frei et al, 1985), menadione (Frei et al, 1986), rotenone and methylphenylpyridinium (MPP+) (Traber, 1991; Cleeter et 2 al, 1992), tetrachlorodibenzo-p-dioxin (Nohl et al, 1989), elevated Ca + (Chacon and Acosta, 1991), or tumor necrosis factor α (Schulze-Osthoff et al, 1992; Hennet et al, 1993a,b) stimulate ROS production by mitochondria, as does ischemia/reperfusion (Turrens et al, 1991; see also Chapter 6). Mitochondrial superoxide and hydrogen peroxide are metabolized by the Mn-containing superoxide dismutase and the Se-containing glutathione peroxidase, respectively (Chance etal, 1979). Furthermore, ROS are scavenged by the vitamin antioxidants (Niki, 1987; Thomas et al, 1989), glutathione (Sies, 1989), and ubiquinol-10 (Frei et al, 1990). Despite these efficient antioxidant defense systems, oxidative damage to mitochondria is notoriously abundant (Miquel et al, 1977). Mitochondria of aged animals produce more ROS than those of young animals both in insects (Sohal, 1991) and in mammals (Nohl et al, 1978; Spoerri, 1984; Sawada and Carlson, 1987; Sohal et al, 1990), species in which maximal life span potential relates inversely to the rate of oxygen consumption and positively to antioxidant capacity (Cutler, 1984; Sohal and Allen, 1985). Mitochondria from older rats show higher levels of lipid peroxides and losses of polyunsaturated fatty acids (Nohl and Hegner, 1978), indicating enhanced oxidative stress at older age. V.

Oxidative Damage to DNA

There is indirect evidence that oxidative DNA damage may be a major cause of aging and age-associated degenerative diseases (Cutler, 1984; Adelman et al, 1988; Ames, 1989). This evidence includes the high level of oxidative damage and its accumulation with age, the correlation between oxidative DNA damage and maximal life span potential, and the increased oxidative damage and premature aging found in people with Down's syndrome. ROS can react with DNA either at the sugar-phosphate backbone or at a base (Imlay and Linn, 1988; Halliwell and Aruoma, 1991). The former reaction leads to strand fragmentation, the latter results in a chemically

6

CHRISTOPH RICHTER

modified base. ROS are, therefore, potent intracellular mutagens ( Joenje, 1989). Numerous base modifications are detectable when ROS react with DNA (von Sonntag, 1987; Steenken, 1989). The most studied oxidized base is 8 0 H d G , formed when DNA is attacked by hydroxyl radicals or singlet oxygen. 8 0 H d G can be measured in the femtomole range by several techniques (Halliwell and Aruoma, 1991). In addition to being a useful marker for oxidative DNA damage, the formation of 8 0 H d G in DNA is also mutagenic (Kuchino et al, 1987; Wood et al, 1990; McBride et al, 1991; Shibutani et al, 1991; Cheng et al, 1992). The steady-state level of 8 0 H d G in nuclear DNA of some organs increases with advancing age (Fraga et al, 1990). Escherichia coli mutants defective in repairing single-strand breaks are vulnerable to being killed by hydrogen peroxide (Imlay and Linn, 1986), indicating that unrepaired oxidative damage to DNA can be lethal. This, and the fact that nuclear enzymes which remove oxidatively damaged bases from DNA are common (Saul et al, 1987), suggests that oxidative DNA damage is an important biological problem. mtDNA does not bind histones, and is at least transiently attached to the inner mitochondrial membrane (Shearman and Kalf, 1977), where large amounts of ROS are produced. Therefore, mtDNA is particularly susceptible to oxidative damage. Indeed, the steady-state level of oxidized bases in mtDNA is about 16 times higher than that in nuclear DNA (Richter et al, 1988; Hruszkewycz and Bergtold, 1990; Chung et al, 1992). In addition, ROS generate strand breaks in mtDNA (reviewed by Richter, 1988). For example, adriamycin and bleomycin introduce nicks in mtDNA in an oxygen-dependent manner in vivo, in cell cultures, or in isolated mitochondria. The "redox cycler" alloxan causes massive DNA strand fragmentation in isolated mitochondria (Meier, 1991). mtDNA is also far more threatened than nuclear DNA by alkylating agents and polycyclic aromatic hydrocarbons (Wunderlich et al, 1970; Backer and Weinstein, 1980). In mitochondria, DNA repair is less efficient than in the nucleus (reviewed by Richter, 1988). The mammalian organelles do not have significant recombinational repair but may excise damaged bases, since they contain uracil DNA glycosylases, AP and UV endonucleases (Tomkinson et al, 1988, 1990), and may repair alkylated DNA bases (Myers et al, 1988; Satoh et al, 1988). The presence of repair activities in mitochondria suggests that DNA repair does occur in the organelles. However, the enzymes may also, or additionally, initiate the degradation of damaged mtDNA in order to ensure the survival of only the population of undamaged mitochondrial genomes (Tomkinson et al, 1990).

mtDNA MODIFICATIONS IN DISEASE AND AGING V.

7

Mutation Mechanisms

As outlined above, ROS cause base modifications and strand breaks in mtDNA. It is very likely, yet presently unproven, that oxidative damage to mtDNA is of great importance for the accumulation of mtDNA alterations in the etiology of degenerative diseases and aging (Wallace, 1992a). The mechanisms by which deletions and duplications are formed are not clear (Lestienne, 1992; Zeviani and Antozzi, 1992). Deletion boundaries are often flanked by direct repeats that vary in length from 5 to 13 base pairs, but there are also deletions that lack detectable repeats. This may be due to different mechanisms by which deletions are formed, such as intramolecular recombination at the direct repeats or slipped mispairing during replication. It is also not known how most offsprings can start out with a "clean" set of mtDNA. Perhaps germ cells with mutated mitochondrial genomes are not competent in gametogenesis or fertilization (Linnane et al., 1992).

VII.

Mitochondrial Diseases: The Introduction

The concept of mitochondrial diseases was introduced by Ernster, Luft, and colleagues (Luft et al., 1962) who described a woman with severe hypermetabolism caused by loose coupling of muscle mitochondria. Ultrastructural studies of muscle (Shy and Gonatas, 1964; Shy et al., 1966) later linked myopathies to abnormal proliferation of mitochondria in affected fibers. In the early 1970s, biochemical investigations of mitochondria isolated from muscle biopsies and postmortem tissues were started (Spiro et al., 1970; French et al, 1972; DiMauro et al., 1973). These investigations helped to identify the sites of the defects in mitochondrial metabolism. Five broad categories of mitochondrial diseases were found (reviewed by DiMauro et al., 1989), based on defects of mitochondrial substrate transport and utilization, coupling, the Krebs cycle, and the respiratory chain. Finally, the 1980s saw the advent of molecular biology and genetics in the area of mitochondrial diseases. Its basis was the elucidation of the complete sequence of the human mtDNA (Anderson et al., 1981). Today, mitochondrial genetics is considered a paradigm for aging and degenerative diseases (Wallace, 1992a), but there are also disorders that involve mitochondria yet they, due to their unknown etiology and mode of inheritance, cannot be currently classified genetically (e.g., Alper's, Leigh's, and Luffs disease; lethal infantile cardiomyopathy; mitochondrial myopathy) (for review, see Shoffner and Wallace, 1990).

8

CHRISTOPH RICHTER VIII.

Changes of Mitochondrial DNA in Degenerative Diseases

In 1988 a number of papers appeared that related alterations of mtDNA to human diseases. One reported (Wallace et al., 1988) that the appearance of Leber's hereditary optic neuropathy (LHON) correlates with a single base alteration in mtDNA. Sequencing the mtDNA of a patient suffering from LHON revealed one missense mutation which resulted in the conversion of the highly conserved 340th amino acid in the ND4 subunit of complex I from an arginine to a histidine. At the same time several other papers (Holt et al, 1988a,b; Lestienne and Ponsot, 1988; Ozawa et al., 1988; Rotig et al, 1988; Zeviani et al, 1988) showed that muscle mtDNA of patients with encephalomyopathies contained deletions. Since then, a tremendous amount of information has been published which strongly suggests that alterations of the mitochondrial genome are causally related to degenerative diseases. Most of the known mutations affect organs with high oxygen consumption and mitochondrial energy supply such as brain, heart, and skeletal muscle. The associated diseases are therefore sometimes called "energy diseases." The affected tissues contain highly differentiated, postmitotic cells. Since these cells cannot be replaced efficiently, mutated mtDNA molecules accumulate in these organs, which may be the explanation for the often observed threshold phenomenon or the progression of the disease. Several excellent books and reviews covering this topic have recently appeared (Lestienne, 1989, 1992; DiMauro etal, 1989; Shoffner and Wallace, 1990; Grossman, 1990; Kadenbach et al, 1991; Sato and DiMauro, 1991; Müller-Höcker, 1992; Tyler, 1992; Wallace, 1989a,b; Wallace, 1992a,b; Zeviani and Antozzi, 1992). They give access to most of the original publications of this rapidly growing field. Two classes of clinical syndromes associated with alterations of human mtDNA have been clearly identified. The first comprises myopathies and encephalomyopathies and is characterized by the presence of ragged-red fibers (RRF). These fibers, detected with Gomori trichrome staining, contain peripheral and intermyofibrillar accumulations of abnormal mitochondria and are histological hallmarks of the disorders. The following syndromes belong to this group: myoclonic epilepsy with ragged-red fibers (MERF), mitochondrial encephalomyopathy with lactic acidosis and strokelike episodes (MELAS), Kearn-Sayre syndrome (KSS), chronic progressive external ophthalmoplegia (CPEO), and maternally inherited myopathy and cardiomyopathy (MMC). The second class is "pure" encephalopathies (no gross morphological abnormalities in muscle). It includes

mtDNA MODIFICATIONS IN DISEASE AND AGING

9

LHON and neurogenic ataxia, neurogenic muscle weakness, and retinitis pigmentosa (NARP). The basis of a genetic classification of the mtDNA mutations can be molecular lesions (point mutations, deletions, insertions), the mode of inheritance, and the extent of heteroplasmy. In the case of molecular lesions it is helpful to use yeast genetics as a model. Thus, there are point mutations affecting mitochondrial protein-encoding genes (corresponding to yeast mit- mutations), point mutations affecting mitochondrial tRNA genes (corresponding to yeast syn- mutations), and large-scale deletions (corresponding to yeast p- mutations). Encephalopathies are frequently associated with mit- mutations, encephalomyopathies with syn- and p- mutations. Whereas point mutations are often maternally inherited and homoplasmic, large-scale rearrangements are often sporadic, heteroplasmic are inherited as a mendelian trait, indicating that they arose after egg fertilization. In the following paragraphs examples of well-characterized clinical syndromes are described.

A.

SYN- MUTATIONS

The MERF syndrome comprises progressive myoclonic epilepsy, mitochondrial myopathy with RRF, and slowly progressive dementia, MELAS is characterized by strokelike episodes and a mitochondrial myopathy with RRF. MERF is caused by a heteroplasmic mutation at nucleotide (NT) L s 8344 in the t R N A y gene. It is maternally inherited. The clinical symptoms are proportional to the accompanying defects in complexes I and IV, and their severity depends on the percentage of mutant DNA and on the age of the affected individual. Also in MELAS (mutation at NT 3243 in the L eu L eu tRNA gene) and in MMC (mutation at NT 3260 in the t R N A gene) variable phenotypes associated with heteroplasmy have been observed.

B.

MIT- MUTATIONS

LHON is a maternally inherited blindness in which central vision is lost but peripheral vision retained. Individuals affected by LHON are generally symptom free during childhood, but lose their vision, on average, at about age 30. Four mtDNA mutations (at NTs 11778, 3460, 4160, 15,257) are sufficient in themselves to cause vision loss. Five other mutations (NTs 4216, 4917, 5244, 13,708) contribute in combination with other LHON

CHRISTOPH RICHTER

10

mutations to the disease. Genetic studies with LHON have shown a clear correlation between the frequency and sites of the mutations and the probability that symptoms will appear. The same statement can be made for NARP (missense mutation at NT 8993).

C.

p- MUTATIONS

These insertion-deletion mutations cause KSS, CEOP, and Pearson's syndrome, and are generally spontaneous, i.e., probably somatic in origin. The deletions are very heterogeneous, but never cover the origins of replication.

D.

OTHER CLINICAL PROBLEMS RELATED TO MITOCHONDRIAL DNA

ALTERATIONS

In addition to the above-mentioned syndromes several other diseases or pathological states may be related to mtDNA alterations. They comprise ischemic heart disease and late-onset diabetes mellitus, and Alzheimer's, Huntington's, and possibly Parkinson's disease. Thus, chronic cardiac ischemia has been associated with a 5-kb deletion in mtDNA and a compensatory induction of some mitochondrial genes (Corral-Debrinski et α/., 1991). Very recently late-onset diabetes was in part attributed to mtDNA mutations (Ballinger et al., 1992). The affected individuals carry a very large (10.4 kb) mtDNA deletion which spans the origin of replication of the light strand. Patients suffering from Alzheimer's disease have in their platelet mitochondria defective in complex IV (Parker et al., 1990b), but whether this is due to mtDNA mutations is not clear. The same is true for Huntington's disease where complex IV and complex I defects have been found in the patients' basal ganglia and platelet mitochondria, respectively (Brennan et al., 1985; Parker et al., 1990a). (For a discussion of Parkinson's disease related to mtDNA mutations see below.)

IX.

Changes of Mitochondrial DNA during Aging

Mitochondrial gene alterations could also make significant contributions to the aging process. Results obtained with various experimental approaches indicate a positive correlation between natural or pathological aging and mtDNA modifications. Early electron microscopy studies (Piko and Matsumoto, 1977; Bulpitt

mtDNA MODIFICATIONS IN DISEASE AND AGING

11

and Piko, 1984; Piko et al., 1988) indicated age-associated deletions/ additions in mtDNA of rodent tissue. Subsequently, molecular biological techniques allowed a more detailed investigation of mtDNA alterations. According to Ozawa et al. (1990a) three types of mtDNA mutations can be distinguished: homoplasmy, where a base transition has occurred, and all mtDNA molecules are alike; heteroplasmy, in which normal mtDNA and one type of deleted mtDNA are present, whereby the population of the deleted type is large enough to be detected by rather insensitive Southern blot analysis; and pleioplasmy, where mutated mtDNAs with various deletions coexist with normal-size mtDNA, and the deleted species can be detected only with the highly sensitive polymerase chain reaction (PCR). Ozawa and colleagues proposed that accumulation of mtDNA mutations and subsequent cytoplasmic segregation of these mutations during the lifetime of an organism may be important for the aging process (Linnane et al., 1989; Ozawa étal, 1991). Recently, several publications in support of the above proposal appeared. Linnane et al. (1990) documented by PCR an age-related 5-kb deletion (between nucleotide positions 8,470 and 13,459) in the human mitochondrial genome of a wide range of tissues. Cortopassi and Arnheim (1990) detected by PCR age-dependent mtDNA deletions in normal heart muscle and brain from adult human individuals, as did Yen et al. (1991) in human liver. Ozawa's group reported age-dependent mtDNA deletions in human skeletal muscle (3,610 base pairs, nucleotide positions 1,837-5,447) (Katayama et al, 1991), and in human heart muscle (7,436 base pairs, nucleotide position 8,649-16,084) (Sugiyama et al, 1991), and suggested deletions in human heart mtDNA to contribute to presbycardia (Hattori et al., 1991). Early reports of Ozawa's group on the occurrence of Parkinson's disease-specific mtDNA deletions in brain (Ikebe et al., 1990; Ozawa et al., 1990b) could not be confirmed by others (Schapira et al., 1990; Mann et al., 1992). According to their findings, these deletions accumulate to a similar extent also in the brains of healthy old subjects. Also at the RNA level age-dependent changes are detectable. When 12S rRNA and the mRNA coding for subunit II of cytochrome oxidase were quantitatively determined in tissues of adult and aging rats (Gadaleta et al., 1990), levels of both RNA species were lower in senescent brain and heart but not in liver. Treatment with acetyl-L-carnitine restored the levels to that of adult tissues. Recent biochemical and biophysical studies also document changes of mtDNA during aging. Ozawa's laboratory reported an age-associated increase in the level of 8 0 H d G in mtDNA of human diaphragm (Hayakawa et al., 1991). A novel type of modified DNA components termed I com3 2 pounds was discovered by a P postlabeling assay for DNA adducts and

CHRISTOPH RICHTER

12

shown to increase with age in rat liver mtDNA (Gupta et al., 1990). Furthermore, liver mtDNA of old rats shows a different buoyant density profile from that of young animals, probably due to permanent covalent attachment of proteins to it (Asano et al, 1991). Finally, mitochondrial enzyme levels may change with age. Histochemical analyses in human heart, diaphragm, and limb muscle revealed an age-associated increase in the number of cells lacking complex IV (Müller-Höcker, 1989, 1990). This is opposed by the report of Byrne et al. (1991) that during aging there is no decrease in the content of human complexes I, III, and IV of the respiratory chain.

X.

Aging Theories Based on Mitochondrial DNA Alterations

Harman (1956) proposed that free radicals might play a role in aging through crosslinking reactions, and later suggested that one of the possible sites of free radical attack is mtDNA of all cell types (Harman, 1972, 1983). A slightly different proposal was made by Miquel and collègues (Miquel et al., 1980; Fleming et al., 1982; Miquel, 1991) who argued that aging results from changes of the mitochondrial genome of differentiated cells. Based on the findings that mtDNA is fragmented by ROS, Richter (1988) suggested that a time-dependent accumulation of mtDNA fragments in nuclear DNA occurs, a process which would progressively change the nuclear DNA information content and thereby cause aging. Unlike other proposals, this hypothesis is explicit without the need to explain that most mitochondria isolated from old individuals are quite "young" (normal), and is in keeping with the finding that gene transfer from mitochondria to the nucleus naturally occurs in eukaryotes (Osiewacz, 1990), is rapid, and is essentially a "one-way traffic" (Thorsness and Fox, 1990). Subsequently, others (Linnane et al, 1989; Trounce et al, 1989) suggested that bioenergetically defective cells are a key factor in the aging process, and that the generation of free radicals in mitochondria could chronically injure mtDNA. According to Kadenbach and Müller-Höcker (1990) the continuous accumulation of respiration-deficient cells during life, mainly in human heart, limits the lifespan of each individual.

XI.

Prevention and Therapy of Mitochondrial Degenerative Diseases and Aging

There are two ways to experimentally extend life expectancy in mammals: caloric restriction (Masoro, 1990) and treatment with deprenyl

mtDNA MODIFICATIONS IN DISEASE AND AGING

13

(Knoll, 1988), an inhibitor of mitochondrial monoamine oxidase B. Caloric restriction also retards most age-associated physiological changes and diseases. In the context of this review it is interesting to note that (i) caloric restriction lowers the steady-state level of DNA oxidation as shown by 8 0 H d G analysis both in the nucleus and in mitochondria (Chung et al., 1992), and (ii) monoamine oxidase Β reduces molecular oxygen to hydrogen peroxide. This is consistent with the idea that ROS are major contributors to the aging process. Attempts at metabolic therapies for diseases related to mitochondrial dysfunction have been made, but they are isolated efforts, and their efficacy was not confirmed in controlled studies (for a review see Shoffner and Wallace, 1990). Coenzyme Q 1 0 (CoQ), an electron carrier in the inner mitochondrial membrane, may stabilize the respiratory chain components and act as an antioxidant. Protection of peroxidation-sensitive cardiolipin may be particularly important in preserving mitochondrial functioning since this lipid facilitates the interaction between cytochrome c and cytochrome oxidase (Vik et al., 1981). CoQ had beneficial effects in KS/CEOP syndromes and in MELAS. Succinate, an electron donor to the mitochondrial respiratory chain, decreased strokelike episodes in a MELAS patient. Vitamin K x (phylloquinone) and vitamin K 3 (menadione) can transfer electrons to cytochrome c by an electron bypass from CoQ. Also, vitamin C alone or together with menadione can reduce cytochrome c. A combination of the latter two reductants improved the bioenergetic capacity of skeletal muscle with a severe defect in complex III of the respiratory chain, as shown by in 31 vivo P-NMR. Phylloquinone improved retinal cone function in CEOP, and thiamine, a cofactor of pyruvate dehydrogenase, improved plasma lactate and pyruvate levels in a KS/CEOP patient. Medication with riboflavin, a precursor of flavin adenine nucleotides, raised the exercise capacity in a patient with complex I dysfunction. The RNA-stabilizing effect of acetyl-L-carnitine is mentioned above. The redox compounds mentioned in the preceeding paragraph are generally thought to act in a bypass reaction as electron donors to the respiratory chain, and thereby maintain its function at least partly, or as oxygen radical scavengers. A principally different role of redox compounds as therapeutic agents was proposed by Linnane, Nagley, and co-workers (Linnane et al., 1992). They argue that cells, particularly those with lower bioenergetic demand, survive despite serious defects in oxidative phosphorylation as long as their glycolytic capacity is maintained. In this scenario redox compounds are proposed to act as "redox sinks" and maintain levels of cytosolic NAD+ adequate for the operation of glycolysis. Indeed, cells lacking mtDNA (p°) can survive, provided pyruvate (an electron acceptor from NADH) and uridine (necessary for maintenance of nucleic

14

CHRISTOPH RICHTER

acid synthesis) are present in the growth medium (Desjardins et al., 1985; King and Attardi, 1989). Also, anaerobically grown cells are viable as long as excess pyruvate is available (Vaillant et al., 1991). Sustained growth and viability of p° human cells can even be achieved in the absence of pyruvate when the extracellular electron acceptor ferricyanide is present. Growth restoration by ferricyanide, and also that reported for CoQ, may be mediated by a NADH-linked plasma membrane oxidoreductase (Linnane et al., 1992).

XII. Conclusions and Future Studies The decline in oxidative phosphorylation capacity in degenerative diseases and during aging is related to changes in the mtDNA. It is very likely, albeit not experimentally demonstrated, that the accumulation of modifications in mtDNA is caused to a large extent by oxidation. 8 0 H d G , one out of many oxidatively generated modified bases, is formed in DNA by ROS in vitro, is present in high amounts in mtDNA, and is an established mutagen. ROS also cause strand breaks in mtDNA in vitro. It is, therefore, reasonable to assume that ROS may be the agents responsible for many of the observed base modifications and fragmentation of mtDNA in vivo. Experimental tests of this assumption are possible. For example, it will be of interest to study mtDNA of animals whose Mn-, Se-, and antioxidant vitamin states are altered. Also, deletions in mtDNA should be investigated in more detail with respect to their size and number and by which mechanism they are formed. Are there indeed hot spots for deletions, as indicated by the (few mtDNA domains analyzed in) PCR studies? Do ROS break mtDNA in certain positions preferentially, possibly due to topological constraints or its attachment at ROS-generating sites in the inner mitochondrial membrane? The lessons learned from basic research and therapeutic approaches with redox active compounds may also be applied to nutritional aspects of disease prevention. A diet well balanced in vitamins and antioxidants, and a reduced caloric intake, should enable humans to reduce the risk of cardiovascular and other degenerative diseases and preserve organ function at older age (Ames, 1983, 1989; Adelman etal., 1988). Another unexplored avenue of considerable interest is the role of nuclear gene products in the decline of mitochondrial functions. It is clear that nuclear DNA mutations contribute to some degenerative diseases. For example, the point mutation in mtDNA in Leber's disease is not sufficient to cause the disease because the mutation is also present in nonaffected individuals (see Lestienne, 1992). Do nuclear mutations interfere directly

mtDNA MODIFICATIONS IN DISEASE AND AGING

15

with mitochondrial energy output, or do they affect the mitochondrial genetic makeup? Molecular biological techniques should also be used more extensively. Since we are faced with some clearly defined mitochondrial diseases, is it possible to apply gene therapy to positively affect these diseases (see Lander and Lodish, 1990)? Finally, an understanding of the role of mtDNA mutations in diseases suggests an improved diagnosis at the molecular level. Both point mutations (Wallace et al., 1990; Hammans et al., 1991) and deletions (Wallace et al., 1990; Poulton et al., 1991) in mtDNA can be analyzed in DNA taken from blood samples. Antenatal diagnosis, however, may not be widely applicable because many mutations occur spontaneously and are often necessary but not sufficient to cause the disease. Nevertheless, it is clear that once a precise understanding of mtDNA mutations is achieved, the application of molecular biological techniques for the analysis and therapy of mitochondrial diseases will be a major contribution to modern medicine.

ACKNOWLEDGMENTS The work done in the author's laboratory was supported by grants from the Schweizerische Nationalfonds, the Schweizerische Krebsliga, and the Krebsliga of the Kanton Zürich.

REFERENCES Adelman, R., Saul, R. L., and Ames, Β. N. (1988). Proc. Natl. Acad. Sei. U.S.A. 85, 27062708. Ames, Β. N. (1983). Science 221, 1256-1264. Ames, Β. N. (1989). Free Radical Res. Commun. 7, 121-128. Anderson, S., Bankier, A. T., Barreil, B. G., DeBruijn, M. H. L., Coulson, A. R., Drouin, J., Eperson, I. C , Nierlich, D. P., Roe, Β. Α., Sariger, F., Schreier, P. Η., Smith, Α. J. Η., Staden, R., and Young, I. G. (1981). Nature (London) 290, 457-465. Asano, K., Amagese, S., Matsuura, E. T., and Yamagishi, H. (1991). Mech. Ageing Dev. 60, 275-284. Attardi, G., and Schatz, G. (1988). Annu. Rev. Cell Biol. 4, 289-333. Backer, J. M., and Weinstein, I. B. (1980). Science 209, 297-299. Baliinger, S. W., Shoffner, J. M., Hedaya, Ε. V., Trounce, I., Polak, Μ. Α., Koontz, D. Α., and Wallace, D. C. (1992). Nat. Genet. 1, 11-15. Brennan, W. Α., Jr., Bird, E. D., and Aprille, J. R. (1985). J. Neurochem. 44, 1948-1950. Brown, W. M., George, M. J. R., and Wilson, A. C. (1979). Proc. Natl. Acad. Sei. U.S.A. 76, 1967-1971. Bulpitt, K. J., and Piko, L. (1984). Brain Res. 300, 41-48. Byrne, E., Trounce, I., and Dennet, X. (1991). Mech. Ageing Dev. 60, 295-302. Cadenas, Ε. (1989). Annu. Rev. Biochem. 58, 79-110.

16

CHRISTOPH RICHTER

Capaldi, R. (1988). Trends Biochem. Sei. 13, 144-148. Chacon, Ε., and Acosta, D. (1991). Toxicol Appl. Pharmacol 107, 117-128. Chance, B., Sies, H., and Boveris, A. (1979). Physiol Rev. 59, 527-605. Cheng, K. C , Cahill, D. S., Kasai, H., Nishimura, S., and Loeb, L. (1992). /. Biol Chem. 267, 166-172. Chung, Μ. H., Kasai, H., Nishimura, S., and Yu, B. P. (1992). Free Radical Biol. Med. 12, 523-525. Clayton. D. A. (1982). Cell (Cambridge, Mass.) 28, 693-705. Clayton, D. A. (1991). Trends Biochem. Sei. 16, 107-111. Cleeter, M. W. J., Cooper, J. M., and Schapira, Α. Η. V. (1992). /. Neurochem. 58, 786-789. Corral-Debrinski, M., Stepien, G., Shoffner, J. M., Lott, M. T., Kanter, Κ., and Wallace, D. C. (1991). JAMA, J. Am. Med. Assoc. 266, 1812-1816. Cortopassi, G. Α., and Arnheim, N. (1990). Nucleic Acids Res. 18, 6927-6933. Cutler, R. G. (1984). In "Free Radicals in Biology" (W. A. Pryor, ed.), Vol. 4, pp. 371-428. Academic Press, Orlando, FL. De Giorgi, C , and Saccone, C. (1989). Cell Biophys. 14, 67-78. Desjardins, P., Frost, E., and Morais, R. (1985). Mol. Cell. Biol. 5, 1163-1169. DiMauro, S., Schotland, D. L., Bonilla, E., Lee, C. P., Gambetti, P. L., and Rowland, L. P. (1973). Arch. Neurol. (Chicago) 29, 170-179. DiMauro, S., Lombes, Α., Nakase, H., Zeviani, M., Rizzuto, R., Moraes, C. T., and Schon, E. A. (1989). In "Molecular Basis of Membrane-Associated Diseases" (A. Azzi, Z. Drahota, and S. Papa, eds.), pp. 157-166. Springer-Verlag, Berlin. Fleming, J. E., Miquel, J., Cottrell, S. F., Yengoyan, L. S., and Economos, A. C. (1982). Gerontology 28, 44-53. Forman, H. J., and Boveris, A. (1982). In "Free Radicals in Biology" (W.A. Pryor, ed.), Vol. 5, pp. 65-90. Academic Press, New York. Fraga, C. G., Shigenaga, M. K., Park, J.-W., Degan, P., and Ames, Β. N. (1990). Proc. Natl Acad. Sei. U.S.A. 87, 4533-4537. Frei, Β., Winterhalter, Κ. Η., and Richter, C. (1985). J. Biol. Chem. 260, 7394-7401. Frei, Β., Winterhalter, Κ. Η., and Richter, C. (1986). Biochemistry 25, 4438-4443. Frei, Β., Kim, M., and Ames, Β. Ν. (1990). Proc. Natl. Acad. Sei. U.S.A. 87, 4879-4883. French, J. H., Sherard, E. S., Lubell, H., Brotz, M., and Moore, C. L. (1972). Arch. Neurol. (Chicago) 26, 229-244. Gadaleta, M. N., Petruzella, V , Renis, M., Fracasso, F., and Cantatore, P. (1990). Eur. J. Biochem. 187, 501-506. Gray, M. W. (1989). Annu. Rev. Cell Biol. 5, 25-50. Gross, N. J., Getz, G. S., and Rabinowitz, M. (1969). J. Biol Chem. 244, 1552-1562. Grossman, L. I. (1990). Am. J. Hum. Genet. 46, 415-417. Gupta, K. P., van Golen, K. L., Randerath, Ε., and Randerath, Κ. (1990). Mutât. Res. 237, 17-27. Gyllensten, U., Wharton, D., Josefsson, Α., and Wilson, A. C. (1991). Nature (London) 352, 255-257. Halliwell, B., and Aruoma, Ο. I. (1991). FEBS Lett. 281, 9-19. Hammans, S. R., Sweeney, M. G., Brockington, M., Morgan-Hughes, J. Α., and Harding, A. E. (1991). Lancet 337, 1311-1313. Harman, D. (1956). /. Gerontol 11, 298-300. Harman, D. (1972). /. Am. Geriatr. Soc. 20, 145-147. Harman, D. (1983). Age 6, 86-94. Hattori, K., Tanaka, M., Sugiyama, S., Obayashi, T., Ito, T., Satake, T., Hanaki, Y , Asai, J., Nagano, M., and Ozawa, T. (1991). Am. Heart J. 121, 1735-1742.

mtDNA MODIFICATIONS IN DISEASE AND AGING

17

Hayakawa, M., Torii, K., Sugiyama, S., Tanaka, M., and Ozawa, T. (1991). Biochem. Biophys. Res. Commun. 179, 1023-1029. Hennet, T., Richter, C , and Peterhans, E. (1993a). Biochem. J. 289, 587-592. Hennet, T., Bertoni, G., Richter, C , and Peterhaus, E. (1993b). Cancer Res. 53, 1456-1460. Holt, I. J., Cooper, J., Morgan-Hughes, J. Α., and Harding, A. E. (1988a). Lancet 1, 1462. Holt, I. J., Harding, A. E., and Morgan-Hughes, J. A. (1988b). Nature (London) 331, 717719. Hruszkewycz, Α. M., and Bergtold, D. S. (1990). Mutat. Res. 244, 123-128. Ikebe, S., Tanaka, M., Ohno, K., Sato, W., Hattori, K., Kondo, T., Mizuno, Y., and Ozawa, T. (1990). Biochem. Biophys. Res. Commun. 170, 1044-1048. Imlay, J. Α., and Linn, S. (1986). J. Bacteriol. 166, 519-527. Imlay, J. Α., and Linn, S. (1988). Science 240, 1302-1309. Joenje, H. (1989). Mutat. Res. 219, 193-208. Jukes, T. H., and Osawa, S. (1990). Experientia 46, 1117-1126. Kadenbach, Β., and Müller-Höcker, J. (1990). Naturwissenschaften 27, 221-225. Kadenbach, Β., Seibel, P., Johnson, Μ. Α., and Turnbull, D. (1991). In "Molecular Basis of Neurological Disorders and their Treatment" (J.W. Gorrod, O. Alband, E. Ferrari, and S. Papa, eds.) pp. 200-208. Springer-Verlag, Berlin. Katayama, M., Tanaka, M., Yamamoto, H., Ohbayashi, T., Nimura, Y., and Ozawa, T. (1991). Biochem. Int. 25, 47-56. King, M., and Attardi, G. (1989). Science 246, 500-503. Knoll, J. (1988). Mech. Ageing Dev. 46, 237-262. Kuchino, Y., Mori, F., Kasai, F., Inoue, H., Iwai, S., Miura, K., Ohtsuka, E., and Nishimura, S. (1987). Nature (London) 327, 77-79. Lander, E. S., and Lodish, H. (1990). Cell (Cambridge, Mass.) 61, 925-926. Lestienne, P. (1989). Biochimie 71, 1115-1123. Lestienne, P. (1992). Biochimie 74, 123-130. Lestienne, P., and Ponsot, G. (1988). Lancet 1, 885. Linnane, A. W., Marzuki, S., Ozawa, T , and Tanaka, M. (1989). Lancet 1, 642-645. Linnane, A. W., Baumer, Α., Maxwell, R. J., Preston, H., Zhang, C , and Marzuki, S. (1990). Biochem. Int. 22, 1067-1076. Linnane, A. W., Zhang, C , Baumer, Α., and Nagley, P. (1992). Mutat. Res. 275, 195-208. Luft, R., Ikkos, D., Palmieri, G., Ernster, L., and Afzelius, B. (1962). /. Clin. Invest. 41, 1776-1804. Mann, V. M., Cooper, J. M., and Schapira, Α. Η. V. (1992). FEBS Lett. 299, 218-222. Margolis, L. (1981). "Symbiosis in Cell Evolution." Freeman, San Francisco. Martin, A. P., Naylor, G. J. P., and Palumbi, S. R. (1992). Nature (London) 357, 153-155. Masoro, E. J. (1990). In "Geriatric Nutrition" (J. E. Morley, Z. Glick, and L. Z. Rubenstein, eds.), pp. 19-25. Raven Press, New York. McBride, T. J., Preston, B. D., and Loeb, L. A. (1991). Biochemistry 30, 207-213. Meier, P. (1991). Diplomarbeit ΕΤΗ. Miquel, J. (1991). Arch. Gerontol. Geriatr. 12, 99-117. Miquel, J., Oro, J., Bensch, K. G., and Johnson, J. E., Jr. (1977). In "Free Radicals in Biology" (W. A. Pryor, ed.), Vol. 3, pp. 133-181. Academic Press, New York. Miquel, J., Economos, A. C , Fleming, J., and Johnson, J. R., Jr. (1980). Exp. Gerontol. 15, 575-591. Müller-Höcker, J. (1989). Am. J. Pathol. 134, 1167-1171. Müller-Höcker, J. (1990). J. Neurol. Sei. 100, 14-21. Müller-Höcker, J. (1992). Brain Pathol. 2, 149-158. Myers, Κ. Α., Saffhill, R., and O'Connor, P. J. (1988). Carcinogenesis (London) 9, 285-292.

18

CHRISTOPH RICHTER

Niki, E. (1987). Chem. Phys. Lipids 44, 227-253. Nohl, H., and Hegner, D. (1978). Eur. J. Biochem. 82, 563-567. Nohl, H., Breuninger, V., and Hegner, D. (1978). Eur. J. Biochem. 90, 385-390. Nohl, H., De Silva D., and Summer, Κ. H. (1989). Free Radical Biol. Med. 6, 369-374. Osiewacz, H. D. (1990). Mutat. Res. 237, 1-8. Ozawa, T., Yoneda, M., Tanaka, M., Ohno, K., Sato, W., Suzuki, H., Nishikimi, M., Yamamoto, M., Nonaka, I., and Horai, S. (1988). Biochem. Biophys. Res. Commun. 154, 1240-1247. Ozawa, T., Tanaka, M., Sato, W., Ohno, K., Sugiyama, S., Yoneda, M., Yamamoto, T., Hattori, K., Ikebe, S., Tashiro, M., and Sahashi, K. (1990a). In "Bioenergetics" (C. H. Kim and T. Ozawa, eds.), pp. 413-427. Plenum, New York. Ozawa, T., Tanaka, M , Ikebe, S., Ohno, K., Kondo, T., and Mizuno, Y. (1990b). Biochem. Biophys. Res. Commun. 172, 483-489. Ozawa, T., Tanaka, M., Hayakawa, M., Sugiyama, S., Sato, W., Ohno, K., Ikebe, S., and Yoneda, M. (1991). Prog. Neuropathol. 7, 141-151. Parker, W. D., Jr., Boyson, S. J., Luder, A. S., and Parks, J. K. (1990a) Neurology 40, 12311234. Parker, W. D., Jr., Filley, C. M., and Parks, J. K. (1990b). Neurology 40, 1302-1303. Piko, L., and Matsumoto, L. (1977). Nucleic Acids Res. 4, 1301-1314. Piko, L., Hougham, A. J., and Bulpitt, K. J. (1988). Mech. Ageing Dev. 43, 279-293. Poulton, J., Deadman, M. E., Turnbull, D. M., Lake, B., and Gardiner, R. M. (1991). Clin. Genet. 39, 33-38. Richter, C. (1988). FEBS Lett. 241, 1-5. Richter, C , Park, J.-W., and Arnes, Β. Ν. (1988). Proc. Natl. Acad. Sei. U.S.A. 85, 64656467. Rotig, Α., Colonna, M., Blanche, S., Fischer, Α., LeDeist, F., Frezal, J., Saudubray, J. M., and Munnich, A. (1988). Lancet 2, 567-568. Sato, T., and DiMauro, S., eds. (1991). "Mitochondrial Encephalomyopathies," Prog. Neuropathol., Vol. 7. Raven Press, New York. Satoh, M. S., Huh, N., Rajewsky, M. F., and Kuroki, T. (1988). /. Biol. Chem. 263, 68546856. Saul, R. L., Gee, P., and Ames, Β. N. (1987). In "Modern Biological Theories of Aging" (H. R. Warner, R. N. Butler, R. L. Sprott, and E. L. Schneider, eds.), pp. 113-129. Raven Press, New York. Sawada, M., and Carlson, J. C. (1987). Mech. Ageing Dev. 41, 125-137. Schapira, Α. H. V., Holt, I. J., Sweeney, M., Harding, A. E., Jenner, P., and Marsden, C. D. (1990). Movement Dis. 5, 294-297. Schulze-Osthoff, K., Bakker, A. C , Vanhaesebroeck, B., Beyaert, R., Jacob, W. Α., and Fiers, W. (1992). J. Biol. Chem. 267, 5317-5323. Shearman, C. W., and Kalf, G. F. (1977). Arch. Biochem. Biophys. 182, 573-586. Shibutani, S., Takeshita, M., and Grollman, A. P. (1991). Nature (London) 349, 431-434. Shoffner, J. M., and Wallace, D. C. (1990). Adv. Hum. Genet. 19, 267-330. Shy, C. M., and Gonatas, Ν. K. (1964). Science 145, 493-496. Shy, C. M., Gonatas, Ν. K., and Perez, M. (1966). Brain 89, 133-158. Sies, H. (1989). Naturwissenschaften 76, 57-64. Sohal, R. S. (1991). Mech. Ageing Dev. 60, 189-198. Sohal, R. S., and Allen, R. G. (1985). Basic Life Sei. 35, 75-104. Sohal, R. S., Arnold, L. Α., and Sohal, Β. H. (1990). Free Radical Biol. Med. 10, 495-500. Spiro, A. J., Moore, C. L., Prineas, J. W., Strasberg, P. M., and Rapin, I. (1970). Arch. Neurol. (Chicago) 23, 103-112.

mtDNA MODIFICATIONS IN DISEASE AND AGING

19

Spoerri, P. E. (1984). Monogr. Dev. Biol. 17, 210-220. Steenken, S. (1989). Chem. Rev. 89, 503-520. Sugiyama, S., Hattori, K., Hayakawa, M., and Ozawa, T. (1991). Biochem. Biophys. Res. Commun. 180, 894-899. Thomas, S. M., Gebicki, J. M., and Dean, R. T. (1989). Biochim. Biophys. Acta 1002, 189197. Thorsness, P. E., and Fox, T. D. (1990). Nature (London) 346, 376-379. Tomkinson, A. E., Bonk, R. T , and Linn, S. (1988). /. Biol. Chem. 263, 12532-12537. Tomkinson, A. E., Bonk, R. T , Kim, J., Bartfeld, Ν., and Linn, S. (1990). Nucleic Acids Res. 18, 929-935. Traber, J. (1991). Diplomarbeit ΕΤΗ. Trounce, I., Byrne, E., and Marzuki, S. (1989). Lancet 1, 637-639. Turrens, J. F., Beconi, M., Barilla, J., Chavez, U. B., and McCord, J. M. (1991). Free Radical Res. Commun. 12-13, 681-689. Tyler, D. (1992). "The Mitochondrion in Health and Disease." VCH Publishers, New York. Vaillant, F., Loveland, Β. Ε., Nagley, P., and Linnane, A. W. (1991). Biochem. Int. 23, 571580. Vik, S. B., Georgevich, G., and Capaldi, R. (1981). Proc. Natl. Acad. Sei. U.S.A. 78, 14581460. von Sonntag, C. (1987). "The Chemical Basis of Radiation Biology." Taylor & Francis, London. Wallace, D. C. (1989a). Cytogenet. Cell Genet. 51, 612-621. Wallace, D. C. (1989b). Trends Genet. 5, 9-14. Wallace, D. C. (1992a). Science 256, 628-632. Wallace, D. C. (1992b) Annu. Rev. Biochem. 61, 1175-1212. Wallace, D. C , Singh, S., Lott, M. T , Hodge, J. Α., Schurr, T. G., Lezza, A. M. S., Elsas, L. J., III, and Nikoskelainen, E. (1988). Science 242, 1427-1430. Wallace, D. C , Lott, M. T , Lezza, A. M. S., Seibel, P., Voljavec, A. S., and Shoffner, J. M. (1990). Pediatr. Res. 28, 525-528. Wood, M. L., Dizdaroglu, M., Gajewski, E., and Essigman, J. M. (1990). Biochemistry 29, 7024-7032. Wunderlich, V., Schutt, M., Bottger, M. and Graffi, A. (1970). Biochem. J. 118, 99-109. Yen, T., Su, J., King, K., and Wei, Y. (1991). Biochem. Biophys. Res. Commun. 178, 124131. Zeviani, M., and Antozzi, C. (1992). Brain Pathol. 2, 121-131. Zeviani, M., Moraes, C. T., DiMauro, S., Nakase, H., Bonilla, E., Schon, Ε. Α., and Rowland, L. P. (1988). Neurology 38, 1339-1346.

This page intentionally left blank

CURRENT TOPICS IN BIOENERGETICS, VOLUME 17

Mitochondrial Myopathies: Biochemical Aspects SARA SHANSKE AND SALVATORE D I M A U R O

H. Houston Merritt Clinical Research Center for Muscular Dystrophy and Related Diseases, Department of Neurology, Columbia Presbyterian Medical Center New York, New York 10032 I.

Introduction A. Defects of Nuclear DNA (nDNA) B. Defects of Mitochondrial DNA (mtDNA) C. DNA/mtDNA Communication II. Mitochondrial Metabolism III. Mitochondrial Disorders A. Defects of substrate transport B. Defects of substrate utilization C. Defects of the Krebs cycle D. Defects of Oxidation-Phosphorylation Coupling E. Defects of the Respiratory Chain and ATP Synthase IV. Concluding Remarks References

I.

Introduction

The concept of mitochondrial disease was introduced in 1962 when Luft et al. described "loose coupling" of muscle mitochondria in a patient with nonthyroidal hypermetabolism. The decades of 1960 and 1970 were dominated by morphological and clinical descriptions, mostly of patients with mitochondrial myopathies. Biochemical studies were not conducted systemically until the 1970s and were often inconclusive, owing both to the difficulty of isolating functionally intact mitochondria from human muscle biopsy specimens and (as we now realize) to the relative insensitivity of polarography in detecting partial metabolic blocks. During the 1970s, specific biochemical defects were described in increasing number, including pyruvate dehydrogenase complex (PDHC) deficiency, carnitine palmitoyltransferase (CPT) deficiency, carnitine deficiencies, 21 Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.

22

SARA SHANSKE AND SALVATORE DIMAURO

and defects of individual complexes of the respiratory chain. In the 1980s, the rapid accumulation of biochemical knowledge led to a rational biochemical classification of mitochondrial diseases, which were divided into five main groups based on the area of mitochondrial metabolism affected, as will be discussed in detail below. Mitochondrial diseases are uniquely interesting from the genetic point of view because mitochondria are endowed with their own DNA. Human mitochondrial DNA (mtDNA) encodes 13 structural proteins, all of them subunits of respiratory chain complexes and ATP synthase, and also two rRNAs and 22 tRNAs needed for translation. Recent advances in mtDNA genetics and in our understanding of diseases related to mtDNA mutations has made possible* a genetic classification of mitochondrial defects (Table I). This classification allows us to formulate some general predictions as to the biochemical consequences of different genetic defects.

A.

DEFECTS OF NUCLEAR

DNA (nDNA)

Mutations of nuclear genes encoding structural mitochondrial proteins can affect any area of mitochondrial metabolism and should result in diseases transmitted by Mendelian inheritance. Most disorders in this group are, in fact, transmitted as autosomal recessive traits. Biochemically, these are usually single enzyme defects (monoenzymopathy) and, in homozygotes, there is almost complete deficiency of the affected enzyme. These defects can involve genes encoding tissue-specific proteins or genes encoding proteins common to all tissues. In the former case, the clinical manifestations should be confined to one or a few tissues, and there should be a specific enzyme defect limited to the same tissues. In the latter case, the disorder should be multisystemic and characterized biochemically by a defect of a single enzyme in all tissues. A special case is represented by nuclear defects of genes involved in the complex translocation system required for the transport and correct assembly of proteins synthesized in the cytoplasm and imported into mitochondria. The biochemical consequences here might be a single enzyme defect or multiple enzyme defects (multienzymopathy), depending on whether the lesion affects a component of the translocation machinery unique to one enzyme or shared by several mitochondrial enzymes.

B.

DEFECTS OF MITOCHONDRIAL

DNA (mtDNA)

Biochemical consequences of alterations in mtDNA should be confined to the respiratory chain linked energy transduction, as all mtDNA genes

TABLE I GENETIC CLASSIFICATION OF MITOCHONDRIAL DISEASES

Site of defect Nuclear DNA (nDNA) Tissue-specific gene Non-tissue-specific gene Mitochondrial DNA (mtDNA) Point mutations

Deletions or duplications nDNA/mtDNA Communication Multiple mtDNA deletions mtDNA depletion

Heredity

Clinical features

Biochemistry

Mendelian Mendelian

Tissue-specific syndrome Multisystemic disorder

Tissue-specific monoenzymopathy Generalized monoenzymopathy

Maternal

Multisystemic, heterogeneous

Sporadic

PEO;KSS; Pearson

Generalized monoenzymopathy (structural genes) Generalized multienzymopathy (tRNA genes) Generalized (±) multienzymopathy

Mendelian (AD) Mendelian (AR)

PEO ± other features Myopathy ± nephropathy; hepatopathy; encephalopathy

Generalized multienzymopathy Tissue-specific multienzymopathy

24

SARA SHANSKE AND SALVATORE DIMAURO

control the synthesis of respiratory chain enzymes and ATP synthase. Alterations in mtDNA consist of point mutations, deletions or duplications, and depletion. Point mutations in mtDNA can affect either structural genes or tRNA genes. Biochemically, mutations in mtDNA genes encoding structural proteins, such as those affecting complex I genes in Leber's optic atrophy, should cause defects of individual enzymes. However, mutations in genes encoding tRNAs, such as those described in MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), and MERRF (myoclonus, epilepsy, and ragged-red fibers) ought to affect translation as a whole and cause multiple enzyme deficiencies. Since mtDNA is contributed almost exclusively by the ovum, disorders due to mtDNA point mutations are transmitted by maternal inheritance. Large-scale deletions and duplications of mtDNA have been associated with Kearns-Sayre syndrome (KSS), progressive external ophthalmoplegia (PEO), and Pearson's marrow/pancreas syndrome. These disorders, when associated with single mtDNA deletions, are invariably sporadic, implying that the deletions arise in the zygote and affect somatic rather than germline cells. Biochemically, multiple enzymes should be affected, because all described deletions encompass at least one tRNA gene, thereby affecting translation.

C.

nDNA/mtDNA

COMMUNICATION

Some disorders might be due to faulty communication between the two genomes, since nuclear DNA encodes numerous factors controlling various aspects of mtDNA function. Two such disorders have now been described. One of these, multiple mtDNA deletions, is transmitted in autosomal dominant fashion, and is attributed to a mutation in an nDNA-encoded factor making mtDNA more prone to deletions. The second disorder, mtDNA depletion, characterized by a quantitative rather than qualitative abnormality in mtDNA, and inherited as an autosomal recessive trait, is believed to be the result of mutation in an nDNA-encoded factor controlling mtDNA replication. II.

Mitochondrial Metabolism

A brief review of mitochondrial metabolism in muscle will be followed by a systematic description of biochemical defects classified according to the area of mitochondrial metabolism specifically affected. In a schematic overview of mitochondrial metabolism (Fig. 1), five main

FIG. 1. Scheme of mitochondrial metabolism. Abbreviations: NADH, nicotinamide adenine nucleotide, reduced; FMN,flavinmononucleotide; FAD,flavinadenine dinucleotide; FeS, non-heme iron-sulfur protein; CoQ, coenzyme Q; Ph inorganic phosphate. Reprinted with permission from DiMauro and Devivo (1989).

26

SARA SHANSKE AND SALVATORE DIMAURO

steps can be recognized. (1) Because the inner mitochondrial membrane is impermeable to anions and neutral metabolites, these have to be transported across the membranes by a set of carriers or translocases. (2) In the matrix, metabolites are further oxidized, pyruvate by the pyruvate dehydrogenase complex and fatty acids through the sequential reactions of the ß-oxidation pathway. (3) The common product of intramitochondrial oxidation, acetyl-CoA, is oxidized in the Krebs cycle. (4) The reducing equivalents produced by the oxidation of acetyl-CoA are passed along a chain of proteins embedded in the inner mitochondrial membrane (the electron transport or respiratory chain) through a series of oxidation/reduction reactions in which the final hydrogen acceptor is molecular oxygen and the final product is water. (5) The energy released in this series of reactions is harnessed to pump protons from one side of the membrane to the other. The resulting electrochemical proton gradient is used to synthesize adenosine triphosphate (ATP) at three sites along the respiratory chain: These are the sites of oxidation/phosphorylation coupling.

III.

Mitochondrial Disorders

To carry out these many metabolic reactions (and others not mentioned in our abbreviated survey), mitochondria contain dozens of enzymes, localized in different compartments. Table II provides a partial list of mitochondrial enzymes and their locations; enzyme defects implicated in human diseases are indicated. Using the five areas of metabolism indicated in Fig. 1, we can classify mitochondrial diseases into five groups (Table III): (1) defects of transport, (2) defects of substrate utilization, (3) defects of the Krebs cycle (4) defects of oxidation/phosphorylation coupling, and (5) defects of the respiratory chain. Groups 1-4 are caused by defects of nuclear DNA (nDNA), whereas group 5, defects of the respiratory chain, may be due to defects of nDNA or mtDNA, as described below.

A.

DEFECTS OF SUBSTRATE TRANSPORT

The better known defects of mitochondrial substrate transport affect lipid metabolism and are due to CPT deficiency and to both primary and secondary carnitine deficiencies. 1.

Carnitine Palmitoyltransferase Deficiency

In order to cross the mitochondrial membranes and undergo ß-oxidation, long-chain fatty acids are first activated by coenzyme A and then

T A B L E II MITOCHONDRIAL ENZYMES 0

Outer membrane NADH-cytochrome b5 oxidoreductase Cytochrome b5 Monoamine oxidase Kynurine hydroxylase Glycerolphosphate acyl transferase Lysophosphatidyl acyl transferase Phosphatidate phosphatase Phospholipase A Nucleoside diphosphokinase Carnitine palmitoyltransferase I

a

Inner membrane

Intermembrane space

Matrix

NADH-coenzyme Q oxidoreductase Succinate-coenzyme Q oxidoreductase Coenzyme QH2-cytochrome c oxidoreductase Cytochrome c oxidase Oligomycin-sensitive ATPase ß-Hydroxybutyrate dehydrogenase Pyridine nucleotide transhydrogenase Carnitine palmitoyltransferase II Ferrochelatase Adenine nucleotide carrier

Adenylate kinase Nucleoside diphosphokinase Nucleoside monophosphokinase

Pyruvate dehydrogenase complex q-Ketoglutarate dehydrogenase complex Citrate synthase Aconitase Malate dehydrogenase Isocitrate dehydrogenase Fumarase Glutamate dehydrogenase Pyruvate carboxylase Aspartate aminotransferase Ornithine carbamyltransferase Carbamyl-phosphate synthetase Fatty acyl-CoA synthetase Enoyl hydrase ß-Hydroxyacyl-CoA dehydrogenase ß-Ketoacyl-CoA thiolase Amino acid activating enzyme DNA polymerase y DNA topoisomerase RNA polymerase RNA processing enzymes

Adapted from E. Kuylenstiema (1970). Enzymes reported defective in human diseases are underlined.

SARA SHANSKE AND SALVATORE DIMAURO

28

T A B L E III BIOCHEMICAL CLASSIFICATION OF THE MITOCHONDRIAL MYOPATHIES

1.

2.

3.

4. 5.

Defects of substrate transport Carnitine palmitoyltransferase deficiency Carnitine deficiency Defects of substrate utilization Pyruvate dehydrogenase complex deficiency Pyruvate carboxylase deficiency Defects of ß-oxidation Defects of the Krebs cycle Fumarase deficiency a-Ketoglutarate dehydrogenase (dihydrolipoyl dehydrogenase) deficiency Aconitase deficiency Defects of oxidation-phosphorylation coupling Luff s syndrome (loose coupling of muscle mitochondria) Defects of the respiratory chain and ATP synthase Complex I deficiency Complex II deficiency Complex III deficiency Complex IV deficiency Complex V deficiency Combined defects of respiratory chain components

reversibly esterified with L-carnitine, a reaction that is catalyzed by the enzyme carnitine palmitoyltransferase (CPT). The CPT system consists of two mitochondrial membrane-bound enzymes, CPT I and CPT II. CPT I is located on the inner side of the outer mitochondrial membrane, is specifically inhibited by malonyl Co A, and exists in tissue-specific isoforms. In contrast, CPT II is located on the inner face of the inner mitochondrial membrane, is insensitive to malonyl Co A inhibition, and is not tissue specific. CPT I deficiency has thus far been described in only three infants and is characterized by hepatomegaly, nonketotic hypoglycemia, hyperammonemia, increased levels of serum transaminases and plasma free fatty acids, and coma (Bougneres et al., 1981; Falik-Borenstein et al., 1992). Clinically, CPT II deficiency can cause two distinct phenotypes, one dominated by myopathy, the other by liver disease. The myopathic form was first described in 1973 in two brothers with myoglobinuria (DiMauro and Melis-DiMauro, 1973), and typically presents in young adults with recurrent myoglobinuria triggered by prolonged exercise, prolonged fasting, or a combination of the two (DiMauro and Papadimitriou, 1986). This disorder is inherited in an autosomal recessive manner, the enzyme defect has been identified in more than 50 patients and, for reasons unknown, there is a marked prevalence of affected males. The infantile "hepatic" form of

MITOCHONDRIAL MYOPATHIES

29

CPT II deficiency causes a life-threatening and often fatal disease characterized by nonketotic hypoglycemia, cardiomegaly, and coma (Demaugre et al., 1991). The phenotypic heterogeneity of CPT II deficiency reflects heterogeneity at the molecular level. Studies aimed at understanding the molecular basis of CPT II deficiency are now possible, because the fulllength cDNA for human CPT has been cloned and the gene has been assigned to chromosome 1, region l q l 2 - l p t e r (Finocchiaro et al., 1991). Molecular analysis of a patient with the early onset, "hepatic" form of CPT II deficiency showed homozygosity for a mutant allele carrying three missense mutations (Taroni et al., 1992). One of these, R631C, appeared to be the crucial mutation, whereas the other two were sequence polymorphisms which did not affect enzyme activity per se, but did exacerbate the effects of the R631C substitution. Biochemical characterization of the mutant CPT in the patient's cells showed severe reduction of Vmax, normal apparent Km values, and decreased protein stability. In contrast to this defect, which is seen in infants with the "hepatic" form, most of the patients with the more typical "myopathic" form of CPT II deficiency have a mutation at codon 113, changing a highly conserved leucine to arginine (Taroni et al., 1993). 2.

Carnitine Deficiency

Carnitine deficiency is a clinically heterogeneous disorder with multiple causes resulting in decreased concentration of carnitine in skeletal muscle (myopathic form) or in muscle, liver, blood, and various other tissues (systemic form). Primary carnitine deficiencies are probably less frequent than secondary forms, which are often seen in the setting of mitochondrial diseases. Myopathic carnitine deficiency is characterized by generalized weakness, starting in childhood in most patients and affecting mainly proximal limb and trunk muscles, but sometimes also facial and pharyngeal muscles, while nonmuscle tissues are clinically spared (DiDonato et al., 1992). The course is usually slowly progressive, but weakness may fluctuate in severity. Transmission appears to be autosomal recessive. Biochemically, there is a severe deficiency of muscle carnitine contrasting with normal levels of plasma carnitine. The primary defect may involve the active transport of carnitine from blood into muscle, but this has not been documented and the very existence of primary myopathic carnitine deficiency is questionable. Systemic carnitine deficiency is more frequently a secondary rather than a primary condition. It can be associated with disorders causing excessive carnitine loss or decreased hepatic synthesis and dietary intake , or it can

30

S A R A SHANSKE A N D SALVATORE

DIMAURO

be secondary to genetic defects of intermediary metabolism, such as defects of ß-oxidation and defects of the respiratory chain. The mechanism of carnitine depletion in metabolic disorders appears to be mediated through excessive accumulation of acyl-coenzyme A thioesters. These potentially toxic compounds are esterified to acylcarnitines and excreted in the urine, resulting in a net loss of carnitine. Regardless of the etiology, secondary systemic carnitine deficiency usually presents in early childhood with recurrent episodes of encephalopathy closely resembling Reye's syndrome. The episodes are often triggered by mild intercurrent illnesses and are accompanied by nausea, vomiting, confusion, or coma. A progressive neuromuscular disorder similar to that of the myopathic form is also common. A special form of systemic carnitine deficiency, and the only one so far that can be considered primary, is associated with childhood cardiomyopathy and is invariably fatal if untreated (Stanley et al., 1991; Tein and DiMauro, 1992). In this condition carnitine concentration is very low in plasma and in muscle and there is severe renal carnitine leak. There is good evidence that this condition is due to a defect of the specific high-affinity, low-concentration, carrier-mediated carnitine uptake mechanism (Eriksson et al., 1988; Treem et al., 1988; Tein et al., 1990). Although the defect has been documented only in cultured fibroblasts, the same uptake system is probably shared by muscle, heart, and kidney, thus explaining the lipid storage myopathy, the cardiomyopathy, and the renal loss of carnitine. Autosomal recessive inheritance was suggested by pedigree analysis and has been confirmed by the observation that asymptomatic parents have intermediate levels of plasma carnitine concentrations and a partial defect of carnitine uptake in cultured fibroblasts (Tein et al., 1990).

Β.

DEFECTS OF SUBSTRATE UTILIZATION

1.

Pyruvate Dehydrogenase Complex Deficiency

The pyruvate dehydrogenase complex (PDHC) catalyzes the conversion of pyruvate to acetyl-coA (Fig. 1) and is dependent on thiamine pyrophosphate and lipoic acid as cofactors. The complex has five enzymes, three with catalytic functions and two with regulatory roles. Defects of PDHC can affect any one of the three catalytic components, Ei (pyruvate decarboxylase), E 2 (dihydrolipoyl transacetylase), or E 3 (dihydrolipoyl dehydrogenase), or either of the two regulatory components, PDH-kinase, which inactivates the enzyme, or PDH-phosphatase, which activates it. By far the most common enzyme defect involves Ej (pyruvate decarbox-

MITOCHONDRIAL MYOPATHIES

31

ylase), which is a tetramer composed of two α and two β subunits. The α subunit is encoded by a gene on the X chromosome, at Xp22.1-p22.2 (Brown et al., 1989), and a total of 20 different mutations in the Ε Γ α gene have been described, including deletions, insertions, and point mutations (Dahl et al., 1992). The β subunit is encoded by a gene on chromosome 3 (Ho et al., 1988). About 100 patients have been reported with PDH-E X deficiency. There is striking clinical heterogeneity, but three main syndromes have emerged (Robinson et al., 1977; Robinson, 1989): (1) A neonatal form, with onset in the first days or weeks of life and death usually before 6 months, is characterized by hypotonia, episodic apnea and lethargy, seizures, failure to thrive, and severe lactic acidosis. Dysmorphic features similar to those of fetal alcohol syndrome and agenesis of the corpus callosum are frequent. There is a marked predominance of affected males, suggesting involvement of the X-linked Ε Γ α subunit in many patients. Genetic heterogeneity is suggested by decreased cross-reacting material (CRM) for El in some patients but not in others and by presence or absence of the mRNA for El in different patients. (2) In the infantile form, symptoms appear before 6 months of age and consist of psychomotor delay, hypotonia, seizures, episodic apnea and lethargy, ataxia, ophthalmoplegia, optic atrophy, and mild to moderate lactic acidosis. Death usually occurs before 3 years of age. In most patients, neuropathology shows the symmetrical necrotic lesions in basal ganglia and brain stem characteristic of Leigh syndrome. There is a slight predominance of affected males, and immunologic studies in seven male patients have shown decreased CRM for both Ε Γ α and E r ß . (3) The benign form has been described in seven male patients with normal psychomotor development but intermittent ataxia or exercise intolerance, apparently responsive to thiamine administration. Western blots in two patients showed normal abundance but abnormal migration of the Ex-a band (Robinson, 1989). Although patients in the neonatal group tend to have the lowest PDHC residual activities, there is not good correlation between clinical severity and residual activity. To explain this, Robinson (1989) suggested that additional factors may be important, such as superimposed perinatal hypoxia, age at diagnosis, ketogenic therapy, or differential involvement of Ε Γ α or -β subunits. A single case of PDHC-E2 (dihydrolipoyl transacetylase) deficiency has been described (Robinson et al., 1990) in a child who presented with hyperammonemia and severe lactic acidosis at 2 weeks of age and, at 3.5 years, had microcephaly and severe psychomotor delay. Ε3 (dihydrolipoyl dehydrogenase) is a homodimer whose subunit is shared by two other α-ketoacid dehydrogenases: a-ketoglutarate dehydrogenase and branched chain ketoacid dehydrogenase. Therefore, defects

32

SARA SHANSKE AND SALVATORE DIMAURO

of PDHC-E 3 are never isolated and result in blood accumulation (and urinary excretion) of pyruvate, lactate, a-ketoglutarate, and branched chain α ketoacids. This pattern is a useful diagnostic clue. Only three patients with dihydrolipoyl dehydrogenase deficiency have been described, one boy and two girls. The boy developed, at 8 weeks of age, a progressive neurologic disorder including labored breathing, inspiratory stridor, lethargy, and hypotonia alternating with irritability. He died at 7 months, and neuropathology showed demyelination and cavitation of basal ganglia, thalamus, and brain stem, sparing the cortex (Robinson et al., 1977). The second patient was floppy at birth, fed poorly, and had a respiratory arrest at 10 weeks. At 5 months, she was hypotonic, and at 2 years she developed seizures (Robinson et al., 1981). By 6 months of age, the third patient had severe development delay, hypotonia, and microcephaly: She died at 18 months (Munnich et al., 1982). PDH-phosphatase deficiency was described in four patients, three of whom had the clinical and neuropathologic features of Leigh syndrome (Robinson, 1989). 2.

Pyruvate Carboxylase Deficiency

Defects of pyruvate carboxylase (PC), the first enzyme in gluconeogenesis, cause two major syndromes. One is a severe disorder manifesting soon after birth and characterized by hepatomegaly, metabolic acidosis, and death before 3 months. The other is a milder syndrome starting in the first months of life and dominated by metabolic acidosis, delayed psychomotor development leading to severe mental retardation, and death in childhood (Robinson, 1989). Lactic acidosis is the main laboratory abnormality in the mild variant, whereas patients with the severe variant also show increased blood levels of ammonia, citrulline, and lysine. The two different clinical phenotypes seem to be related to presence or absence of residual enzyme activity. Western blot in fibroblasts from patients with the mild variant consistently showed presence of the 125-kDa PC single subunit (the holoenzyme is a tetramer containing four biotin molecules). The subunit was absent in some, but not all, patients with the severe variant and it is assumed that, when present, the protein is totally devoid of enzyme activity. 3.

Defects of ß-Oxidation

The first description of a defect in a fatty acid ß-oxidation enzyme was that of medium chain acyl-CoA dehydrogenase (MCAD) deficiency (Kolvraa et al., 1982; Rhead et al., 1983). Since then, a number of other

MITOCHONDRIAL MYOPATHIES

33

ß-oxidation enzyme defects have been well characterized. Myopathy is rarely the predominant feature in this group of disorders, so we will only briefly describe the various entities and further details can be found in recent reviews (Roe and Coates, 1989; Bennett and Hale, 1992). The three acyl-CoA dehydrogenases (short chain, SCAD; medium chain, MC A D ; long chain, LCAD) can be affected separately or together. Combined defects of the three dehydrogenases (multiple acyl-CoA dehydrogenase deficiency, MAD, or glutaric aciduria type 2) can be due to defects of the electron transfer factor (ETF) per se or of ETF-dehydrogenase and can be responsive to riboflavin. Patients with deficiencies of the 3-hydroxyacylCoA dehydrogenases, both short chain (SCHAD) and long chain (LCHAD) have also been described (Hale and Thorpe, 1989; Bertini et al, 1990; Rocchiccioli et al., 1990; Wanders et al., 1990; Tein et al, 1991). Ail of the defects share certain clinical and laboratory findings, including fasting intolerance, lipid accumulation in tissues, hypoglycemia, hypoketonuria, serum fatty acid elevations, secondary carnitine deficiency, and characteristic dicarboxylic aciduria. Several clinical and laboratory features aid in distinguishing the various defects from one another. Careful analysis of organic acids in the urine provides the most helpful information in localizing the site of the defect. Unique clinical presentations include cardiomyopathy in patients with LCAD (Treem et al, 1991). MCAD appears to be the most prevalent of the fatty acid oxidation disorders and has been studied at the molecular level. Its frequency has been estimated as 1 in 10,000 to 1 in 20,000 in Caucasian populations of Northern European origin (Bennett et al, 1987), but this is probably a rare disorder in other population groups. Approximately 90% of MCAD patients have a single common mutation: An A to G nucleotide transition at position 985 in the cDNA, resulting in a lysine to glutamate substitution at amino acid position 304 of the mature MCAD protein (Kelly et al, 1990). This finding raises the possibility of a common ancestral heritage for this mutation and probably explains the high prevalence of MCAD deficiency in a single ethnic group.

C.

DEFECTS OF THE KREBS CYCLE

1.

Fumarase Deficiency

Disorders of the Krebs cycle are best illustrated by fumarase deficiency, a devastating encephalomyopathy of infancy first described in 1986 (Zinn et al, 1986) and characterized by poor feeding, lethargy, persistent vomiting, microcephaly, hypotonia, and hyporeflexia. The laboratory hall-

34

SARA SHANSKE AND SALVATORE DIMAURO

mark of the disease is the excretion of large amounts of fumaric acid and, to a lesser extent, succinic acid in the urine. Biochemical studies showed markedly decreased fumarase activity (below 10% of normal) in all tissues, including fibroblasts. The defect involves both cytoplasmic and mitochondrial enzyme activities, consistent with the fact that both isozymes are encoded by a single gene on the long arm of chromosome 1 (Tolley and Craig, 1975). Western blots using antibodies against pig heart fumarase showed almost complete lack of cross-reacting material (CRM) in tissues from one patient (Gellera et al., 1990). A partial fumarase deficiency in fibroblasts from the asymptomatic parents of the same patient suggests autosomal recessive transmission. 2.

a-Ketoglutarate Dehydrogenase Deficiency

a-Ketoglutarate dehydrogenase deficiency is accompanied by deficiencies of dihydrolipoyl dehydrogenase (PDH-E 3) and branched chain ketoacid dehydrogenase because the three enzymes share the same subunit, which has been described above. 3.

Aconitase Deficiency

Aconitase deficiency has been described in a patient with exercise intolerance and myoglobinuria, who also had complex II deficiency (Haller et al., 1991), as described below.

D.

DEFECTS OF OXIDATION-PHOSPHORYLATION COUPLING

Only two patients with nonthyroidal hypermetabolism (Luft disease) have been reported (Luft et al., 1962; Haydar et al., 1971; DiMauro et al., 1976). Both were sporadic with negative family histories and no known consanguinity. Symptoms began in childhood or adolescence with fever, heat intolerance, excessive perspiration, resting tachypnea, and dyspnea, polydipsia, and polyphagia. There was exercise intolerance, but weakness was only mild to moderate. Although basal metabolic rate was greatly increased, thyroid function was normal. Muscle biopsy specimens showed numerous ragged red fibers (RRF) and many enlarged mitochondria with tightly packed cristae and dark osmiophilic inclusions. Studies of oxidative phosphorylation in isolated muscle mitochondria from both patients showed maximal respiratory rate even in the absence of ADP, an indication that respiratory control was lost. Respiration proceeded at a high rate indepen-

MITOCHONDRIAL MYOPATHIES

35

dent of phosphorylation, and energy was lost as heat, causing hypermetabolism and hyperthermia. The genetic basis for Luft disease is unknown, but circumstantial evidence suggests that it may be due to a nuclear gene defect.

E.

DEFECTS OF THE RESPIRATORY CHAIN

The respiratory or electron transfer chain is composed of four multimeric enzymatic complexes (complexes I, II, III, and IV) and two mobile carriers (coenzyme Q and cytochrome c), all embedded in the inner mitochondrial membrane. The electron transfer generates a pH gradient across the inner membrane which creates a proton flow through a fifth enzyme complex (complex V or ATP synthase) promoting ATP synthesis from AD Ρ and P{. Complexes I, III, IV, and V contain some subunits encoded by mtDNA and others encoded by nDNA (Table IV). Accordingly, respiratory chain and ATP synthase defects may result from genetic errors in either the nuclear or the mitochondrial genome. Thus, mitochondrial disorders may manifest as mendelian traits when caused by nDNA mutations, or as sporadic or maternally inherited traits when caused by mtDNA mutations. The usually heteroplasmic nature of mtDNA mutations (i.e. the coexistence of both normal and mutated mtDNA in the same tissue) is one factor responsible for the frequent observation of partial defects of respiratory chain enzymes. Traditional biochemistry has limitations in detecting such defects, as measurement of activity in homogenates gives an average value. In addition, deletions of mtDNA or point mutations in tRNA genes appear to affect mtDNA translation as a whole (see Chapter 3), thus involving multiple enzymes containing mtDNA-encoded subunits. As a result, patients with such defects have deficiencies of multiple respiratory chain complexes. The clinical heterogeneity observed in mitochondrial disorders caused by mtDNA mutations can be explained both by heteroplasmy, as defined above, and by the threshhold effect (i.e. a certain minimum proportion of mutant mtDNA must be present for the disease to be expressed in a given tissue). This threshold may vary in different tissues according to the dependence of each tissue on oxidative metabolism. As a result of heteroplasmy, defects of the respiratory chain cause complex, variable, and sometimes undetectable biochemical changes. Recent applications of new techniques in histochemistry, immunohistology, and in situ hybridization have helped define defects of the respiratory chain by allowing studies of single fibers. Nevertheless, even traditional biochemistry can identify deficiencies of respiratory enzymes in a number of patients.

TABLE IV RESPIRATORY CHAIN ENZYMES AND THEIR PROPERTIES

Complex

I

II

Ill

IV

V

Synonym

NADH-CoQ oxidoreductase

Succinate-CoQ oxidoreductase

CoQH2-cytochrome c oxidoreductase

Cytochrome c oxidase

ATP synthetase

nDNA subunits mtDNA subunits Prosthetic groups

>30 7 FMN, FeS

4 0 FAD, FeS heme

8 1 6562, 6566 [2Fe-2S]

10 2 Adenine nucleotide

Proton pumping ATP synthesis Inhibitor

+

-

+

10 3 aa 3hemes, Cu a, Cu e3 +

-

-

-

-

Rotenone Piericidin Amobarbital

-

Antimycin A

Cyanide CO

^560

+ + Oligomycin

MITOCHONDRIAL MYOPATHIES I.

37

Defects of Complex I

Complex I, or NADH-coenzyme Q oxidoreductase, the largest complex of the respiratory chain, contains at least 35 different polypeptides, 7 of which are encoded by mtDNA. In addition, there are several nonprotein components, including flavin mononucleotides (FMN), 6 nonheme ironsulfur clusters, and phospholipid (Hatefi, 1985; Walker, 1992). The complex can be resolved into three fractions, a hydrophobic fraction containing 16 subunits, a hydrophilic fraction containing 6 subunits, and a hydrophilic flavoprotein fraction containing 3 polypeptides. There is no chromosomal assignment for any of the nDNA-encoded human complex I subunits, but genetic mapping of complementation groups for complex I deficient Chinese hamster fibroblasts suggested that some subunits are encoded by genes on the X chromosome (Day and Scheffler, 1982). A block at the level of complex I impedes the oxidation of NADH formed in the Krebs cycle, whereas oxidation of FADH derived from the succinate dehydrogenase (SDH) reaction should not be affected because it is mediated by complex II. This has been an important diagnostic clue to complex I deficiency using Polarographie studies of freshly isolated mitochondria. Defects of complex I have also been documented by measurement of partial enzyme reactions, such as rotenone-sensitive N A D H - C o Q oxidoreductase, N A D H cytochrome c oxidoreductase, or NADH-dehydrogenase in tissue extracts or in mitochondrial fractions. When one considers the large number of proteins comprising complex I, it is hardly surprising that complex I deficiency is very heterogeneous from a clinical point of view. Defects of complex I have been described in approximately 25 patients and seem to cause two main syndromes: a pure myopathy with exercise intolerance and myalgia, or a multisystem disorder. Myopathy, with exercise intolerance followed by fixed weakness, can start in childhood or adult life (Morgan-Hughes et al., 1988). The tissuespecific nature of this disorder suggests that one or more of the nDNAencoded complex I subunits may be tissue specific, a concept reinforced by the demonstration that complex I activity was normal in the liver of a patient with myopathy (Watmaugh et al., 1989). Immunoblot analysis in a few patients, however, has shown a generalized decrease of all bands rather than a selective defect of any one band (Morgan-Hughes et al., 1988; Bet et al., 1990). In agreement with the postulated nuclear nature of these disorders, family history is usually negative or compatible with mendelian inheritance. One exception is a patient whose mother and two siblings had died of cardiorespiratory failure (Watmaugh et al., 1990). Patients with multisystem disorders are clinically heterogeneous, some presenting in infancy with a fatal infantile form of the disease and others

38

SARA SHANSKE AND SALVATORE DIMAURO

with a less severe encephalomyopathy with onset in childhood or adult life. The fatal infantile form is characterized by severe congenital lactic acidosis, psychomotor delay, diffuse hypotonia and weakness, cardiopathy, and cardiorespiratory failure causing death in the neonatal period (Moreadith et al., 1984; Hoppel et al., 1987; Robinson et ai, 1986). In two patients, complex I deficiency was documented in multiple organs (Moreadith et al., 1984; Hoppel et al., 1987). In one infant, the biochemical defect was further localized between FMN and CoQ, and electron paramagnetic resonance (EPR) spectroscopy of liver submitochondrial particles showed almost complete absence of the iron-sulphur clusters of complex I (Moreadith et al., 1984). Immunological studies using antibodies against the whole complex or against the four main subunits of the iron-protein fragment showed selective absence of the 75- and 13 kDa polypeptides (Moreadith et al., 1987). In one patient, a deficiency of a 20 kDa nDNAencoded subunit of complex I was demonstrated in fibroblasts (Slipetz et al., 1991b). The molecular defect is not known in any of these cases, but circumstantial evidence indicates that it is due to nDNA defects. This is based on the lack of maternal inheritance, asymptomatic parents, in one case consanguineous parents (Robinson et ai, 1986, case 2), and presence of both affected and nonaffected siblings. Mitochondrial encephalomyopathy with onset in childhood or adult life can occur with variable combinations of multiple symptoms and signs, including ophthalmoplegia, seizures, dementia, ataxia, neurosensory hearing loss, pigmentary retinopathy, sensory neuropathy, and involuntary movements (Morgan-Hughes et al., 1988). This heterogeneous group is likely to include some patients with nDNA and others with mtDNA defects because most cases were reported before systematic analysis of mtDNA was conducted. Complex I deficiency has also been reported in disorders known to be associated with mutations in mtDNA. Leber's hereditary optic neuropathy (LHON) was the first disorder in which a point mutation in mtDNA was documented: This G to A transition results in the substitution of a histidine for a highly conserved arginine in subunit 4 of complex I (Wallace et al., 1988a). A total of 11 different mtDNA point mutations have now been reported to be associated with LHON, 8 of which affect subunits of complex I (Wallace et al., 1991). This disorder exemplifies the difficulties in correlating mtDNA mutations with biochemical phenotype, as there have been only a few instances where a decrease in complex I activity could be demonstrated in patients with LHON (Parker et al., 1989; Howell et al., 1991; Larsson et al., 1991). A specific defect of complex I has also been suggested in patients with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) (Kobayashi etal., 1987; Ichiki et

mtDNA MODIFICATIONS IN DISEASE AND AGING

39

al., 1988; Koga et al., 1988). However, other reports have described a more widespread biochemical impairment of the respiratory chain (Ciafaloni et al., 1992; Goto et al., 1992), which would be in keeping with the molecular defect, a point mutation in the tRNALeu (UUR) gene of mtDNA. Other clinical phenotypes associated with complex I deficiency include a case of Alper's disease (Prick et al., 1981) and a patient with the clinical diagnosis of Leigh syndrome, but without neuropathologic or magnetic resonance imaging (MRI) documentation of the characteristic lesions (van Erven et al., 1987). In patients with typical Parkinson's disease, a defect of complex I was found in several tissues, including the substantia nigra and the striatum (Schapira et al., 1992), but the pathogenic significance of these findings remains uncertain. Complex I activity was also decreased in platelets from five patients with Huntington's disease (Parker et al., 1990a), but it remains to be established whether the defect is primary or secondary and whether it is expressed in brain. 2.

Defects of Complex II

Complex II, or succinate-coenzyme Q oxidoreductase is composed of four nuclear-encoded polypeptides, and is the only respiratory chain enzyme complex that does not have any subunits encoded by mtDNA. In five patients with encephalomyopathy and reported complex II deficiency, the biochemical defect has not been fully characterized and diagnosis was based on decreased succinate-cytochrome c oxidoreductase activity (Sengers et al., 1983; Behbehani et al., 1984; Riggs et al., 1984; Sped et al., 1988). More convincing biochemical evidence of complex II deficiency was provided in two patients with myopathy, who also showed complete lack of SDH stain in muscle biopsies. One was a 22-year-old man with exercise intolerance and myoglobinuria (Haller et al., 1991), in whom studies of isolated muscle mitochondria showed impaired succinate, but normal glutamate, oxidation. A defect of complex II was confirmed by the documentation of SDH deficiency, with low amounts of the 30-kDa (iron-sulfur) and the 13.5-kDa proteins. There was also an associated defect of aconitase but not of other Krebs cycle enzymes. The other patient was a 14-year-old girl with weakness and exercise intolerance, but no myoglobinuria (Garavaglia etal., 1990). 3.

Defects of Complex III

Complex III, or reduced CoQ-cytochrome c oxidoreductase is composed of 11 subunits, including two high molecular weight core proteins, the apoprotein of cytochrome cl, a non-heme iron-sulfur protein known as

40

SARA SHANSKE AND SALVATORE DIMAURO

the Rieske protein, and the apoprotein of cytochrome b, which is encoded by mtDNA (Hatefi, 1985). A block at the level of complex III ought to impair utilization of both NAD-linked and FAD-linked substrates, and this has been confirmed by Polarographie studies. Enzymatic analyses show defects of both succinate-cytochrome c oxidoreductase and N A D H cytochrome c oxidoreductase activities. Some patients show lack of reducible cytochrome b, whereas others have normal cytochrome spectra. In patients with normal amount of reducible cytochrome b, the defect may involve the Rieske protein or coenzyme Q. As for complex I defects, the clinical picture of complex III disorders also falls into two groups: a pure myopathy with onset in childhood or adolescence, or an encephalomyopathy. Patients with myopathy are characterized by exercise intolerance with premature fatigue, often followed by fixed weakness. In two patients, measurement of oxygen consumption during incremental exercise showed minimal increase of oxygen uptake despite higher than normal minute ventilation (Morgan-Hughes et al, 1985; Kennaway, 1988). Reduced minus oxidized spectra have shown low levels of cytochrome b in most (Morgan-Hughes et al., 1977; Hayes et al., 1984; Kennaway et al., 1984), but not all (Reichmann et al., 1986), patients. Immunoblot analysis in muscle from one patient showed decreased amounts of cytochrome b, core proteins 1 and 2, the Rieske protein, and peptide VI, whereas cytochrome cx was present in normal amount (DarleyUsmar et al., 1983). The existence of tissue-specific isoforms is suggested by the observation that complex III activity was normal in fibroblasts and lymphoid cells from a patient with pure myopathy (Darley-Usmar et al., 1986). The tissue specificity of the disease and the lack of maternal inheritance suggest that in patients with myopathy the defect resides in a nDNA gene encoding a muscle-specific subunit. However, in two sporadic patients with myopathy, PEO, and deletions in mtDNA, there was an apparently isolated defect of complex III (Holt et al, 1988; Kennaway, 1988). Among the encephalomyopathies, a fatal infantile form has been described in a child who died at 53 hr (Birch-Machin et al, 1989). Muscle biopsy was histochemically normal, but biochemical studies showed an isolated defect of complex III in muscle, heart, and liver, and a 75% decrease of cytochrome b in muscle. Immunoblot analysis showed a defect of the Rieske protein in muscle, liver, and heart mitochondria, but apparently normal amounts of core proteins, cytochrome cx, and subunit VI in muscle. Both parents were asymptomatic and two siblings were normal, so a defect in a nDNA-encoded subunit appears more likely than an mtDNA mutation. Encephalomyopathies of later onset have been characterized by various combinations of weakness, short stature, dementia, ataxia, sensorineural deafness, pigmentary retinopathy, sensory neuropathy, and pyr-

MITOCHONDRIAL MYOPATHIES

41

amidal signs (Morgan-Hughes et al., 1985; Kennaway, 1988). Family history was noninformative except for two pairs of patients. One involved a father and a son (Spiro et al., 1970), implying a nDNA defect; the other involved a mother and a daughter (Morgan-Hughes et al., 1985), raising the possibility of a mtDNA defect. Complex III deficiency has also been reported in one patient with facioscapulohumeral muscular dystrophy (Slipetz et al., 1991a) and in one patient with a rare and fatal cardiomyopathy known as histiocytoid cardiomyopathy of infancy (Papadimitriou et al., 1984). In the latter case, there was no block of the respiratory chain in muscle or liver from the same patient, again suggesting the existence of tissue-specific isozymes. Defect of Coenzyme Q10. In one family, a primary defect of a tissuespecific isozyme in the C o Q 1 0 synthetic pathway was postulated (Ogasahara et al., 1989). Two sisters had a marked decrease (approximately 5% of normal) in C o Q 1 0 in muscle, with normal levels in serum and cultured fibroblasts. Polarographic analysis showed decreased respiration with both NAD-dependent substrates and succinate, but cytochrome spectra were normal. Clinically, both sisters had normal early milestones, but developed exercise intolerance and slowly progressive weakness of axial and proximal limb muscles, sparing facial and extraocular muscles. Around age 5, brain involvement was manifested by learning disability in both sisters, seizures in one, and cerebellar syndrome in the other. 4.

Defects of Complex IV

Complex IV, or cytochrome c oxidase (COX), the last enzyme complex of the respiratory chain, catalyzes the transfer of reducing equivalents from cytochrome c to molecular oxygen. The energy generated by the reaction sustains a transmembrane proton-pumping activity. The complex contains as redox centers two copper atoms and two unique heme a iron porphyrins bound to a multisubunit protein frame embedded in the inner mitochondrial membrane. The apoprotein is composed of 13 polypeptides. The three largest subunits (I, II, and III) are encoded by mtDNA, are synthesized in mitochondria, and perform both catalytic and proton-pumping activities. The 10 smaller subunits are encoded by nDNA and are synthesized in the cytoplasm. It is believed that these smaller nDNA-encoded subunits have a regulatory role, adjusting COX activity to the metabolic requirements of different tissues. This concept is supported by the demonstration (based on electrophoretic mobility, amino acid sequences, and antibody specificity) that some COX subunits are tissue specific and developmen-

42

SARA SHANSKE AND SALVATORE DIMAURO

tally regulated (Kadenbach et al., 1987). Full-length cDNAs have been obtained for all subunits of human COX (DiMauro et al., 1990; Koga et al., 1990a) and Northern analysis using these cDNAs as probes has shown that only subunits Via and Vila (according to the nomenclature of Kadenbach et al., 1983) are tissue specific. The clinical phenotypes associated with COX deficiency again fall into two main categories, one in which myopathy is the predominant or exclusive manifestation and another in which brain dysfunction predominates. In the first group, the most common disorder is fatal infantile myopathy, characterized by generalized weakness, respiratory insufficiency, and death before 1 year of age. Although heart, liver, and brain are clinically spared, many patients have renal disease with glycosuria, phosphaturia, and aminoaciduria (DeToni-Fanconi-Debre syndrome). In these patients, COX deficiency is confined to skeletal muscle (Bresolin et al., 1985), a finding which supports a defect of a tissue-specific isozyme. Immunologic studies using antibodies against human heart COX holoenzyme showed decreased amount of CRM in muscle from patients, both by enzyme-linked immunosorbent assay (ELISA) and by immunocytochemistry. The subunit composition of the mutant COX appeared normal when examined by S D S PAGE after immunoprecipitation. However, using antibodies against the individual subunits, a selective defect of COX VIIa,b was found by immunocytochemistry in four patients (Tritschler et al., 1991). Pedigree analysis in informative families suggests autosomal recessive transmission. In contrast to this invariably fatal condition, some children with severe myopathy and lactic acidosis at birth improve spontaneously and are vitually normal by age 2 or 3 years (DiMauro et al., 1983; Zeviani et al., 1987; Servidei et al., 1988). Although potentially benign, this myopathy is life threatening in the first few months of life, and patients require vigorous life-sustaining measures. This benign infantile mitochondrial myopathy is due to a reversible COX deficiency; the enzyme activity is markedly decreased (less than 10% of normal) in muscle biopsies taken soon after birth but returns to normal in the first year of life. Immunocytochemistry and immunotitration using antibodies against the holoenzyme show normal amounts of enzyme protein in all muscle biopsies. This finding differs from the decreased CRM in patients with fatal infantile myopathy and may represent a useful prognostic test. Another differential feature has been observed using immunocytochemistry with antibodies against individual subunits of COX, where patients with the benign myopathy paradoxically showed absence of subunit II as well as subunits 7a,b, while those with the fatal myopathy showed absence of only 7a,b (Tritschler et al., 1991). The reversibility of the enzyme defect in these patients suggests that the genetic defect may affect a nDNA-encoded COX subunit which is not only tissue specific, but

mtDNA MODIFICATIONS IN DISEASE AND AGING

43

also development ally regulated. A mutation of a fetal or neonatal isozyme would correct spontaneously when the mature isozyme begins to be expressed. Among the encephalomyopathies associated with COX deficiency, the most important is Leigh syndrome (subacute necrotizing encephalomyelopathy). This is a devastating encephalomyopathy of infancy or childhood (rarely of adult age), characterized by psychomotor retardation, signs of brainstem dysfunction, and abnormal respiration. The pathological hallmark consists of focal, symmetrical necrotic lesions extending from thalamus to pons and involving the inferior olives and the posterior columns of the spinal cord. Microscopically, these "spongy" brain lesions show demyelination, vascular proliferation, and astrocytosis. Muscle histochemistry is normal, but electron microscopy may show increased number of mitochondria. About 40 patients with Leigh syndrome and COX deficiency have been reported, making this the most common biochemical cause of this disorder (DiMauro et al., 1990; Van Coster et al., 1991). The COX deficiency is generalized, but partial, residual activities vary in different tissues yet tend to be similar in the same tissue from different patients (DiMauro et al., 1987; Koga et al., 1990b). Immunologic studies showed that the amount of CRM was variably decreased in different tissues (DiMauro et al., 1987; Koga et al., 1990b), and in fibroblasts the decrease seemed proportional to the loss of COX activity (Glerum et al., 1988). No alteration of the subunit pattern was found in mitochondria isolated from brain or cultured fibroblasts (DiMauro et al., 1987; Glerum et al., 1988). Northern analysis of mRNA extracted from different tissues and hybridized with cDNA probes for eight nDNA-encoded subunits showed messages of normal size and abundance (Lombes et al., 1991). Thus, Leigh syndrome associated with COX deficiency seems to be due to the mutation of a nuclear regulatory gene that controls the assembly or stability of the complex, rather than to the mutation of a gene encoding a specific COX subunit. That the defect involves a nuclear gene was documented by experiments in which COX-deficient fibroblasts from a patient with Leigh syndrome were fused with a variant strain of HeLa cells (Miranda et al., 1989). Prolonged cultivation of the hybrids in appropriate media led to the preferential loss of HeLa cell mtDNA with retention of the patient's mitochondria. Because COX activity was normal in cell clones that had lost almost all the HeLa cell mtDNA, it was concluded that the enzyme defect was corrected by a nDNA-encoded factor from the HeLa parental cell. The involvement of siblings of both sexes and the occurrence of parental consanguinity suggests autosomal recessive transmission (Van Coster et al., 1991). Because the defect is expressed in fibroblasts in most (but not all) patients, prenatal diagnosis ought to be possible in families with one af-

44

SARA SHANSKE AND SALVATORE DIMAURO

fected child and has been performed by enzyme analysis of chorionic villi (Ruitenbeek et al., 1988). COX deficiency has also been described in muscle biopsy specimens from two unrelated cases of Alper's disease (Prick et al., 1983) and in three patients (two of them siblings) with myoneurogastrointestinal disorder and encephalopathy, or MNGIE syndrome (Bardosi et al., 1987; Blake et al., 1990). Partial COX deficiency was also described in platelets from four of five patients with Alzheimer disease (Parker et al., 1990b), but this finding remains to be confirmed in brain. 5.

Defects of Complex V

Complex V (ATP synthase) converts the transmembrane proton gradient generated in the respiratory chain into chemical energy by synthesizing ATP from adenosine diphosphate (ADP) and Pr The complex consists of two parts: a membrane portion F 0 and a catalytic portion Fx, joined by a stalk. Complex V is composed of 12 to 14 subunits, 2 of which (subunits 6 and 8) are encoded by mtDNA (Hatefi, 1985). Defects of ATPase were demonstrated indirectly in two patients by Polarographie analysis of isolated muscle mitochondria (Schotland et al., 1976; Clark et al., 1983). Respiration with different substrates was impaired but returned to normal 2 after addition of the uncoupling agent 2,4-dinitrophenol or Ca +, suggesting that the limiting step involved phosphorylation rather than the respiratory chain. In addition, ATPase activity was decreased and responded poorly to dinitrophenol stimulation. The two patients had different phenotypes. One was a 37-year-old woman with congenital, slowly progressive myopathy, RRF, and a profusion of paracrystalline inclusions in virtually all mitochondria (Schotland et al., 1976). The other was a 17-year-old boy with a multisystem disorder characterized by weakness, ataxia, retinopathy, dementia, and peripheral neuropathy (Clark et al., 1983). Although family history was noninformative in both cases, the syndrome in the second patient is similar to that seen in a family with an mtDNA mutation in subunit 6 of complex V , as described below. A point mutation in the mtDNA gene for subunit 6 of ATP synthase was first described in four members of a family who had a variable combination of symptoms including neuropathy, ataxia, and retinitis pigmentosa, or NARP (Holt et al., 1990). This mutation, a Τ to G substitution at nucleotide 8993, results in an arginine instead of a leucine at codon 156 in ATP synthase 6, a subunit of the F 0 portion. When this mutation is present at high levels (greater than 90%), it manifests as Leigh syndrome (Tatuch et al., 1992, Shoffner et al., 1992; Ciafaloni et al., 1993; Santorelli et al., 1992). To date, no biochemical defect has been associated with this muta-

MITOCHONDRIAL MYOPATHIES

45

tion, but Tatuch and co-workers have pointed out (Tatuch et al., 1992) that the mutation occurs within F 0 , in a region postulated to be directly apposed to a glutamic acid residue of subunit 9 that is required for proton conducting activity. Thus, this mutation may result in altered proton translocation through F 0 , but this phenomenon or any other effect on ATP synthase activity remains to be proven. 6.

Combined Defects of the Respiratory Chain

Potential mechanisms whereby nDNA mutations might cause defects of multiple respiratory chain complexes include mutations of regulatory genes controlling more than one complex; mutations of subunits shared by two or more complexes; mutations affecting the milieu of the complexes, such as the phospholipid composition of the inner membrane; and defects of mitochondrial protein import affecting subunits of multiple complexes. Although none of these mechanisms has been documented in humans, an autosomal recessive nuclear mutation has been shown to cause a combined defect of complexes I and IV in Chinese hamster cells (Malczewski and Whitfield, 1984). A combined defect of complexes I and IV affecting muscle, heart, and liver but sparing brain and kidney was reported in an infant with growth retardation, diffuse weakness, progressive cardiomyopathy, hepatic insufficiency, severe lactic acidosis, and RRF (Zheng et al., 1989). A mutation in the mature form of a nDNA-encoded, tissue-specific, and developmentally regulated subunit shared by complexes I and IV was postulated. Conversely, it was suggested that a mutation affecting the fetal isoform of the same subunit might explain the benign, spontaneously reversible myopathy described by Roodhooft et al., (1986), which was also accompanied by a defect of complexes I and IV. A combined defect of complexes III and IV was reported in an infant who died at 5 months with diffuse weakness, lactic acidosis, and cardiorespiratory insufficiency (Takamiya et al., 1986). Because mtDNA encodes subunits of four of the five complexes, combined defects can be explained by mutations of mtDNA affecting its overall function, such as deletions, point mutations in tRNA genes, or mtDNA depletion. In fact, most of the mtDNA-related diseases are characterized biochemically by combined defects of the respiratory chain. mtDNA Point Mutations ΜΕRRF (Myoclonic Epilepsy with Ragged Red Fibers). MERRF is a mitochondrial encephalomyopathy characterized by myoclonic epilepsy, ataxia, and myopathy with RRF. Maternal inheritance was first suggested by the pattern of transmission (Rosing etal., 1985) and the molecular basis

46

SARA SHANSKE AND SALVATORE DIMAURO

for this disorder has now been established as a point mutation in mtDNA. The majority of patients have an A to G transition at position 8344 in the tRNA Lys (Shoffner et al., 1990; Silvestri et al., 1993),whereas two families have been reported with a Τ to C transition at position 8356 in the same gene (Silvestri et al., 1992; Zeviani et al., 1993). Biochemical studies of muscle have given inconsistent results, with defects of complex III (Berkovic et al., 1989; Morgan-Hughes et al., 1982); complexes II and IV (Berkovic et al., 1989); complexes I and IV (Wallace et al., 1988b); complexes I, III,and IV (Desnuelle et al., 1989); or complex IV alone (Lombes et al., 1989). In one large series, many patients showed partial deficiencies of one or more of the three complexes containing mtDNA-encoded subunits (i.e. complexes I, III, and IV) (Silvestri et al., 1993). However, the data were statistically significant only for complex IV, confirming the selective vulnerability of this complex (Lombes et al., 1989). Combined partial defects of all complexes containing mtDNA-encoded subunits probably occur and can explain the apparent discrepancies among different reports. Consistent with the theory that a mutation in a tRNA gene would result in a defect in protein synthesis, Noer et al., (1991) observed abnormal patterns of mitochondrial translation products in skeletal muscle mitochondria isolated from patients with MERRF. In a study by Chomyn et al., (1991), human cell lines completely depleted of mtDNA by long-term exposure to ethidium bromide (rho° cells, King and Attardi, 1989) were repopulated with mitochondria containing wild-type or mutant mitochondrial genomes. A severe defect in protein synthesis was documented both in the myoblasts of a patient with MERRF and in rho° cells repopulated with mitochondria from the patient's myoblasts. There was a good correlation between the defect of protein synthesis, the deficiencies of respiration and COX activity, and the proportion of mitochondrial genomes with the MERRF mutation. MELAS {Mitochondrial Encephalomyopathy, Lactic Acidosis, and StrokeLike Episodes). MELAS is a maternally inherited mitochondrial disorder defined by recurrent cerebral insults resembling strokes, lactic acidosis and/or RRF, seizures, dementia, recurrent headache and vomiting. As with MERRF, MELAS is associated with a highly specific, although not exclusive, mtDNA point mutation, a G to A transition at nucleotide 3243 in the tRNA Leu (UUR) gene (Goto etal, 1990a; Kobayashi etal., 1990). As in the case of MERRF, biochemical studies have given variable results. A number of investigators have shown complex I deficiency (Kobayashi et al., 1987; Ichiki et al., 1988; Koga et al., 1988), and immunoblot analysis and electron paramagnetic resonance spectra have suggested a disproportionate deficiency of some subunits (including the 75- and the 51-kDa

MITOCHONDRIAL MYOPATHIES

47

subunits) and of the iron-sulfur clusters (Ichiki et al., 1988; MorganHughes et al., 1988). A combined defect of complexes I and IV was found in identical twins that had features of MERRF and MELAS (Byrne et al., 1988). In two large series of MELAS patients, multiple deficiencies were described. Ciafaloni and co-workers (1992) normalized respiratory chain enzyme activities to citrate synthase activity as a measure of mitochondrial volume and found defects of complexes I, III, and especially IV. In the second study, 13 patients had a deficiency of complex I and 7 of complex IV, and 4 had combined defects of complexes I and IV (Goto et al., 1992). Kobayashi et al., (1991) reported a correlation between respiratory impairment (measured by inability to survive in glucose-deficient medium) or COX deficiency (evaluated by cytochemical staining) and the proportion of mtDNA 3243 mutation in muscle cell lines from a patient with MELAS. When cytoplasts from two patients with MELAS were fused to rho° cells (King and Attardi, 1989), it was found that cybrids containing more than 95% mutant mtDNA showed the following alterations: decreased rates of synthesis and steady-state levels of mitochondrial translation products, slightly altered mobility of NADH dehydrogenase subunit I on Polyacrylamide gel electrophoresis, and severe respiratory chain deficiency (King et al., 1992). However, in contrast to in vitro observations (Hess et al., 1991), there was no evidence for any major decrease in the proper termination of transcription at the end of the rRNA genes. A second mtDNA mutation has been described in a small number of patients with MELAS: a Τ to C transition at nucleotide 3271, in the same tRNA Leu (UUR) gene (Goto et al., 1991), but biochemical studies in these cases have not yet been reported. In addition, an A to G transition at nucleotide 11,084, leading to a Thr to Ala amino acid replacement in the ND4 subunit of complex I, has been reported in one family (Lertrit et al., 1992), but this awaits confirmation. Leber's Optic Atrophy. Leber's hereditary optic neuroretinopathy (LHON), a maternally transmitted disease of young adults, is the first disorder in which a point mutation in mitochondrial DNA was documented (Wallace et al., 1988a). The G to A transition at nucleotide 11,778 results in the replacement of a highly conserved arginine in subunit 4 of complex I (ND4). To date, a total of 11 different mtDNA point mutations, affecting subunits of complexes I, III, and IV, have been associated with LHON (Wallace et al., 1991). While genetic linkage studies indicate that the presence of one of the LHON mutations is a necessary condition to produce the clinical symptomatology, additional factors, either genetic or environmental, seem to be required for phenotypic expression. The mechanisms by

48

SARA SHANSKE AND SALVATORE DIMAURO

which so many different mutations can result in the same phenotype of late-onset loss of central vision is unclear. The deleterious consequences of these mutations at the biochemical level are also unclear. Complex I deficiency was reported in one family, but the clinical diagnosis of LHON was uncertain and no molecular genetic studies were performed (Parker et al, 1989). A specific defect of complex I was found in platelets isolated from several individuals of a LHON family with the nucleotide 3460 mutation (G to A), but the biochemical defect was present in both affected and asymptomatic individuals (Howell et al., 1991). Larsson et al. (1991) demonstrated complex I deficiency in five members of a family with the nucleotide 11,778 mutation in ND4 (both affected and asymptomatic) by Polarographie measurement in intact muscle mitochondria while spectrophotometric measurements were normal. Another study failed to demonstrate any biochemical defect in muscle or platelet mitochondrial enzymes in three siblings of a family with the nucleotide 11,778 (G to A) mutation (Cortelli et al., 1991). Therefore, the pathogenic mechanism(s) and biochemical consequences of the mtDNA mutations described in LHON remain to be elucidated. mtDNA Deletions Since they were first described by Holt and co-workers in 1988, largescale single deletions of mtDNA have been associated with three main clinical syndromes: KSS (Zeviani et al., 1988), sporadic PEO with RRF (Moraes et al., 1989), and Pearson's marrow-pancreas syndrome (Rotig et al., 1990). Prior to the description of mtDNA deletions, a number of studies had shown impairment of mitochondrial respiratory chain linked functions in patients with KSS, including generalized deficiency of respiratory chain complexes (Lee et al., 1989). In one study, where intact mitochondria were isolated from muscle, three patients with KSS were found to have a mitochondrial electron transport defect and COX deficiency associated with an excess of the c-cytochromes (Martens et al., 1988). Following the description of mtDNA deletions in patients with KSS (Zeviani et al., 1988), a number of studies found variable defects of respiratory chain complexes in patients, but no consistent correlation was found between site or size of the deletion and biochemical data (Romero et al., 1989; Gerbitz et al., 1990; Goto et al, 1990b; Trounce et al, 1991). Moraes et al (1989) noted that of six respiratory chain enzymes studied, the four containing mtDNA-encoded subunits (COX, succinate-cytochrome c oxidoreductase, rotenone-sensitive NADH-cytochrome c oxidoreductase and NADH dehydrogenase) were significantly lower in patients with mtDNA deletions, whereas succinate dehydrogenase and citrate synthase, enzymes entirely encoded by nDNA, had normal activities. They suggested that any dele-

MITOCHONDRIAL MYOPATHIES

49

tion in the mtDNA could impair mitochondrial protein synthesis as a whole, probably because even the smallest deletion includes one or more tRNA genes. This concept is supported by the demonstration that translation rather than transcription of deleted genomes was impaired in cultured muscle and fibroblasts from patients with KSS (Nakase et al, 1990). However, a few reports suggest that there was a correlation between location of the deletions and biochemical defects, at least in some patients (Shoffner et al., 1989; Holt et al., 1989; Yamamoto étal, 1991; Hammans étal, 1992). For example, four patients with deletions involving complex I subunit genes, together with tRNA or rRNA genes, had Polarographie changes suggesting isolated complex I deficiency (Holt et al, 1989; Hammans et al., 1992). Multiple deletions of mtDNA were first described by Zeviani et al, (1989) in members of a family with adult onset familial PEO and mitochondrial myopathy and have since been described in additional patients, some of whom had heterogeneous clinical features (Zeviani et al, 1990; Servidei et al, 1991; Mizusawa et al, 1988; Cormier et al, 1991; Ohno et al, 1991; Suomalainen et al, 1992). Some patients had onset in childhood (Mizusawa et al, 1988; Cormier et al, 1991), but most did not develop clinical manifestations until adult age (Zeviani et al, 1990; Servidei et al, 1991). Common clinical features are bilateral ptosis and PEO without pigmentary changes of the retina, and generalized muscle weakness. However, two Japanese brothers presented with recurrent myoglobinuria (Ohno et al, 1991). In most cases, transmission of this disorder is autosomal dominant, suggesting that a defect in a nuclear gene affects the mitochondrial genome. Biochemical analysis showed generalized and partial decreases of respiratory chain enzymes containing mtDNA-encoded subunits. In the Italian pedigrees, COX activity was the most severely reduced (50-70% of normal), while the nuclear-encoded citrate synthase activity was unaffected (Zeviani et al, 1990; Servidei et al, 1991). In muscle mitochondria isolated from two patients, rotenone-sensitive NADH cytochrome c reductase activity was most severely affected (10-20% of normal), followed by COX and succinate-cytochrome c reductase (Mizusawa et al, 1988). One boy had markedly reduced NADH-coenzyme Q oxidoreductase activity in muscle mitochondria and decreased oxygen consumption by intact lymphocytes (Cormier et al, 1991). Depletion of mtDNA Depletion of mtDNA is the first hereditary mitochondrial disease characterized by a quantitative rather than a qualitative abnormality of mtDNA. It is also one of only two disorders apparently due to faulty communication between the nuclear and the mitochondrial genomes (i.e., a nDNA defect

50

SARA SHANSKE AND SALVATORE DIMAURO

transmitted by mendelian inheritance but manifesting as an mtDNA alteration) (Table 1). Depletion of mtDNA has been associated with two clinical phenotypes, a congenital mitochondrial disease which is fatal in early infancy (Moraes et al., 1991) and a mitochondrial myopathy of childhood (Tritschler et al., 1992). There is good correlation between clinical severity and degree of mtDNA depletion, with affected infants having less than 10% of the normal quantity of mtDNA and patients with the milder form having 15-35%. Disorders asssociated with mtDNA depletion have variable tissue involvement, and both the mtDNA defect and biochemical abnormalities are observed only in affected tissues. Of the four infants studied by Moraes et al. (1991), one had myopathy, two had myopathy and nephropathy, and one had liver disease. Biochemical studies of isolated muscle mitochondria in the patient with myopathy showed COX deficiency and absence of cytochromes b and aa3 (Boustany et al., 1983). Liver, kidney, brain, heart, and lungs were clinically unaffected and had normal COX activity and cytochrome contents. One infant with myopathy and nephropathy, from whom multiple tissues were available for study, had mtDNA depletion and COX deficiency in muscle and kidney, while liver and brain were normal (Moraes et al., 1991). Combining the data obtained by Moraes et al. (1991) and Tritschler et al. (1992), we can conclude that there is marked COX deficiency (0-14% of normal activity) in affected tissues of infants with mtDNA depletion (more than 90%). When studied, complexes I and III are variably decreased. Patients with later clinical onset, slower progression, and milder depletion (65-85%) also had reduced activities for complexes I, III, and IV, especially when these were normalized to either citrate synthase or SDH. In one patient, muscle biopsies obtained at different stages of the disorder showed a progressive decrease of COX activity, consistent with the clinical downhill course (Tritschler et al., 1992). Transmission of this disorder appears to be autosomal recessive as four pedigrees included more than one affected sibling and parents were normal.

IV.

Concluding Remarks

Great strides have been made in our understanding of mitochondrial diseases in the last three decades and a complementary, mirror-image situation has emerged with respect to nuclear-encoded versus mitochondrialencoded defects. For nuclear defects, it is usually a single enzyme that is affected, and the biochemical classification is clear and unequivocal. However, due to the complexity of nuclear DNA, most of these disorders have not yet been defined at the molecular genetic level. In contrast, great

MITOCHONDRIAL MYOPATHIES

51

progress has been made in the past 5 years in defining disorders of mitochondrial DNA at the molecular level, while the biochemical classification has been problematic. Because of the heteroplasmic nature of these defects, biochemistry alone has not been definitive for diagnostic purposes. Still, certain clues as to what to look for in mtDNA defects have emerged and, together with the clinical picture, have provided patterns useful for diagnosis.

ACKNOWLEDGMENTS This work was supported by Center Grant NS11766 from the National Institutes of Health, a grant from the Muscular Dystrophy Association, and by a generous donation from Libero and Graziella Danesi.

REFERENCES Bardosi, Α., Creutzfeldt, W., DiMauro, S., Felgenhauer, Κ., Friede, R. L., Goebel, H. H., Kohlschutter, Α., Meyer, G., Rahlf, G., Servidei, S., Van Lessen, G., and Wetterling, T. (1987). Acta Neuropathol 74, 248-258. Behbehani, A. W., Goebel, H., Osse, G., Gabriel, M., Langenbeck, U., Berden, J., Berger, R., and Schutgens, R. B. (1984). Eur. J. Pediatr. 143, 67-71. Bennett, M. J., and Hale, D. E. (1992). In "L-Carnitine and its Role in Medicine: From Function to Therapy" (R. Ferrari, S. DiMauro, and G. Sherwood, eds.), pp. 187-206. Academic Press, London. Bennett, M. J., Worthy, E., and Pollitt, R. J. (1987). J. Inherited Metab. Dis. 10, 241-242. Berkovïc, S. F., Carpenter, S., Evans, Α., Karpati, G., Shoubridge, Ε. Α., Andermann, F., Meyer, Ε., Tyler, J. L., Diksic, M., Arnold, D., Wolfe, L. S., Andermann, Ε., and Hakim, A. M. (1989). Brain 112, 1231-1260. Bertini, E., Sabatelli, M., Garavaglia, Β., Burlina, Α. Β., Rimoldi, M., Ricci, E., DionisiVici, C , Bartuli, Α., Sabetta, G., and DiDonato, S. (1990). J. Neurol. Sei. 98, 273. Bet, L., Bresolin, N., Moggio, M., Meola, G., Prelle, Α., Schapira, A. H., Binzoni, T., Chomyn, Α., Fortunato, F. Cerretelli, P., and Scarlato, G. (1990). J. Neurol. 237, 399404. Birch-Machin, M. Α., Shepherd, I. M., Watmaugh, N. J., Sherratt, H. S. Α., Bartlett, K., Darley-Usmar, V. M., Milligan, D. W. Α., Welch, R. J., Aynsley-Green, Α., and Turnbull, D. M. (1989). Pediatr. Res. 25, 553-559. Blake, D., Lombes, Α., Minetti, C , Bonilla, E., Hays, A. P., Lovelace, R. E., Butler, L, and DiMauro, S. (1990). Neurology 40, Suppl. 1, 294. Bougneres, P. F., Saudubray, J. M., Marsac, C , Bernard, O., Odievre, M., and Girard, J. (1981). J. Pediatr. 98, 742-746. Boustany, R. N., Aprille, J. R., Halperin, J., Levy, H., and DeLong, G. R. (1983). Ann. Neurol. 14, 462-470. Bresolin, N., Zeviani, M., Bonilla, E., Miller, R. H., Leech, R. W., Shanske, S., Nakagawa, M., and DiMauro, S. (1985). Neurology 35, 802-812. Brown, R. M., Dahl, H.-H. M., and Brown, G. K. (1989). Genomics 4, 174-181.

52

SARA SHANSKE AND SALVATORE DIMAURO

Byrne, E., Trounce, I., Dennet, X., Gilligan, B., Morley, J. B., and Marzuki, S. (1988). /. Neurol. Sei. 88, 327-337. Chomyn, Α., Meola, G., Bresolin, N., Lai, S. T., Scarlato, G., and Attardi, G. (1991). Mol. Cell. Biol. 11, 2236-2244. Ciafaloni, E., Ricci, E., Shanske, S., Moraes, C. T., Silvestri, G., Hirano, M., Simonetti, S., Angelini, C , Donati, Α., Garcia, C , Martinuzzi, Α., Mosewich, R., Servidei, S., Zammarchi, E., Bonilla, E., DeVivo, D. C , Rowland, L. P., Schon, Ε. Α., and DiMauro, S. (1992). Ann. Neurol. 31, 391-398. Ciafaloni, E., Santorelli, F. M., Shanske, S., Deonna, T., Roulet, E., Janzer, C , Pescia, G., and DiMauro, S. (1993). J. Pediatr. 122, 419-422. Clark, J. B., Hayes, D. J., Byrne, E., and Morgan-Hughes, J. A. (1983). Biochem. Soc. Trans. 11, 626-627. Cormier, V., Rotig, Α., Tardieu, M., Colonna, M., Saudubray, J.-M., and Munnich, A. (1991). Am. J. Hum. Genet. 48, 643-648. Cortelli, P., Montagna, P., Avoni, P., Sangiorgi, S., Bresolin, N., Moggio, M., Zaniol, P., Mantovani, V., Barboni, P., Barbiroli, B., and Lugaresi, E. (1991). Neurology 41, 1211— 1215. Dahl, H.-H. M., Brown, G. K., Brown, R. M., Hansen, L. L., Kerr, D. S., Wexler, I. D., Patel, M. S., De Meirleir, L., Lissens, W., Chun, K., McKay, N., and Robinson, B. H. (1992). Hum. Mutât. 1, 97-102. Darley-Usmar, V. M., Kennaway, N. G., Buist, N. R., and Capaldi, R. A. (1983). Proc. Natl. Acad. Sei. U.S.A. 80, 5103-5106. Darley-Usmar, V. M., Watanabe, M., Uchiyama, Y., Kondo, L, Kennaway, N. G., Gronke, L., and Hamaguchi, H. (1986). Clin. Chim. Acta 158, 253-261. Day, C. E., and Scheffler, I. E. (1982). Somatic Cell Genet. 8, 691-707. Demaugre, F., Bonnefont, J. P., Colonna, M., Cepanec, C , Leroux, J. P., and Saudubray, J. M. (1991). J. Clin. Invest. 87, 859-864. Desnuelle, C , Birch-Machin, M., Pellissier, J. F., Bindoff, L. Α., Ackrell, A. C , and Turnbull, D. M. (1989). Biochem. Biophys. Res. Commun. 163, 695-700. DiDonato, S., Garavaglia, B., Rimoldi, M., and Carrara, F. (1992). In "L-Carnitine and its Role in Medicine: From Function to Therapy" (R. Ferrari, S. DiMauro, and G. Sherwood, eds.), pp. 81-98. Academic Press, London. DiMauro, S., and DeVivo, D. C. (1989). In "Basic Neurochemistry: Molecular, Cellular, and Medical Aspects" (G. J. Siegel, B. W. Agranoff, R. W. Albers, and P. Β. Molinoff, eds.), pp. 647-670. Raven Press, New York. DiMauro, S., and Melis-DiMauro, P. (1973). Science 182, 929-931. DiMauro, S., and Papadimitrou, A. (1986). In "Myology" (A. G. Engel and B. Q. Banker, eds.), pp 1697-1708. McGraw-Hill, New York. DiMauro, S., Bonilla, E., Lee, C. P., Schotland, D. L., Scarpa, Α., Conn, H., and Chance, B. (1976). J. Neurol. Sei. 27, 217-232. DiMauro, S., Nicholson, J. F., Hays, A. P., Eastwood, A. B., Papadimitriou, Α., Koenigsberger, R., and DeVivo, D. C. (1983). Ann. Neurol. 14, 226-234. DiMauro, S., Servidei, S., Zeviani, M., DiRocco, M., DeVivo, D. C , DiDonato, S., Uziel, G., Berry, K., Hoganson, G., Johnsen, S. D., and Johnson, P. C. (1987). Ann. Neurol. 22, 498-506. DiMauro, S., Lombes, Α., Nakase, H., Mita, S., Fabrizi, G. M., Tritschler, H.-J., Bonilla, E., Miranda, A. F., DeVivo, D. C , and Schon, E. A. (1990). Pediatr. Res. 28, 526-541. Eriksson, Β. Ο., Gustafson, Β., Lindstedt, S., and Nordin, I. (1988). Eur. J. Pediatr. 147, 662-663.

MITOCHONDRIAL MYOPATHIES

53

Falik-Borenstein, Ζ., Jordan, S. C , and Saudubray, J. M. (1992). N. Engl. J. Med. 327, 24-27. Finocchiaro, G., Taroni, F., Rocchi, M., Martin, A. L., Colombo, I., Tarelli, G. T., and DiDonato, S. (1991). Proc. Natl. Acad. Sei. U.S.A. 88, 661-665. Garavaglia, Β., Antozzi, C , Girotti, F., Paluchetti, D., Mimoldi, M., Zeviani, M., and DiDonato, S. (1990). Neurology 40, Suppl. 1, 294. Gellera, C , Uziel, G., Rimoldi, M., Zeviani, M., LaVerda, Α., Carrara, F., and DiDonato, S. (1990). Neurology 40, 495-499. Gerbitz, K. D., Obermaier-Kusser, B., Zierz, S., Pongratz, D., Muller-Hocker, J., and Lestienne, P. (1990). /. Neurol. 237, 5-10. Glerum, M., Yanamura, W., Capaldi, R. Α., and Robinson, Β. H. (1988). FEBS Lett. 236, 100-104. Goto, Y.-I., Nonaka, I., and Horai, S. (1990a). Nature (London) 348, 651-653. Goto, Y.-I., Koga, Y , Horai, S., and Nonaka, I. (1990b). J. Neurol. Sei. 100, 63-69. Goto, Y.-I, Nonaka, I., and Horai, S. (1991). Biochim. Biophys. Acta 1097, 238-240. Goto, Y.-I, Horai, S., Matsuoka, T., Koga, Y , Nihei, K., Kobayashi, M., and Nonaka, I. (1992). Neurology 42, 545-550. Hale, D. E., and Thorpe, C. (1989). Pediatr. Res. 25, 199A. Haller, R. G., Henriksson, K. G., Jorfeldt, L., Hultman, E., Wibon, R., Sahlin, K., Areskog, N.-H., Gunder, M., Ayyad, K., Blomqvist, C. G., Hall, R. E., Thuillier, P., Kennaway, N. G., and Lewis, S. F. (1991). J. Clin, invest. 88, 1197-1206. Hammans, S. R., Sweeney, M. G., Holt, I. J., Cooper, J. M., Toscano, Α., Clark, J. B., Morgan-Hughes, J. Α., and Harding, A. E. (1992). /. Neurol. Sei. 107, 87-92. Hatefi, Y (1985). Annu. Rev. Biochem. 54, 1015-1069. Haydar, Ν. Α., Conn, H. L., Afifi, Α., Wakid, N., Ballas, S., and Fawaz, K. (1971). Ann. Intern. Med. 74, 548-558. Hayes, D. J., Lecky, B. R. F., Landon, D. N., Morgan-Hughes, J. Α., and Clark, J. B. (1984). Brain 107, 1165-1177. Hess, J. F., Parisi, Μ. Α., Bennet, J. L. and Clayton, D. A. (1991). Nature (London) 351, 236-239. Ho, L., Javed, Α. Α., Pepin, R. Α., Thekkumkara, T. J., Raefsky, C , and Mole, J. E. (1988). Biochem. Biophys. Res. Commun. 150, 904-908. Holt, I. J., Harding, A. E., and Morgan-Hughes, J. A. (1988). Nature (London) 331, 717719. Holt, I. J., Harding, A. E., Cooper, J. M., Schapira, Α. H. V., Toscano, Α., Clark, J. B., and Morgan-Hughes, J. A. (1989). Ann. Neurol. 26, 699-708. Holt, I. J., Harding, A. E., Petty, R. K., and Morgan-Hughes, J.A. (1990). Am. J. Hum. Genet. 46, 428-433. Hoppel, C. L., Kerr, D. S., Dahms, B., and Roessman, U. (1987). J. Clin. Invest. 80, 71-77. Howell, N., Bindoff, L., McCullogh, D. Α., Kubacka, I., Poulton, J., MacKey, D., Taylor, L., and Turnbull, D. M. (1991). Am. J. Hum. Genet. 49, 939-950. Ichiki, T., Tanaka, M., Nishikimi, M., Suzuki, H., Ozawa, T., Kobayashi, M., and Wada, Y (1988). Ann. Neurol. 23, 287-294. Kadenbach, B., Ungibauer, B. M., Jarousch, J., Buge, U., and Kuhn- Nentwig, L. (1983). Trends Biochem. Sei. 8, 398-400. Kadenbach, Β., Kuhn-Nentwig, L., and Buge, U. (1987). Curr. Top. Bioenerg. 15, 114-161. Kelly, D. P., Whelan, A. J., Ogden, M. L., Alpers, R., Zhang, Ζ., Bellus, G., Gregersen, Ν., Dorland, L., and Strauss, A. W. (1990). Proc. Natl. Acad. Sei. U.S.A. 87, 9236-9240. Kennaway, N. G. (1988). /. Bioenerg. Biomembr. 20, 325-352.

54

SARA SHANSKE AND SALVATORE DIMAURO

Kennaway, N. G., Buist, N. R., Darley-Usmar, V. M., Papadimitriou, Α., DiMauro, S., Kelley, R. I., Capaldi, R. Α., Blank, Ν. K., and D'Agostino, A. (1984). Pediatr. Res. 18, 991-999. King, M. P., and Attardi, G. (1989). Science 246, 500-503. King, M. P., Koga, Y., Davidson, M., and Schon, Ε. Α. (1992). Mol. Cell. Biol. 12, 480-490. Kobayashi, M., Morishita, H., Sugiyama, Ν., Yokochi, Κ., Nakano, M., Wada, Y , Hotta, Y , Terauchi, Α., and Nonaka, I. (1987). J. Pediatr. 110, 223-227. Kobayashi, Y , Momoi, M. Y , Tominaga, K., Momoi, T., Nihei, K., Yanagisawa, M., Kagawa, Y , and Ohta, S. (1990). Biochem. Biophys. Res. Commun. 173, 816-822. Kobayashi, Y , Momoi, M. Y , Tominaga, K., Shimoizumi, H., Nihei, K., Yanagisawa, M., Kagawa, Y , and Ohta, S. (1991). Am. J. Hum. Genet. 49, 590-599. Koga, Y , Nonaka, L, Kobayashi, M., Toiyo, M., and Nihei, K. (1988). Ann. Neurol. 24, 749756. Koga, Y , Fabrizi, G. M., Mita, S., Arnaudo, E., Lomax, M. I., Aqua, M. S., Grossman, L. L, and Schon, E. A. (1990a). Nucleic Acids Res. 18, 684. Koga, Y , Nonaka, L, Nakao, M., Yoshino, M., Tanaka, M., Ozawa, T., Nakase, H., and DiMauro, S. (1990b). J. Neurol. Sei. 95, 63-76. Kolvraa, S., Gregersen, N., Christensen, Ε., and Holbolth, Ν. (1982). Clin. Chim. Acta 126, 53-67. Kuylenstierna, E. L. (1970). "Outer Membrane of Mitrochondria," pp. 172-210. Van Nostrant-Reinhold, New York. Larsson, N.-G., Andersen, O., Holme, E., Oldfors, Α., and Wahlström, J. (1991). Ann. Neurol. 30, 701-708. Lee, C. P., Martens, M. E., Peterson, P. L., Tsang, S. H., and Sciamanna, M. (1989) In "Molecular Basis of Membrane-Associated Diseases" (A. Azzi, Z. Drahota, and S. Papa, eds.), pp. 167-182. Springer-Verlag, Berlin. Lertrit, P., Noer, A. S., Jean-François, M. J. Β., Kapsa, R., Dennett, X., Thyagarajan, D., Lethlean, K., Byrne, E., and Marzuki, S. (1992). Am. J. Hum. Genet. 51, 457-468. Lombes, Α., Mendell, J. R., Nakase, H., Barohn, R. J., Bonilla, E., Zeviani, M., Yates, A. J., Omerza, J., Gales, T. L., Nakahara, K., Rizzuto, R., Engel, W. K., and DiMauro, S. (1989). Ann. Neurol. 26, 20-33. Lombes, Α., Nakase, H., Tritschler, H.-J., Kadenbach, Β., Bonilla, E., DeVivo, D. C , Schon, Ε. Α., and DiMauro, S. (1991). Neurology 41, 491-498. Luft, R., Ikkos, D., Palmieri, G., Ernster, L., and Afzelius, B. (1962). /. Clin. Invest. 41, 1776-1804. Malczewski, R. M., and Whitfield, C. D. (1984). /. Biol. Chem. 259, 11103-11113. Martens, M. E., Peterson, P. L., Lee, C. P., Nigro, Μ. Α., Hart, Z., Glasberg, M., Hatfield, J. S., and Chang, C. H. (1988). Ann. Neurol. 24, 630-637. Miranda, A. F., Ishii, S., DiMauro, S., and Shay, J. W. (1989). Neurology 39, 697-702. Mizusawa, H., Watanabe, M., Kanazawa, L, Nakanishi, T , Kobayashi, M., Tanaka, M., Suzuki, H., Nishikimi, M., and Ozawa, T. (1988). /. Neurol. Sei. 86, 171-184. Moraes, C. T , DiMauro, S., Zeviani, M., Lombes, Α., Shanske, S., Miranda, A. F., Nakase, Η., Bonilla, E., Werneck, L. C , Servidei, S., Nonaka, L, Koga, Y , Spiro, A. J., Brownell, A. K. W., Schmidt, B., Schotland, D. L., Zupanc, M., DeVivo, D. C , Schon, Ε. Α., and Rowland, L. P. (1989). N. Engl. J. Med. 320, 1293-1299. Moraes, C. T., Shanske, S., Tritschler, H.-J., Aprille, J. R., Andreeta, F., Bonilla, E., Schon, Ε. Α., and DiMauro, S. (1991). Am. J. Hum. Genet. 48, 492-501. Moreadith, R. W., Batshaw, M. L., Ohnishi, T., Kerr, D., Knox, B., Jackson, D., Hruban, R., Olson, J., Reynafarje, B., and Lehninger, A. L. (1984). /. Clin. Invest. 74, 685-697.

MITOCHONDRIAL MYOPATHIES

55

Moreadith, R. W., Cleeter, M. W., Ragan, C. I., Batshaw, M. C , and Lehninger A. L. (1987). J. Clin. Invest. 79, 463-467. Morgan-Hughes, J. Α., Darveniza, P., Kahn, S. Ν., Landon, D. Ν., Sherratt, R. M., Land, J. M., and Clark, J. B. (1977). Brain 100, 617-640. Morgan-Hughes, J. Α., Hayes, D. J., Clark, J. B., Landon, D. N., Swash, M., Stark, R. J., and Rudge, P. (1982). Brain 105, 553-582. Morgan-Hughes, J. Α., Hayes, D. J., Cooper, M., and Clark, J. B. (1985). Biochem. Soc. Trans. 13, 648-650. Morgan-Hughes, J. Α., Schapira, Α. H. V., Cooper, J. M., and Clark, J. B. (1988). J. Bioenerg. Biomembr. 20, 365-382. Munnich, Α., Saudubray, J. M., Taylor, J., Charpentier, C , Marsac, C , Rocchiccioli, F., Amedee-Manesme, O., Coude, F. X., Frezal, J., and Robinson, B. H. (1982). Acta Paediatr. Scand. 71, 167-171. Nakase, H., Moraes, C. T., Rizzuto, R., Lombes, Α., DiMauro, S., and Schon, E. A. (1990). Am. J. Hum. Genet. 46, 418-427. Noer, A. S., Sudoyo, H., Lertrit, P., Thyagarajan, D., Utthanaphol, P., Kapsa, R., Byrne, E., and Marzuki, S. (1991). Am. J. Hum. Genet. 49, 715-722. Ogasahara, S., Engel, A. G., Frens, D., and Mack, D. (1989). Proc. Natl. Acad. Sei. U.S.A. 86, 2379-2382. Ohno, K., Tanaka, M., Sahashi, K., Ibi, T., Sato, W., Yamamoto, T., Takahashi, Α., and Ozawa, T. (1991). Ann. Neurol. 29, 364-369. Papadimitriou, Α., Neustein, H. B., DiMauro, S., Stanton, S., and Bresolin, N. (1984). Pediatr. Res. 18, 1023-1028. Parker, W. D., Oley, C. Α., and Parks, J. A. (1989). N. Engl. J. Med. 320, 1331-1333. Parker, W. D., Boyson, S. J., Luder, A. S., and Parks, J. K. (1990a). Neurology 40, 12311234. Parker, W. D., Filley, C. M., and Parks, J. K. (1990b). Neurology 40, 1302-1303. Prick, M. J. J., Gabreels, F. J. M., Renier, W. O., Trijbels, J. M. F., Sengers, R. C. Α., and Slooff, J. L. (1981). Arch. Neurol. (Chicago) 38, 767-772. Prick, M. J. J., Gabreels, F. J. M., Trijbels, J. M. F., Janssen, A. J. M., LeCoultre, R., Van Dam, K., Jaspar, H. H. T., Ebels, E. J., and op de Coul, A. A. W. (1983). Clin. Neurol. Neurosurg. 85, 57-70. Reichmann, H., Rohkamm, R., Zeviani, M., Servidei, S., Ricker, K., and DiMauro, S. (1986). Arch. Neurol. (Chicago) 43, 957-961. Rhead, W. J., Amendt, Β. Α., Fritchman, K. S., and Felts, S. J. (1983). Science 221, 73-75. Riggs, J. E., Schochet, S. S., Jr., Fakadej, Α. V., Papadimitriou, Α., DiMauro, S., Crosby, T. W., Gutmann, L., and Moxley, R. T. (1984). Neurology 34, 48-53. Robinson, Β. H. (1989). In "The Metabolic Basis of Inherited Disease" (C. R. Scriver, A. L. Beaudet, W. Sly, and D. Valle, eds.), 6th ed., pp. 869-888. McGraw-Hill, New York. Robinson, Β. H., Taylor, J., and Sherwood, W. G. (1977). Pediatr. Res. 11, 1198-1202. Robinson, Β. H., Taylor, J., Kahler, S. G., and Kirkman, Η. Ν. (1981). Eur. J. Pediatr. 136, 35-39. Robinson, Β. H., Ward, J., Goodyer, P., and Beaudet, A. (1986). J. Clin. Invest. 77, 14221427. Robinson, B. H., MacKay, N., Petrova-Benedict, R., Ozalp, I., Coskun, T , and Stacpoole, P. W. (1990). J. Clin. Invest. 85, 1821-1824. Rocchiccioli, F., Wanders, R. J. Α., Aubourg, P., Vianey-Liaud, C , Ijlst, L., Fabre, M., Cartier, Ν., and Boughneres, P.-F. (1990). Pediatr. Res. 28, 657-662. Roe, C. R., and Coates, P. M. (1989). In "The Metabolic Basis of Inherited Diseases" (C. R.

56

SARA SHANSKE AND SALVATORE DIMAURO

Scriver, A. L. Beaudet, W. S. Sly, and D. Valle, eds.), 6th ed., pp. 889-914. McGrawHill, New York. Romero, N., Lestienne, P., Marsac, C , Paturneau-Jovas, M., Nelson, I., François, D., Eymard, B., and Fardeau, M. (1989). /. Neurol Sei. 93, 297-309. Roodhooft, A. M., Van Acker, K. J., Martin, J. J. Ceuterick, C , Schölte, H. R., and LuytHouwen, I. Ε. M. (1986). Neuropediatrics (Stuttgart) 17, 221-226. Rosing, H. S., Hopkins, L. C , Wallace, D. C , Epstein, C. M., and Weidenheim, Κ. (1985). Ann. Neurol. 17, 228-237. Rotig, Α., Cormier, V., Blanche, S., Bonnefont, J. P., Ledeist, F., Romero, N., Schmitz, J., Rustin, P., Fisher, Α., Savdubray, J. M., and Munnich, A. (1990). J. Clin. Invest. 86, 1601-1608. Ruitenbeek, W., Sengers, R., Albani, M., Trijbels, F., Janssen, Α., Van Diggelen, Ο., and Bakkeren, J. (1988). N. Engl. J. Med. 319, 1095. Santorelli, F. M., Shanske, S., Sciacco, M., Ciafaloni, E., Bonilla, E., DeVivo, D. C , and DiMauro, S. (1992). Ann. Neurol. 32, 467-468. Schapira, Α. Η. V., Mann, V. M., Cooper, J. M., Krige, D., Jenner, P. J., and Marsden, C. D. (1992). Ann. Neurol. (Chicago) 32, S116-S124. Schotland, D. L., DiMauro, S., Bonilla, E., Scarpa, Α., and Lee, C. P. (1976). Arch. Neurol. (Chicago) 33, 475-479. Sengers, R. C. Α., Fischer, J. C , Trijbels, J. M. F., Ruitenbeek, W., Stadhouders, A. M., ter Laak, H. J., and Jaspar, H. H. (1983). Eur. J. Pediatr. 140, 332-337. Servidei, S., Bertini, Ε., Dionisi-Vici, C , Miranda, A. F., Ricci, E., Silvestri, G., Bonilla, Ε., Tonali, P., and DiMauro, S. (1988). Clin. Neuropathol. 7, 209-210. Servidei, S., Zeviani, M., Manfredi, G, Ricci, E., Silvestri, G., Bertini, E., Gellera, C , DiMauro, S., DiDonato, S., and Tonali, P. (1991). Neurology 41, 1053-1059. Shoffner, J. M., Lott, M. T., Voljavec, A. S., Soueidan, S. Α., Costigan, D. Α., and Wallace, D. C. (1989). Proc. Natl. Acad. Sei. U.S.A. 86, 7952-7956. Shoffner, J. M., Lott, M. T., Lezza, A. M. S., Seibel, P., Ballinger, S. W., and Wallace, D. C , (1990). Cell (Cambrigde, Mass.) 61, 931-937. Shoffner, J. M., Fernhoff, P. M., Krawiecki, N. S. Caplan, D. B., Holt, P. J., Koontz, D. Α., Takei, Y., Newman, N. J., Ortiz, R. G., Polak, M., Ballinger, S. W., Lott, M. T., and Wallace, D. C. (1992). Neurology 42, 2168-2174. Silvestri, G., Moraes, C. T., Shanske, S., Oh, S. J., and DiMauro, S. (1992). Am. J. Hum. Genet. 51, 1213-1217. Silvestri, G., Ciafaloni, E., Santorelli, F. M., Shanske, S., Servidei, S., Graf, W. D., Sumi, M., and DiMauro, S. (1993). Neurology 43, 1200-1206. Slipetz, D. M., Aprille, J. R., Goodyer, P. R., and Rozen, R. (1991a). Am. J. Hum. Genet. 48, 502-510. Slipetz, D. M., Goodyer, P. R. and Rozen, R. (1991b). Am. J. Hum. Genet. 48, 1121-1126. Sped, W., Ruitenbeek, W., and Trijbels, J. M. F. (1988). Eur. J. Pediatr. 147, 418-421. Spiro, A. J., Moore, C. L., Prinaes, J. W., Strasberg, P. M., and Rapin, I. (1970). Arch. Neurol. (Chicago) 23, 103-112. Stanley, C. Α., De Leeuw, S., Coates, P. Α., Vianey-Liaud, C , Divry, P., Bonnefont, J.-P., Saudubray, J.-M., Haymond, M., Trefz, F. K., Breningstall, G. N., Wappner, R. S., Byrd, D. J., Sansaricq, C , Tein, L, Grover, W., Valle, D., Rutledge, S. L., and Treem, W. R. (1991). Ann. Neurol. 30, 709-716. Suomalainen, Α., Majander, Α., Haltia, M, Somer, H., Lonnquist, J., Savontaus, M. L., and Peltonen, L. (1992). /. Clin. Invest. 90, 61-66. Takamiya, S., Yanamura, W., Capaldi, R. Α., Kennaway, N. G., Bart, R., Sengers, R. C. Α., Trijbels, J. M. F., and Ruitenbeek, W. (1986). Ann. N.Y. Acad. Sei. 488, 33-43.

MITOCHONDRIAL MYOPATHIES

57

Taroni, F., Verderio, E., Fiorucci, S., Cavadini, P., Finnocchiaro, G., Uziel, G., Lamantea, E., Gellera, C., and DiDonato, S. (1992). Proc. Natl. Acad. Sei. U.S.A. 89, 84298433. Taroni, F., Verderio, E., Dworzak, F., Willelms, P., Cavadini, P., and DiDonato, S. (1993). Nat. Genet. 4, 314-319. Tatuch, Y., Christodoulou, J., Feigenbaum, Α., Clarke, J. T. R., Wherret, J., Smith, C , Rudd, N., Petrova-Benedict, R., and Robinson, B. H. (1992). Am. J. Hum. Genet. 50, 852-858. Tein, L, and DiMauro, S. (1992). In "L-Carnitine and its Role in Medicine: From Function to Therapy" (R. Ferrari, S. DiMauro, and G. Sherwood, eds.), pp. 155-184. Academic Press, London. Tein, L, De Vivo, D. C , Bierman, F., Pulver, P., DeMeirleir, L. J., Civitanovic-Sojat, L., Pagon, R. Α., Bertini, Ε., Dionisi-Vici, C , Servidei, S., and DiMauro, S. (1990). Pediatr. Res. 28, 247-255. Tein, L, De Vivo, D. C , Hale, D. E., Clarke, J. T. R., Zinman, H., Laxer, R., Shore, Α., and DiMauro, S. (1991). Ann. Neurol. 30, 415-419. Tolley, E., and Craig, I. (1975). Biochem. Genet. 13, 866-883. Treem, W. R., Stanley, C. Α., Finegold, D. N., Hale, D. E., and Coates, P. M. (1988). N. Engl. J. Med. 319, 1331-1336. Treem, W. R., Stanley, C. Α., Hale, D. E., Leopold, Η. B., and Hyams, J. S. (1991). Pediatrics 87, 328-333. Tritschler, H.-J., Bonilla, E., Lombes, Α., Andretta, F., Servidei, S., Schneyder, Β., Miranda Α. F., Schon, Ε. Α., Kadenbach, Β., and DiMauro, S. (1991). Neurology 41, 300305. Tritschler, H.-J., Andreeta, F., Moraes, C. T., Bonilla, E., Arnaudo, E., Danon, M. J., Glass, S., Zelaya, B. M., Vamos, E., Telerman-Toppet, N., Shanske, S., Kadenbach, Β., DiMauro, S., and Schon, Ε. Α. (1992). Neurology 42, 209-217. Trounce, I., Byrne, E., Marzuki S., Dennet, X., Sudoyo, H., Mastaglia, F., and Berkovic, S. F. (1991). /. Neurol. Sei. 102, 92-99. Van Coster, R., Lombes, Α., DeVivo, D. C , Chi, T. L., Dodson, W. E., Rothman, S., Orrecchio, E. J., Grover, W., Berry, G. T., Schwartz, J. F., Habib, Α., and DiMauro, S. (1991). /. Neurol. Sei. 104, 97-111. Van Erven, P. M. M., Gabreels, F. J. M., Ruitenbeek, W., Renier, W. O., and Fischer, J. C. (1987). Arch. Neurol. (Chicago) 44, 775-778. Walker, J. E. (1992). Q. Rev. Biophys. 25, 253-324. Wallace, D. C , Singh, G., Lott, M. T., Hodge, J. Α., Schurr, T. G., Lezza, A. M. S., Elsas, L. J., and Nikoskelainen, Ε. K. (1988a). Science 242, 1427-1430. Wallace, D. C , Zheng, X., Lott, M. T., Shoffner, J. M., Hodge, J. Α., Kelley, R. L, Epstein, C. M., and Hopkins, L. C. (1988b). Cell (Cambridge, Mass.) 55, 601-610. Wallace, D. C , Lott, M. T., Torroni, Α., and Shoffner, J. M. (1991). Cytogenet. Cell Genet. 58, 1103-1123. Wanders, R. J. Α., Ijlst, L., Van Gennip, A. H., Jakobs, C , De Jager, J. P., Dorland, L., Van Sprang, F. J., and Duran, M. (1990). /. Inherited Metab. Dis. 13, 311-314. Watmaugh, N. J., Birch-Machin, M. Α., Bindoff, L. Α., Aynsley-Green, Α., Simpson, Κ., Ragan, C. L, Sherratt, H. S. Α., and Turnbull, D. M. (1989). Biochem. Biophys. Res. Commun. 160, 623-627. Watmaugh, N. J., Bindoff, L. Α., Birch-Machin, M. Α., Jackson, S., Bartlett, K., Ragan, C. L, Poulton, J., Gardiner, R. M., Sherratt, H. S. Α., and Turnbull, D. M. (1990). J. Clin. Invest. 85, 177-184. Yamamoto, M., Clemens, P. R., and Engel, A. G. (1991). Neurology 41, 1822-1828.

58

SARA SHANSKE AND SALVATORE DIMAURO

Zeviani, M., Peterson, P., Servidei, S., Bonilla, E., and DiMauro, S. (1987). Neurology 37, 64-67. Zeviani, M., Moraes, C. T., and DiMauro, S (1988). Neurology 38, 1339-1346. Zeviani, M., Servidei, S., Gellera, C , Bertini, Ε., DiMauro, S., and DiDonato, S. (1989). Nature (London) 339, 309-311. Zeviani, M., Bresolin, N., Gellera, C , Bordoni, Α., Pannacci, M., Amati, P., Moggio, M., Servidei, S., Scarlato, G., and DiDonato, S. (1990). Am. J. Hum. Genet. 47, 904-914. Zeviani, M., Muntoni, F., Savarese, N., Serra, G., Tiranti, V., Carrara, F., Mariotti, C , and DiDonato, S. (1993). Eur. J. Hum. Genet. 1, 80-87. Zheng, X., Shoffner, J. M., Lott, Μ. Α., Voljavec, A. S., Krawiecki, N. S., Winn, K., and Wallace, D. C. (1989). Neurology 39, 1203-1209. Zinn, A. B., Kerr, D., and Hoppel, C. L. (1986). N. Engl. J. Med. 315, 469-475.

CURRENT TOPICS IN BIOENERGETICS, VOLUME 17

Mitochondrial Myopathies: Genetic Aspects SCOTT W . BALLINGER, JOHN M . SHOFFNER, A N D DOUGLAS C . WALLACE

Emory University School of Medicine Department of Genetics and Molecular Atlanta, Georgia 30322 I.

II.

III.

IV.

V.

Medicine

Introduction A. Mitochondrial OXPHOS B. Mitochondrial DNA C. mtDNA Replication and Transcription D. The Mitochondrial Genetic Code E. Endosymbiotic Origin Human Mitochondrial Genetics A. Unique Characteristics of the mtDNA B. A New Class of Genetic Disease mtDNA Mutations Associated with Mitochondrial Myopathy A. The tRNA Point Mutations B. mtDNA Rearrangement Mutations Contributing Factors A. Genetic Features and Heteroplasmy B. Clinical Features of Protein Synthesis Defects Caused by Deletions and Point Mutations Future Implications References

I.

Introduction

Mendel's laws provide precise rules of segregation and assortment for nuclear genes. Until recently, all forms of genetic disease had been assumed to be explained by Mendel's laws. However, this has changed with the discovery of hereditary diseases due to mitochondrial DNA (mtDNA) mutations (Wallace, 1992a,b). The mitochondrial DNA (mtDNA) was discovered in the early 1960s (Nass and Nass, 1963a,b), and the rules of human mtDNA genetics were defined in the 1970s and 1980s (Wallace et ai, 1975; Giles et al., 1980; 59 Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.

H+ H+

H+

H+

INTERMEMBRANE SPACE

MATRIX

~!t l ~ANT

~m!li .i.i ;ml INTERMEMBRANE SPACE

ANT

~ _ _- - - - - - - - - -...~~

~~e;~f~~~~4~~~:1*1

ANT ATp 4 -

~~fi;I~i.:~]IANT

3ADP + PI

ATp 4 -

~Wj~~~

MITOCHONDRIAL MYOPATHIES: GENETIC ASPECTS

61

Wallace, 1986). Patients with deleterious mtDNA mutations exhibit dysfunction of the tissues with high energetic demands—the brain, heart, skeletal muscle, and pancreas—and so mitochondrial myopathy is a common clinical manifestation of mtDNA diseases. Mitochondrial myopathy is associated with histological abnormalities in skeletal muscle (DiMauro et al., 1985; Petty et al., 1986) termed ragged-red muscle fibers (RRF) where muscle fibers tear during staining and stain red with Gomori modified trichrome due to the proliferation of abnormal mitochondria, frequently with paracrystalline arrays (Engel and Cunningham, 1963). Patients with mitochondrial myopathy may also have a variety of other symptoms including ophthalmoplegia, ptosis, myoclonus, seizures, ataxia, pigmentary retinopathy, optic atrophy, cardiac conduction defects, deafness, and dementia. The onset of disease can range from early childhood to adulthood. Previously, the modes of genetic transmission and pathophysiology of mitochondrial diseases were unclear. However, with the elucidation of the biochemistry and physiology of mitochondrial oxidative phosphorylation (OXPHOS) and the development of mitochondrial genetic paradigms, the nature of these diseases is rapidly being defined.

A.

MITOCHONDRIAL OXPHOS

Mitochondria are the major producers of cellular adenosine triphosphate (ATP) by the process of OXPHOS. The mitochondrial OXPHOS system encompasses five multiple subunit enzyme complexes plus the adenine nucleotide translocator (ANT) (Neckelmann et al., 1987), all of which are embedded in the mitochondrial inner membrane (Fig. 1). Four of these five OXPHOS enzyme complexes contain polypeptide subunits encoded by both the nucleus and the mitochondria. Complex I (NADH dehydrogenase) represents approximately 40 subunits, 7 of which are encoded by the mtDNA. Complex II (succinate dehydrogenase) is composed of only 4 nuclear encoded subunits. Complex III (ubiquinol:cytochrome c oxidoreductase) has 1 mtDNA and 9 nuclear encoded subunits. Complex IV FIG. 1. A schematic of mitochondrial OXPHOS. Electrons entering at complexes I (NADH dehydrogenase) and II (succinate dehydrogenase) are shuttled on to complex III (ubiquinolxytochrome c oxidoreductase) by coenzyme Q. Cytochrome c then shuttles electrons from complex III to complex IV (cytochrome c oxidase) where they are finally donated to molecular oxygen, forming water. ATP synthase (complex V) utilizes the potential energy of the proton gradient created by complexes I, III, and IV to form ATP, which is translocated across the inner membrane through exchange with ADP by the adenine nucleotide translocator (ANT). Abbreviations: F 0, F 0 subunit; F 1? Fj subunit; CoQ, coenzyme Q; cyt c, cytochrome c.

SCOTT W. BALLINGER et al

62

(cytochrome c oxidase) has 3 mtDNA subunits and 10 nuclear subunits. Complex V (ATP synthase) has 2 mtDNA encoded subunits and 10 nuclear subunits (Fig. 1). The first four enzyme complexes (I-IV) comprise the electron transport chain (Fig. 1). Electrons are contributed to the OXPHOS pathway by NADH + H at complex I (NADH dehydrogenase), and via succinate at complex II (succinate dehydrogenase). The electrons are then transferred to the electron carrier coenzyme Q (CoQ or ubiquinone), and reduced CoQ ( Q H 2 or ubiquinol) is oxidized at complex III (ubiquinol: cytochrome c oxidoreductase). The electrons are next shuttled through cytochrome c, then to complex IV (cytochrome c oxidase), and finally to oxygen to form water. The energy released by electron transfer is used to pump protons across the inner membrane at complexes I, III, and IV. This creates a proton (electrochemical) gradient that is utilized by complex V (ATP synthase) as a source of potential energy to condense ADP with inorganic phosphate (Pi) to make ATP (Fig. 1). Each pair of electrons that enters the OXPHOS pathway by way of N A D H + H generates an average of three molecules of ATP. However, electrons donated by the oxidation of succinate (FADH 2) move through complexes II, III, and IV and hence generate approximately two ATP molecules per succinate (FADH 2) oxidized. The generated matrix ATP is exchanged for cytosolic ADP by the ANT (Klingenberg, 1980) and then used for a variety of cellular functions, such as muscle contraction, active transport, cellular repair, or biochemical reactions.

B.

MITOCHONDRIAL

DNA

Using a variety of electron microscopy staining techniques Nass and Nass (1963a,b) first observed mtDNA in chick embryo mitochondria. Moreover, these authors (Nass and Nass, 1963a,b) concluded that mtDNA had properties more similar to that of bacteria than to the chromosomes of the nucleus. This hypothesis was confirmed when the mtDNA was shown to be circular (Nass, 1966; van Brüggen et al, 1966). Almost two decades later, the human mitochondrial genome was sequenced (Anderson et al, 1981), and shown to be 16,569 np in length and to encode 37 genes essential to OXPHOS (Fig. 2): 13 protein coding genes, 2 rRNAs, and 22 tRNAs. The strands of the mtDNA are markedly different. The guanosine rich, heavy (H)-strand encodes 12 OXPHOS polypeptide genes: the ND 1,2,3,4,4L, and 5 genes of complex I; cytochrome b of complex III; the CO I, II, and III genes of complex IV; and the ATPase 6,8 genes of complex V. It also encodes both rRNAs (12S and 16S rRNAs) and

MITOCHONDRIAL MYOPATHIES: GENETIC ASPECTS \ D-Loop Region

63

/

ND5 NDl

ND2

-

COII

r

1

Complex I genes

1

1

(NADH

FEssgra fffiffffia

dehydrogenase)

Complex IV genes (cytochrome c oxidase )

ATPase6 ATPase8

C o m p l e x III g e n e s (ubiquinol : cytochrome c oxidoreductase)

V7À

Complex V genes ( A T P synthase)

|

|

ι I

ι A

Transfer R N A genes

Ribosomal

RNA

genes

FIG. 2. A diagram depicting the organization of the human mtDNA. Genes encoded by the heavy strand are labeled outside of the circle, whereas genes encoded by the light strand are labeled within the circle. Abbreviations: ND, NADH dehydrogenase subunit; C O , cytochrome oxidase subunit; cyt b, cytochrome b\ ATPase, ATP synthase subunit; O h , origin of heavy (H)-strand synthesis; O l , origin of light (L)-strand synthesis; P H, heavy-strand promoter (HSP); P L, light strand promoter (LSP). The 22 tRNA genes are designated by the single letter codes for amino acids (figure reproduced with permission from D . C . Wallace). U U A / G

14 tRNAs (phenylalanine, valine, l e u c i n e , isoleucine, methionine, A G U / c tryptophan, aspartate, lysine, glycine, arginine, histidine, s e r i n e , CUN l e u c i n e , threonine). The cytosine rich, light (L)-strand encodes the remaining protein coding gene (ND 6 of complex I), and 8 tRNAs (proline, UCN glutamate, s e r i n e , tyrosine, cystiene, asparagine, alanine, glutamine).

64

SCOTT W. BALLINGER et al

The human mtDNA also has a 1123 nucleotide noncoding region called the displacement loop (D-loop). The D-loop contains the origin of heavystrand synthesis ( O h ; nucleotides 110-441), and the transcriptional promoters for both heavy (HSP; nucleotides 545-567) and light strands (LSP; nucleotides 392-445). The origin for L-strand synthesis ( O l ; nucleotides 5721-5781) is located within a cluster of tRNAs two thirds of the way around the mtDNA, in the direction of Η-strand synthesis. Since each cell can contain hundreds of mitochondria and there are multiple copies of mtDNA per mitochondrion, there are thousands of mtDNA per cell (Bogenhagen and Clayton, 1974; Oliver and Wallace, 1982; Shuster et al, 1988).

C.

M T D N A REPLICATION AND TRANSCRIPTION

DNA replication commences within the D-loop (Fig. 3) where mitochondrial RNA (mtRNA) polymerase initiates RNA primer synthesis at LSP (Chang and Clayton, 1985). RNA synthesis extends to a series of three conserved sequence blocks (CSBs I, II, and III) near O h . Here the RNA transcript is cleaved at stretches of G residues within CSB II and III by the site-specific endoribonuclease, RNase MRP (mitochondrial RNA processing) enzyme (Chang and Clayton, 1987a,b; Bennett and Clayton, 1990; Stohl and Clayton, 1992). The resulting RNA 3' OH serves as a primer for the mtDNA polymerase (gamma). Once Η-strand DNA synthesis is initiated, the daughter Η-strand is extended to nucleotides 16,157-16,172, where it terminates at the termination associated sequence (TAS). The resulting triple-stranded region contains the new "7S DNA," a metastable intermediate which appears to serve as a replication initiation complex. Further Η-strand elongation is initiated at the 7S DNA and continues around the parental L-strand, displacing the parental Η-strand until two-thirds of the molecule is traversed and O l is exposed. O l forms a hairpin loop structure within the Η-strand (Wong and Clayton, 1985; Hixson etal, 1986) (Fig. 3) and L-strand synthesis begins at a conserved thymidine-rich region (nucleotides 5747-5752) within the loop. A RNA primer is synthesized by mtDNA primase, and RNA polymerization continues until it reaches the highly conserved DNA sequence 3-GGCCG-5' (nucleotides 5766-5770). Then mtDNA polymerase initiates L-strand DNA synthesis (Wong and Clayton, 1985, 1986; Hixson et al, 1986). This transition site is present in human and bovine mtDNAs, but is not seen in rodents (Wong and Clayton, 1985, 1986; Hixson et al, 1986), chickens (Desjardins and Morais, 1990), sea urchins (Jacobs et al, 1988), or Drosophila yakuba (Clary and Wolstenholme, 1985).

MITOCHONDRIAL MYOPATHIES: GENETIC ASPECTS

65

H - Strand Initiation

RNase MRP RNA priming

Transition to H-strand DNA synthesis

•mm - Light Strand Promoter I

I - Conserved Sequence Block

I-Strand Initiation DNA

( Poly-T^ I I Region J \ g f *

Transition to L-Strand DNA synthesis

I.

H I

- Polythymidlne Region I - RNA/DNA Transition Sequence

FIG. 3. The origins of H - and L-strand synthesis. (A) Schematic representation of O H . Mitochondrial RNA polymerase initiates RNA primer synthesis at the light-strand promoter. RNA synthesis continues until it reaches the second conserved sequence block, where transition to DNA synthesis begins. The RNA primer is cleaved from the nascent RNA transcript by RNase MRP near conserved sequence block II. MRP, mitochondrial RNA processing. (B) Schematic representation of O l . When Η-strand synthesis is two thirds complete, a stemloop structure is formed on the displaced parental Η-strand. DNA primase initiates RNA primer synthesis within a polythymidine stretch within the loop. RNA synthesis continues until reaching a conserved RNA/DNA transition sequence, where DNA synthesis of the L-strand begins.

66

SCOTT W. BALLINGER et al

This bidirectional, yet asynchronous replication continues until both daughter strands are complete. DNA ligase then converts them to closed circles (Bogenhagen and Clayton, 1978) and DNA gyrase introduces about 100 negative superhelical turns to the closed circular molecules (Bogenhagen and Clayton, 1978) completing the cycle. One cycle of mtDNA replication requires about 1 hr, with the completed superhelical mtDNAs appearing after about 2 hr (Clayton, 1987). MtDNA replication occurs throughout the cell cycle and all the proteins responsible for these processes are encoded by nucleus. The two promoters (LSP and HSP) of mtDNA transcription are adjacent to each other within the D-loop. Inverted sequences located upstream from both promoters bind mitochondrial transcription factor (mtTFl). MtTFl is required in addition to the mitochondrial RNA (mtRNA) polymerase for efficient transcription (Fisher et al, 1987, 1992; Parisi and Clayton, 1991; Xu and Clayton, 1992). Potential mtTFl binding sites are also located near the CSBs and O h (Clayton, 1992). An additional factor (D'Agostino and Nass, 1992) may facilitate transcriptional initiation by unwinding the duplex molecule. Both strands are transcribed in their entirety, creating polycistronic RNA molecules, which are cleaved into mature species by a RNAse P-like activity (Ojala et al, 1980, 1981; Attardi et al, 1985; Doerson et al, 1985). At high ATP concentrations, the Η-strand transcript can undergo premature termination at a promoter-independent, bidirectional L eu termination site located at the 16S r R N A / t R N A boundary (nucleotide 3229). This results in the preferential synthesis of the 12S and 16S rRNAs that are located between HSP and the terminator (Christianson and Clayton, 1986,1988). All of the factors for mtDNA transcription are encoded within the nucleus. For example, the RNAse MRP is a riboprotein, with both the RNA and protein components encoded in the nucleus. The RNA gene is located on chromosome 9 (Hsieh et al, 1990) and transcribed from a promoter containing the mitochondrial-specific eis element NRF-1 (Evans and Scarpulla, 1990; Topper and Clayton, 1990). Hence, the expression of nuclear encoded mitochondrial genes are controlled primarily by nuclear gene factors.

D.

THE MITOCHONDRIAL GENETIC CODE

The genetic code of the mtDNA differs from nucleus and virtually all other organisms. The mtDNA genetic code is highly degenerate, so that only 22 tRNAs are required for translation (Table I). When uridine is in the wobble position, all 4 members of a codon family can be read by 1 mito-

MITOCHONDRIAL MYOPATHIES: GENETIC ASPECTS

67

TABLE I THE GENETIC CODE OF THE MITOCHONDRIAL D N A °

Codon

fe

Amino acid

AAPur AAPyr ACN AGPur AGPyr AUPur AUPyr

Lysine Asparagine Threonine Termination Serine Methionine Isoleucine

CAPur CAPyr CCN CGN CUN

Glutamine Histidine Proline Arginine Leucine

GAPur GAPyr GCN GGN GUN

Glutamic acid Aspartic acid Alanine Glycine Valine

UAPur UAPyr UCN UGPur UGPyr UUPur UUPyr

Termination Tyrosine Serine Tryptophan Cystiene Leucine Phenylalanine

a

In the nucleus, AGPur codes for arginine, AUA for isoleucine, and UGA for termination. fo The mtDNA genetic code differs from the nuclear code in that AUPyr codes for isoleucine, AUPur codes for methionine, and UGPur codes for tryptophan. Termination codons are UAPur and AGPur. The codons are arranged in alphabetical order. Abbreviations: Pur, purine (A or G); Pyr, pyrimidine (U or C); N, purine or pyrimidine.

chondrial tRNA (Barrell et al., 1980). Pairs of codons can be read using tRNAs with either guanine or uridine in the wobble position. Hence, 8 mitochondrial tRNAs recognize 4 member codon families, while 14 tRNAs recognize codon pairs (Barrell et al., 1979, 1980; Anderson et al., 1981). Specific codons of the mtDNA genetic code have altered function, AUU and AUC code for isoleucine, AUA and AUG for methionine, and UGG

68

SCOTT W. BALLINGER et al.

and UGA for tryptophan (Barrell et al., 1980; Anderson et al, 1981). The termination codons are UAA, UAG, AGA, and AGG. The mtDNAs use the nuclear-cytosol stop codon UGA for tryptophan, and the arginine codons AGA and AGG for stop codons means that mitochondrial genes and mRNAs cannot function in the nuclear-cytosol compartment. This has blocked mitochondrial-to-nuclear gene transfer and served to stabilize the mitochondrial genome (Wallace, 1982).

E.

E N D O S Y M B i O T i c ORIGIN

The identification and characterization of the mitochondrion's independent genome and biosynthetic system has led to the proposal that it arose from an endosymbiotic association between an oxidative photosynthetic bacterium and a nucleated host cell (Margulis, 1970, 1981). Although the endosymbiotic theory of organelle development was originally proposed almost a century ago (Schimper, 1883; Wilson, 1925; Wallin, 1927, Lederberg, 1952), it was not until the discovery of organelle genomes in the 1960s (Nass and Nass, 1963a,b), and until the mitochondria's similarity to prokaryotic eubacteria was reported (Lederberg, 1952), that the theory was seriously considered (Margulis, 1970, 1981). The transfer of genes to the nucleus has decreased the mitochondrial genome complexity, putting many of the critical mitochondrial genes into the nucleus. Consequently, a mitochondrial protein targeting system has evolved to redirect the protein back into the mitochondria. This involves protein genes acquiring a mitochondrial targeting peptide that interacts with a mitochondrial receptor system permitting import. The targeting sequence is then cleaved and the mitochondrial respiratory complexes are assembled on chaperone proteins (Glick and Schatz, 1991; Glick et al., 1992). This symbiotic relationship became permanent with the transfer of genes from the protomitochondria to the protoeukaryotic cell nucleus. Hence, only a few of the OXPHOS subunits and structural RNAs remain in the mtDNA while all other OXPHOS genes and all of the biosynthetic apparatus are encoded by the nucleus.

II.

Human Mitochondrial Genetics

The extra-nuclear location and high copy number of the mtDNA results in a unique genetics. Since the rules of mtDNA inheritance have only recently been elucidated, genetic diseases of the mtDNA were largely

MITOCHONDRIAL MYOPATHIES: GENETIC ASPECTS

69

overlooked and their unusual hereditary behavior was attributed to a variety of Mendelian transmission patterns.

A.

UNIQUE CHARACTERISTICS OF THE M T D N A

Five novel features of the mtDNA result in its unique genetics (Fig. 4). First, the mtDNA is maternally inherited (Dawid and Blackler, 1972; Giles et al., 1980; Case and Wallace, 1981). Only females will pass their mtDNAs to their children, and of those children, only the daughters will transmit their mtDNAs to the next generation. Second, the mtDNA in mammals has a 10- to 20-fold higher rate of mutation than the nucleus (Brown et al., 1979; Miyata et al., 1982; Vawter and Brown, 1986; Neckelmann et al., 1987; Wallace et al., 1987). Hence, gene for gene, the mtDNA is more susceptible to deleterious gene mutations than its nuclear counterpart. The higher rate of mutation is thought to be due to the lack of an efficient DNA repair system, increased susceptibility to oxidative damage (Richter et al., 1988), rapid mutation fixation rates (Hauswirth and Laipis, 1982; Laipis et al., 1988; Koehler etal., 1991), and the relaxed constraints placed upon the mtDNA by its degenerate genetic code. Third, when a new mtDNA mutation occurs, the cell acquires a mixture of mutant and normal molecules, a condition known as "heteroplasmy." During cell division, these mtDNAs are randomly distributed to the daughter cells. As a result, the mtDNA genotype of heteroplasmic cells can drift by random segregation along radiating cell lineages. This process has been studied in detail in somatic cell hybrids using the mtDNA marker for chloramphenicol resistance, which is due to a mutation in the mtDNA 16S rRNA (Wallace, 1986). Fourth, the phenotypic consequence of mtDNA mutations are related to the energetic requirements of the cell or tissue harboring the mutation and the severity of the OXPHOS defect. The most oxidative tissues are the brain, heart, skeletal muscle, and endocrine system. As the OXPHOS capacity of a tissue declines, the energy output ultimately falls below the minimum necessary for cell function. Consequently, the "energetic threshold" is traversed in heteroplasmic cells when the proportion of mutant mtDNAs increase to a level such that ATP output is no longer adequate. Hence, the same proportion of a heteroplasmic mtDNA can create significantly different effects on different tissues and organs sensitive to OXPHOS dysfunction. Moreover, maternal relatives in heteroplasmic families can have quite different proportions of mutant mtDNAs and, hence, very different phenotypes. Finally, OXPHOS capacities decline with age (Trounce et al., 1989; Byrne et al., 1991; Cooper et al., 1992). This is

A

Maternal Inheritance of the MtpNA

D 30

20

10

o

D- MIDHA Type A

II- MIDHA Type B II- MIDHA Type C

B

Repllcatlye segregation

Replication

ATPB ATP I aynt.... (mtONA)

(nDNA)

i

ATP 8 (mtONA)

QXPHQS Declines with Age

E

%OXPHOS

100 r w ""·'

TIuue

Threshold

Level

OXPHOS

CNS

capacity

HHrt/Muacle

(%)

KIdney

Liver - - -.... Smooth Muaci

o

-35 Age (years)

-85

MITOCHONDRIAL MYOPATHIES: GENETIC ASPECTS

71

believed to be the result of accumulated oxidative damage to the mitochondria and its DNA over time (Linnane et al, 1989, 1990; Cortopassi et al., 1992; Corral-Debrinski et al., 1991, 1992; Soong et al, 1992; Wallace, 1992a). Thus, the clinical phenotype of a patient with a mtDNA mutation is a consequence of the nature and severity of the mtDNA mutation, the proportion of mutant mtDNAs, the OXPHOS energy requirements of the tissue, and the age of the individual. B.

A NEW CLASS OF GENETIC DISEASE

Proof that mtDNA mutations cause disease first came from papers published in 1988. One of these papers showed that a mtDNA missense mutation caused Leber's hereditary optic neuropathy (LHON) (Wallace et al., 1988a; Newman, 1991; Brown et al, 1992a), an adult onset form of sudden central vision loss due to optic nerve death. This missense mutation at nucleotide 11,778 (MTND4*LHON 11778) changed a highly conserved arginine to a histidine in the ND4 subunit of complex I (Wallace et al., 1988a). The other papers reported heteroplasmic mtDNA deletions of varying lengths in some patients with mitochondrial myopathy (Holt et al., 1988; Lestienne and Ponsot, 1988). Subsequently, a variety of other deleterious mtDNA mutations have been described. MtDNA rearrangements are now known to be a major cause of Pearson's pancreas and marrow syndrome (Rotig et al., 1988), and the ocular myopathies (Moraes etal, 1989). MtDNA point mutations have been associated with LHON (Wallace et al, 1988a; Howell et al, 1991a,b; Huoponen et al, 1991; Brown et al, 1992b); myoclonic epilepsy and ragFIG. 4. Novel characteristics of the mtDNA. (A) A pedigree showing the maternal transmission of the mtDNA. (B) A diagram depicting replicative segregation from a heteroplasmic progenitor cell with a deleterious mtDNA mutation. Resultant cells can have varying proportions of mutant (black) versus wild-type (white) mtDNAs, while the nuclear component (gray circle) remains constant. (C) When the proportion of mutant mtDNAs are sufficient to inhibit OXPHOS (e.g., 80%), the mitochondrial energy output can fall below the minimal level necessary for normal tissue function resulting in a clinical phenotype. (D) A histogram presenting the relative rates of mutation between mtDNA and nDNA encoded OXPHOS genes. Genes encoded by the mtDNA (ATP 6 and 8) have significantly higher rates of synonymous (open columns) and missense (solid columns) mutations compared to nuclear encoded genes (ATP ß-synthase). (Ε) A hypothetical graph representing OXPHOS decline with age and its progressive effect upon tissues. As OXPHOS capacities decline, the energetic thresholds of each tissue will ultimately become compromised. Individuals born with all normal OXPHOS genes start with a high energetic capacity. Hence, many years are required before the OXPHOS capacity of their tissues falls below any of the energetic thresholds of the various organs (dotted lines). By contrast, individuals born with a mtDNA mutation start at a lower initial OXPHOS level and may cross expression thresholds much earlier in life, creating progressive, degenerative disease. Abbreviation, CNS, central nervous system.

72

SCOTT W. BALLINGER et al

ged red fiber disease, MERRF (Shoffner et al., 1990); mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes, MELAS (Goto et al, 1990; Letrit et al, 1992); neurogenic muscle weakness, ataxia, and retinitis pigmentosa, NARP, together with subacute necrotizing encephalomyelopathy (SNE) or Leigh's syndrome (Holt et al., 1990; Shoffner et al, 1992; Tatuch et al, 1992); adult onset mitochondrial myopathy and cardiomyopathy, MMC (Zeviani et al, 1991); and mitochondrial myopathy, MM (Goto et al, 1992). Most recently, both rearrangement and point mutations have been shown to be associated with diabetes mellitus and deafness (Ballinger et al, 1992a, 1994; van den Ouweland et al, 1992). mtDNA mutations and/or OXPHOS defects may also be associated with some forms of heart disease (Corral-Debrinski et al, 1991; Cortopassi et al, 1992), Alzheimer's disease (Shoffner et al, 1993; Parker et al, 1990), Parkinson's disease (Ikebe et al, 1990; Ozawa et al, 1991; Shoffner et al, 1991, 1993), cancer (Gianni et al, 1980; Abken et al, 1986; Shay and Werbin, 1987; Corral et al, 1989; Heerdt et al, 1990; Zinkewich-Peotti et al, 1991), and even the aging process itself (Miquel et al, 1980; Fleming et al, 1982; Miquel and Fleming, 1986; Piko et al, 1988; Trounce et al, 1989; Cortopassi and Arnheim, 1990; Corral-Debrinsky et al, 1991, 1992a,b; Larsson et al, 1990; Byrne et al, 1991; Wallace, 1992a).

III. mtDNA Mutations Associated with Mitochondrial Myopathy MtDNA mutations that result in mitochondrial myopathy can be caused by either mtDNA point mutations or rearrangements. Point mutations which cause mitochondrial myopathy can include either tRNA mutations (Goto etal, 1990, 1991, 1992; Shoffner etal, 1990; Zeviani et al, 1991) or missense mutations (Letrit et al, 1992). A.

THE T R N A POINT MUTATIONS

Point mutations which cause mitochondrial myopathy are most frequently found in tRNA genes. This is not surprising since tRNA mutations would inhibit mitochondrial translation and thus affect the expression of all mtDNA genes. 1.

Myoclonic Epilepsy and Ragged Red Fiber Disease (MERRF).

MERRF is a chronic, neurodegenerative disease with onset from late childhood to adolescence. It is characterized by myoclonic epilepsy, mito-

MITOCHONDRIAL MYOPATHIES: GENETIC ASPECTS

4T£ III

73

I 3

4

5

6

Myoclonus and Mt myopathy EEG and VER aberrations, Hearing loss, Mt myopathy EEG and VER aberrations, Mt myopathy EEG and VER aberrations Unaffected FIG. 5. A pedigree depicting maternal transmission of myoclonic epilepsy and ragged red fiber disease (MERRF), adapted from Shoffner et al., 1990. Only maternal lineage relatives are affected (II-1 through II-5, and III-l through II-3). Paternal lineage relatives (III-4 through III-6) are not affected. Abbreviations: EEG, electroencephalogram; VER, visual evoked response; Mt, mitochondrial.

chondrial myopathy (RRF), neurosensory hearing loss, and progressive dementia (Fukuhara et al., 1980; Shoffner and Wallace, 1990). Mendelian inheritance of MERRF was excluded in a large pedigree (Fig. 5) by showing that the transmission and characteristics of the disease all met the requirements of a mtDNA mutation (Wallace etal., 1988b). In this pedigree, the proband (III-l) and mother (Π-2) were shown to have OXPHOS deficiencies by three separate modes of analysis. First, the mitochondrial energy generating capacities were measured using exercise stress 31 tests to evaluate anaerobic thresholds. Second, P-NMR spectroscopy was used to determine the ratio of phosphocreatine to inorganic phosphate (PCr/Pj), an indication of the high-energy phosphate metabolism in the muscle. Third, the OXPHOS defect was characterized biochemically

74

SCOTT W. BALLINGER et al.

through respiration studies and OXPHOS enzyme analysis. Moreover, mitochondrial protein synthesis studies in patients lymphoblast lines revealed that synthesis of the larger polypeptides were inhibited (Wallace et al., 1988b). No evidence of a major mtDNA deletion or insertion could be found. Therefore, it was concluded that this disease was caused by a mtDNA point mutation in a tRNA or a rRNA gene (Wallace et al., 1988b). Since variable OXPHOS deficiencies were observed in the maternal lineage relatives, it was concluded that this mutation was heteroplasmic and that replicative segregation was giving the variable OXPHOS defects and different phenotypes. DNA sequencing of the proband's mtDNA identified seven base substitutions that resulted in either OXPHOS gene missense mutations or nucleotide changes in tRNAs (Shoffner et al., 1990). Of these, six could be eliminated by showing that the mtDNA mutation already existed as naturally occurring polymorphisms in human populations (Ballinger et al., 1992b; Brown et al., 1992b; Torroni et al, 1992, unpublished data). Only a nucleotide 8344 A to G transition in a highly conserved nucleotide in the L tRNA ys gene (MTTK*MERRF 8334) remained. This mutation was associated with the disease in 3 independent MERRF pedigrees, but was not found in 75 control mtDNAs (41 Caucasians, 13 African American, and 21 Asians; Shoffner et al., 1990). Furthermore, this mutation was heteroplasmic as determined by direct PCR sequencing and by restriction enzyme analysis using CviJI (Shoffner et al, 1990; Xia et al, 1987). The severity of the MERRF patient phenotype was shown to be related to both the percentage of mutant mtDNAs and the age of the individual. The percentage of mutant mtDNA present in the proband's family (Fig. 5 and Table II) ranged from 73%-97% (Shoffner et al., 1990). While there was a rough association between genotype and phenotype, an additional factor was required to explain the marked phenotypic variability. This proved to be patient age (Shoffner et al., 1990). When family members with similar genotypes were compared, a striking decline was seen with age in the anaerobic threshold and in all of the OXPHOS enzymes. When the samples were classified by age, the phenotype correlated with genotype. For example, for family members 60 to 73 years of age (1-2 and II-l), individual II-1 with 84% mutant mtDNA was more severely affected than individual 1-2 with only 73% mutant mtDNA. For family members 44 to 50 years of age (II-2 through II-5), individuals II-2 and II-3 with 94% to 97% mutant were more affected than their brother, II-5, with 90% mutant. Finally, for individuals in the 19- to 24-year-old group (III-l through III-3), the proband (III-l) and her cousin (III-2), with 94% and 96% mutant mtDNAs, were severely affected, while individual III-3 with 85% mutant was essentially normal. Hence, both the level of heteroplasmy and the age

MITOCHONDRIAL MYOPATHIES: GENETIC ASPECTS

75

TABLE II CLINICAL PHENOTYPE IS RELATED TO PERCENTAGE NUCLEOTIDE 8 3 4 4 PRESENT

Age group 60-73 44-50

19-24

Pedigree member

Percentage nucleotide 8 3 4 4

II-1

84

1-2

73

II-2

94

II-3

97

II-4

96

II-5

90

III-l

94

III-2

96

III-3

85

0

Clinical phenotype Myoclonus, mt myopathy EEG and VER aberrations, EEG and VER aberrations, EEG and VER aberrations, EEG and VER aberrations, EEG and VER aberrations, Myoclonus, mt myopathy EEG and VER aberrations, EEG and VER aberrations

hearing loss, mt myopathy hearing loss, mt myopathy hearing loss, mt myopathy hearing loss, mt myopathy mt myopathy mt myopathy

a

Data from Shoffner et al. ( 1 9 9 0 ) . The percentage nucleotide 8 3 4 4 present and age of the patient relate to the disease phenotype (see text for discussion). The pedigree member numbering corresponds to that of Fig. 5 . Abbreviations: EEG, electroencephalogram; VER, visual evoked response; mt, mitochondrial.

of the individual must be considered in the relationship between OXPHOS defects and phenotypic expression in MERRF. The MTTK*MERRF8344 mutation occurs in the T ^ C loop of the L vs t R N A and thus may affect mitochondrial protein biosynthesis. There is a general correlation between reduction in the mitochondrial polypeptides and the proportion of lysines in each mitochondrial translation product (Wallace et al., 1988b; Shoffner et al., 1990). This is consistent with the concept that the ΎΨ€ loop is important in tRNA-ribosome interaction (Rich and RajBhandary, 1976), and thus its mutation could affect polypeptide elongation by changing the structure of the incoming aminoacyl L vs t R N A bound/elongation factor-GTP complex. Proof that the MERRF protein synthesis defect was caused by the MTTK*MERRF 8344 mutation came from cybrid transfer studies (Bunn et al., 1974) of patient mtDNAs to cells lacking mtDNA (rho° cells) (King and Attardi, 1988, 1989). Transfer of mutant mtDNAs was accompanied by transfer of the cellular defects in respiration, protein synthesis, and OXPHOS function (Chomyn et al., 1991). The transfer of the complete biochemical defect with the mtDNAs thus confirmed that the MTTK*MERRF 8344 mutation causes MERRF. L vs Recently, a second mutation in the t R N A has been reported associated with a MERRF/MELAS overlap syndrome (Silvestri et al., 1993; Zeviani et al., 1993). This is a heteroplasmic Τ to C transition at nucleotide Lvs 8356 (MTTK*MERRF/MELAS 8356) in the T - C stem of t R N A . Simi-

SCOTT W. BALLINGER et al

76

lar to MERRF, the proportion of mutant mtDNA present in patient muscle positively correlated with the disease severity. The MTTK*MERRF/MELAS8356 Τ to C transition was absent in 327 control samples. 2.

Leu UUR

The tRNA ( >

Mutations

The MELAS, MMC, and MM mutations are associated with point mutaL e u U U R tions within the t R N A ( ) gene: MELAS (MTTL1*MELAS3243) at nucleotide 3243 (Goto etal, 1990), MM (MTTL1*MM3250) at nucleotide 3250 (Goto et al, 1992), and MMC (MTTL1*MMC3260) at nucleotide 3260 (Zeviani et al, 1991). MELAS is distinctive among the mitochondrial encephalomyopathies in combining mitochondrial myopathy with transient stroke-like episodes accompanied by MRI and CT structural abnormalities (Goto et al, 1990). Initially, 26 of 31 independent MELAS pedigrees were found to have this heteroplasmic A to G transition at nucleotide 3243, a highly conserved nucleotide in the dihydrouridine loop. Subsequently, MELAS has also been associated with a heteroplasmic Τ to C mutation at nucleotide 3271 (MTTL1 * MEL AS3271) within the anticodon stem in 3 of 40 MELAS patients (Goto et al, 1991). These mutations were absent in all controls. MMC has been associated with a heteroplasmic A to G transition Leu UUR at nucleotide 3260, in the anticodon stem of t R N A ( ) ( Z e v i a n i et al, 1991). This mutation was found in a single family with adult onset myopathy and cardiomyopathy and was absent in 5 normal and 31 disease controls. MM has been associated with a Τ to G mutation at nucleotide 3250, a moderately conserved nucleotide within the dihydrouridine loop (Goto et al, 1992). This mutation has only been seen in a single family. It is not clear why the MTTL1*MELAS3243 mutation creates the MELAS phenotype (Goto et al, 1990), while the MTTL1*MM3250 and MTTL1*MMC3260 mutations in the same gene cause MM (Goto et al, 1992) and MMC (Zeviani etal, 1991). This relationship is made even more complex by the recent report that the nucleotide 3243 mutation can also be associated with diabetes mellitus and deafness (MTTL1*NIDDMD3243) (van den Ouweland et al, 1992). The variation of clinical presentations could reflect differences in the biochemical effect of each mutation, variation in the level and distribution of tissue heteroplasmy, or differences in nuclear-cytoplasmic interactions. Cybrid studies on the MTTL1*MELAS3243 mutation have confirmed the cytoplasmic transmission of the protein synthesis defect. However, this may not be the only pathological feature of this mutation, since it also affects the mtDNA transcription terminator. Both the nucleotide MTTL 1 * MEL AS3243 and MTTL1*MM3250 mutations occur within the L e u U U R transcription terminator binding region at the 16S r R N A / t R N A ( )

MITOCHONDRIAL MYOPATHIES: GENETIC ASPECTS

77

boundary. This has led to the suggestion that these mutations may alter the ratio of rRNAs to mRNAs (Hess et al, 1991; Goto et al., 1992). Support for this hypothesis was obtained by showing that the affinity for the termination factor was reduced in vitro (Hess et al., 1991), though significant deviations from normal Η-strand rRNA to mRNA ratios have not been observed (Chomyn et al., 1992; King et al., 1992; Moraes et al., 1992a). L e u U U R Thus, the pathogenicity of the t R N A ( ) mutation is clearly related to its affect on protein synthesis, but the nature and extent of this abnormality is still unclear.

B.

M T D N A REARRANGEMENT MUTATIONS

The first mtDNA rearrangements discovered in human mtDNA inL vs volved the loss of a 9-bp repeat between COII and t R N A at nucleotides 8272-8289 in certain Asian mtDNAs (Cann and Wilson, 1983; Wrischnik et al., 1987). Since this length polymorphism was within a noncoding intergenic region of the mtDNA, it probably has little affect upon OXPHOS. Subsequently, much larger mtDNA deletions were found associated with Pearson's syndrome (Rotig et al., 1988, 1990; Cormier et al., 1990) and the ocular myopathies (Kearns-Sayre syndrome, KSS; and progressive external ophthalmoplegia, PEO) (Lestienne and Ponsot, 1988; Schon et al., 1989; Shoffner et al., 1989). Pearson's syndrome is a fatal childhood disorder of the bone marrow and pancreas characterized by pancytopenia, sideroblastic anemia, vacuolization of the marrow precursors, and exocrine pancreatic failure (Pearson et al., 1979). Children who survive early pancytopenia usually progress to the severe ocular myopathy (KSS) phenotype (McShane et al., 1991; Baerlocher et al., 1992). Ocular myopathy patients encompass a continuum of phenotypic spectrums. Mildly affected patients with PEO manifest mitochondrial myopathy with RRF and abnormal mitochondria, ophthalmoplegia (paralysis of the eye muscles), and ptosis (droopy eyelids). More severely affected patients with KSS may additionally have pigmentary retinopathy, cardiac conduction defects, cerebellar syndrome, elevated CSF protein, ataxia (poor muscle coordination), hearing loss (neurosensory), and dementia. mtDNA rearrangements are often associated with direct nucleotide repeats (Schon et al., 1989; Shoffner et al., 1989). This has led to two alternative proposals as to their origin. One hypothesis is that direct repeats facilitate slip mispairing (Shoffner et al., 1989), a deletion mechanism also proposed for Shizosaccharomyces pombe mtDNA (Ahne et al., 1988). This mechanism was initially proposed for nuclear frameshift mutations (Streisinger et al., 1966), small deletions and insertions (Farabaugh et al.,

78

SCOTT W. BALLINGER et al. A

Sllp-Raolication Modal of MtDNA Deletions

Segment Degradation

FIG. 6. Possible mechanisms that create mtDNA deletions. (A) Slip-replication mechanism, (1) MtDNA replication illustrating the displacement of the parental Η-strand (heavy line) and the synthesis of the nascent Η-strand (thin line with arrow). The open and shaded boxes indicate direct repeats (DR) 1 and 2, respectively. (2) Nascent Η-strand elongation and parental Η-strand displacement continues, exposing both DRs on the parental H-strand. (3) Pairing of the parental Η-strand DR1 with the L-strand DR2, with strand breakage of the parental Η-strand occurring immediately downstream of DR1. (4) The parental H-strand elongates until it reaches the double-stranded molecule with the 5' phosphate end. Ligation completes the cycle. (5) Completed mtDNA synthesis, yielding deleted and wild-type molecules. (B) Illegitimate elongation model for creating deletions. (1) MtDNA replication illus-

MITOCHONDRIAL MYOPATHIES: GENETIC ASPECTS Β

79

Illegitimate Elongation Model of MtDNA Deletions

trating the displacement of the parental H-strand (heavy line) and the synthesis of the nascent H-strand (thin line with arrow). The open and shaded boxes indicate direct repeats (DR) 1 and 2, respectively. (2) The nascent H-strand becomes displaced when DR1 of the parental H-strand pairs with the complementary DR1 on the L-strand. (3) DR1 of the nascent H-strand pairs with DR2 of the L-strand, creating a deletion. (4) Completion of the replication cycle yields two molecules, a wild-type molecule (left), and a molecule with a deletion in its H-strand and a wild-type L-strand that is folded upon itself. (5) The next replication cycle results in a double-stranded version of the mtDNA deletion.

80

SCOTT W. BALLINGER et al

1978), deletions in ß-globin genes (Efstratiadis et al, 1980), and deletions in strains of Escherichia coli (Albertini et al, 1982). The slip-replication mechanism suggests that direct repeats on the H-strand are displaced during mtDNA replication and mispair with complementary L-strand sequences downstream (Fig. 6a). Subsequent H-strand breakage, 3'-OH extension and ligation results in the mtDNA deletion. An extension of this concept is the "illegitimate elongation" proposal (Buroker et al, 1990) which predicts that a competition exists between the displaced (parental) and nascent (daughter) Η-strands for base pairing with the L-strand template. If the parental H-strand successfully displaces the elongating daughter H-strand and reanneals with the L-strand, a cruciform structure is formed, allowing the displaced daughter H-strand to reanneal with a downstream repeat. This effectively creates a mtDNA deletion (Fig. 6b). These mechanisms account for the fact that 95% of known deletions occur within the two-thirds of genome between O h and O L . The alternative hypothesis is that deletions result from erroneous recombination. Both recombination (Schon et al, 1989) and topoisomerase (Zeviani et al, 1989) mechanisms have been postulated. Both deletion mechanisms remain to be confirmed at the molecular level. The enzymology of the recombinational process has yet to be demonstrated, and the reason for the initiation of the slip mispairing process is unclear. One mechanism which might initiate slip-mispairing is the stalling of H-strand replication by oxidative damage. The mtDNA is particularly susceptible to oxidative damage (Richter et al, 1988) due to its lack of histones and efficient DNA repair. The inhibition of replication by oxidative damage has been demonstrated through the development of PCR artifacts when amplifying damaged mtDNA (Paabo et al, 1990). When Taq I polymerase encounters DNA damage (i.e., UV induced or ancient DNA), it stalls at the modified sites, falls off the template, and reattaches to another template. Continued elongation yields a recombination product. It may be that slip replication or illegitimate recombination may be the only means by which mtDNA replication can bypass DNA damage. The inhibition of OXPHOS function by mtDNA deletion has been confirmed by introducing deleted mtDNAs from a KSS patient to rho° HeLa cells (Hayashi et al, 1991). When the proportion of deleted mtDNAs in the cybrid clones exceeded 60%, protein synthesis and cytochrome c oxidase (COX) activity were inhibited. However, the precise mechanism by which mtDNA deletions create OXPHOS dysfunction remains unclear. Deleted mtDNAs may alter the ratio between tRNAs and mRNAs, limiting mitochondrial protein synthesis (Larsson et al, 1990; Nakase et al, 1990; Shoubridge et al, 1990; Hayashi et al, 1991). When the percentage of deleted molecules is low, the tRNAs

MITOCHONDRIAL MYOPATHIES: GENETIC ASPECTS

81

from the normal molecules would be sufficient to translate the mRNAs from both genomes. However, when the deleted molecules come to predominate, competition for the tRNAs may limit translation (Hayashi etal., 1991). Alternatively, the deleted mtDNAs may displace normal mtDNAs by "out replicating" them. This would effectively limit the number of wild-type mtDNAs containing the deleted region of the mtDNA, thus restricting the copy number of certain genes. This model was previously proposed for yeast petite mutants (Fukuhara, 1969; Nagley and Linnane, 1972). Thus, under this scenario, wild-type mtDNAs would fail to sustain normal respiration. The association of mitochondrial myopathy with the loss of wild-type mtDNA is consistent with mtDNA depletion studies (Arnaudo et al., 1991), which report the formation of RRFs in patient muscle when mtDNA replication is inhibited. However, this hypothesis does not easily explain the increased levels of COX negative fibers that occur even when the mtDNA deletion spares all mtDNA encoded COX subunits. Hence, until it is known how mtDNA deletions inhibit protein synthesis and consequently OXPHOS, either or both mechanisms may be plausible. 1.

Three Classes of mtDNA

Rearrangements

Over 100 different mtDNA rearrangements have been reported (Wallace et al., 1991), most associated with Pearson's syndrome and ocular myopathy. These fall into three molecular classes: deleted plus wild-type molecules, duplicated plus wild-type molecules, and deleted plus duplicated plus wild-type molecules. 2.

Deleted plus Wild-type Molecules

Patients harboring heteroplasmic deletions generally present with either Pearson's syndrome (pancytopenia) or ocular myopathy (ptosis, ophthalmoplegia, mitochondrial myopathy). The deletions in these patients remove multiple tRNAs and several OXPHOS genes, but retain both origins of replication ( O l and O h ) and thus occur with two arcs of the mtDNA ( O h to O l and O l to O h ) . Most cases are spontaneous, although autosomal-dominant ocular myopathy associated with multiple mtDNA deletions also occurs (Zeviani et al., 1989), which could be due to a nuclear mutation in a mtDNA replication factor (Zeviani et al., 1989, 1990). A Pearson's syndrome child will provide an example of a heteroplasmic deletion. The patient's mother (individual II-2, Fig. 7) had a full term pregnancy with a normal delivery. At 3 months of age, the proband (III-l) was diagnosed with a severe sideroblastic anemia which required blood

82

SCOTT W. BALLINGER et al.

Ό 2

Ill

1

2

ô

Q

-Unaffected

φ

- Pearson's syndrome

FIG. 7. A pedigree depicting the spontaneous occurrence of Pearson's syndrome. The proband (III-l) is the only affected individual within the pedigree, and the only maternal relative harboring a deletion. This is typical of the spontaneous nature of most mtDNA deletions.

transfusions every 4-6 weeks. This condition resolved spontaneously at 2 years of age. For the next 5 years she did well, though she fatigued easily and experienced hearing loss at 7 years of age. She then manifested intention tremor (trembling with voluntary movements) at 8.5 years, ataxia at 9 years, and atypical retinitis pigmentosa at 10 years of age. Symptoms worsened with exercise or fever producing illness. Intellect was initially preserved, but deteriorated later in her course. The proband's mother (II-2) was unaffected, as were the proband's two older siblings (III-2 and III-3). Laboratory and metabolic evaluations revealed that the proband had a generalized aminoaciduria indicative of proximal tubule dysfunction, elevated triglycerides, mild lactic acidosis, and histochemistry characteristic of mitochondrial myopathy (RRF). OXPHOS enzyme analysis (complexes I V) revealed significant reduction of enzymatic activities at complexes I and IV (Table III). Coenzyme Q10 therapy was ineffective in alleviating the disease and the proband died at age 12 of respiratory arrest. Two forms of mtDNA were detected in the proband's muscle by Southern blot analysis. Digestion with restriction enzymes Xhol and BamHl, which linearize mtDNA, revealed a large mtDNA fragment in addition to the 16.5-kb wild-type mtDNA (Fig. 8a). Additional digestions with FvwII, hybridization with regional probes (Fig. 8b), and comparison with undigested patient and control mtDNAs (Fig. 8c) revealed that the mtDNA

MITOCHONDRIAL MYOPATHIES: GENETIC ASPECTS

83

harbored a deletion located between ND4 and the D-loop that removed the Xhol and BamHl sites. Direct DNA sequencing of the breakpoint region revealed a 5,179-np deletion between two 8-np direct repeats (5'CAACAACC-3') at nucleotides 10,897-10,904 and 16,076-16,083, one of A G Y which was lost (Fig. 9). This deletion removes 6 tRNAs (His, S e r , C U N L e u , Gin, Thr, and Pro), all or part of 4 OXPHOS genes (ND4, 5, 6, and cytochrome b), and 52 nucleotides of the D-loop. Both origins of replication ( O h and O l ) were retained, as were both rRNAs, the N D 1 4L, COI-III, ATPase 6, 8 genes, and 16 tRNA genes. The deletion was absent in lymphoblast cell lines and only barely detectable in myoblast cells by using Southern blot and PCR analyses. To exclude the possibility that mtDNA point mutations were also contributing to this disease, the proband's mtDNA was screened for common mtDNA mutations (Wallace et al., 1988a; Goto et al., 1990; Holt et al., 1990; Shoffner et al., 1990) as well as for unusual restriction site variants by PCR amplification and digestion with 14 restriction enzymes (Ballinger et al., 1992b; Torroni et al., 1992). Only previously identified mtDNA polymorphisms were found. Furthermore, all of the tRNA genes were sequenced, but no new mutations were found. Therefore, the deletion appears to be the primary cause of the disease. The 3' direct repeat of the proband's 5.1-kb deletion (nucleotides

TABLE III OXIDATIVE PHOSPHORYLATION ENZYME ANALYSIS

0

Controls Complex \

b

I + IIF II + III«* III' IV (FT)/ IV (SON)/ Fragility Index« a

Proband

Mean

SD

5% level

3 11 236 1420 794 134

194 229 605 1952 1426 1615

60 149 164 700 433 309

54 0 223 323 373 896

1.24

0.35

0.44

OXPHOS defect in the proband's muscle mitochondria. Complex I, NADH-H-decyl coenzyme Q oxidoreductase. c Complex I -I- III, NADH-Cytochrome c oxidoreductase (rotenone-sensitive fraction). Complex II + III, succinate-cytochrome c oxidoreductase. 'Complex III, reduced Λ-decyl coenzyme Q-cytochrome c oxidoreductase. /Complex IV, cytochrome c oxidase; FT, freeze-thaw; SON, sonicated. « Fragility Index, complex IV (sonicated)/complex IV (freeze-thaw). b

84

SCOTT W. BALLINGER et al. -16.5 kb (undigested)

16.5 kb (linear)

3

1 2

B

4

«•é · · I f e 1 1

2 2

3 3

8

7

β

9

m%

5

4

φ

s

10

—16.5 kb (linear)

£

— 1 6 3 kb (linear)

11

16.5 kb (undigested)

16.5 kb (linear)

FIG. 8. Southern blot analysis of muscle mtDNA from a Pearson's syndrome patient. (A) BamHl digests hybridized with 32P-dATP-labeled mtDNA revealed two bands, one equivalent to linearized mtDNA at 16.5 kb (compare to lane 1), and a slower migrating band which comigrated with the proband's undigested mtDNA (lane 3). Lane 1, control-ZtaraHI digest; lane 2, proband-ZtaraHI digest; lane 3, proband-undigested mtDNA; lane 4, controlundigested mtDNA. (B) Hybridization of patient DNA Pvull digests with regional mtDNA probes. The region between ND4 and the D-loop was absent in the lower band. Lane 1, control; lanes 2 through 11, proband. Hybridization probes for lane 1, linearized HeLa mtDNA; lane 2, mtDNA nucleotides 16,287-2074; lane 3, mtDNA nucleotides 1,562-3,717; lane 4, mtDNA nucleotides 3,108-5,917; lane 5, mtDNA nucleotides 5,317-7,628; lane 6, mtDNA nucleotides 7,367-9,172; lane 7, mtDNA nucleotides 8,264-10,107; lane 8, nucleotides 9,802-11,873; lane 9, nuleotides 11,673-13,950; lane 10, nucleotides 13,914-16,543; lane 11, linearized HeLa mtDNA. (C) Exclusion of a mtDNA duplication by testing with undigested mtDNAs and PCR probes. Lane 1, BamHl digest of proband mtDNA hybridized

MITOCHONDRIAL MYOPATHIES: GENETIC ASPECTS

85

A A

3'

Τ Τ G G Τ Τ Τ Α G Τ Τ G Τ Τ G Τ Τ G G_ G

A

Τ

np 10,904 ND4 Deletion breakpoint •np 16,083 D-Loop

A

C

A

Τ

A A A

FIG. 9. Direct mtDNA deletion junction sequence analysis of the Pearson's patient. Sequencing revealed a 5179-np deletion associated with an 8-np repeat (boxed region:L-strand sequence) between the ND4 gene and the D-loop.

16,076-16,083) has been associated with a relatively common 7.4-kb dele: tion (Ozawa et al., 1990). This same 3' region has been shown to be involved with some of the deletions in autosomal dominant ocular myopathy patients (Yuzaki etal., 1989; Zeviani etal., 1989). Thus, this region may be a 3' hot spot for some mtDNA deletions.

with mtDNA nucleotides 11,673-13,950. Only the 16.5-kb fragment is seen, which excludes a tandem duplication. Lane 2, undigested proband mtDNA hybridized with linearized mtDNA (see also part A, lane 3). Lane 3, BamHl digested and hybridized with full length mtDNA. Notice the appearance of the higher molecular weight fragment in addition to the 16.5-kb fragment. Lane 4, Pvull digest of proband mtDNA hybridized with full-length mtDNA. Both forms of linearized mtDNA (16.5-kb wild-type and 11.4-kb deletion mutant) are observed demonstrating a deletion.

86 3.

SCOTT W. BALLINGER et al. Duplicated Plus Wild-type Molecules

Several patients with heteroplasmic duplications have been reported. Most of these presented with ocular myopathy together with diabetes mellitus (Poulton et al., 1989a,b; Dunbar et al, 1993), some with just diabetes (Dunbar et al, 1993). 4.

Deleted Plus Duplicated Plus Wild-type Molecules

Combinations of related deleted and duplicated molecules have recently been observed in some ocular myopathy patients (Poulton et al., 1993), in a maternally inherited diabetes and deafness pedigree (Ballinger et al., 1992, 1994), and possibly in three patients with spontaneous childhood encephalomyopathy (Miyabayashi et al, 1991). The maternally inherited diabetes mellitus and deafness pedigree (Fig. 10) provides an example of this class of mutations. In this family diabetes mellitus (DM) and/or deafness was maternally inherited over three generations, with a total of nine individuals being affected (individual II-7 not shown in pedigree). Analysis of the proband's muscle mtDNA revealed a trimolecular heteroplasmy involving related insertion and deletion molecules. The deleted molecule lacked 10,423 np of mtDNA, spanning from G ln nucleotide 4,398 in the t R N A gene to nucleotide 14,822 in the cytochrome b gene (Fig. 11). The deletion removed 2 np at the 5' end of the G ln t R N A gene, 11 of 13 OXPHOS subunit genes, 15 of 22 tRNAs, the O L , and 75 np at the 5' end of cytochrome b. The duplicated molecule had a tandem repeat of exactly the same mtDNA sequences retained in the deleted molecules. These encompassed the D-loop including the heavystrand replication origin ( O h ) , 6 tRNAs [Phe, Val, Leu (UAA/G), He, Thr, and Pro], the 12S and 16S rRNA genes, and the ND1 gene. All examined maternal relatives (II-l through II-5, III-l) had the same rearrangements (nucleotides 4,398-14,822), with the same breakpoint junction. This rearrangement was flanked by two 10-np direct repeats (5'CACCCCATCC-3') at nucleotides 14,812-14,821 and 4,389-4,398, one of which was lost. In three family members who were biopsied (II-l, Π-4, and II-5) no evidence of ragged red muscle fibers or mitochondrial accumulation was detected by modified Gomori trichrome or other standard histochemical stains, and ultrastructural study (electron microscopy) showed normal muscle fibers and mitochondria structure. Protein synthesis studies of proband cell lines heteroplasmic for the rearrangement revealed that the deletion-duplication was associated with severe inhibition of mitochondrial protein synthesis. The proband's lymphoblast cell line, containing 53% rearranged mtDNAs, had a marked impair-

87

MITOCHONDRIAL MYOPATHIES: GENETIC ASPECTS

' 1

2

3

4

S

I

mutations causing, 76-77 Mitochondrial metabolism, 24-26 Mitochondrial myopathies, 21-92 biochemical aspects, 21-51

L e u t ( )U U R

and cardiomyopathy, t R N A mutations causing, 76-77 genetic aspects, 59-92 genetic classification, 23 from Krebs cycle defects, 33-34 mtDNA mutations and, 72-89 from oxidation-phosphorylation coupling defects, 34-35 from respiratory chain defects, 35-50 from substrate transport defects, 26-30 from substrate utilization defects, 30-33 Leut(UUR) tRNA , mutations causing, 76-77 Mitochondrial oxidative phosphorylation, 60, 61-62 Mitochondrial respiratory chain, 2-3 Muscle mitochondrial metabolism, 24-26 skeletal, mitochondrial diseases, 106-115 exercise, 110-113 recovery from exercise, 106-110 resting state, 113-115 Mutations mechanisms, 7; see also specific mutations Myocarditis, acute, anti-M7 ΑΜΑ in, 130131 Myoclonic epilepsy with ragged red fibers, 45-46 tRNA point mutations causing, 72-76 Myopathy, mitochondrial, 21-92; see also Mitochondrial myopathies Myophosphorylase deficiency, mitochondrial disorders and, 121

Ν NADH-coenzyme Q oxidoreductase, defect of, 37-39 NADH dehydrogenase, loss in ischemiareperfusion, 183-184 Necrotizing encephalomyelopathy, subacute, 43 Nicotinamide, for mitochondrial diseases, 119 Nuclear DNA, defects of, 22, 23 Nuclear DNA/mitochondrial DNA communication, 23, 24

ο Ocular myopathy, mtDNA rearrangements causing, 77

INDEX °H+>

° L + deletions,

81-85

deletions, 8 6 - 8 9 Optic neuropathy, Leber's hereditary, see Leber's hereditary optic neuropathy Organelle development, endosymbiotic theory, 0

H+

> 0 L-

68

ß-Oxidation, defects of, 3 2 - 3 3 Oxidation-phosphorylation coupling, defects of,

35-50

Oxidative damage, to DNA, 5 - 6 Oxidative phosphorylation, mitochondrial, 6 0 , 61-62

Oxygen delivery, decreased, in mitochondrial diseases, conditions characterized by, 120-121

supply, ethanol treatment and,

sera, reactivity against e 3, 143-144 Τ cells in pathogenesis of, 160-162 Progressive external ophthalmoplegia, mtDNA rearrangements causing, 77 Proteins imported to mitochondria, ethanol treatment and, 231-232 synthesis defects from mtDNA mutations, clinical features, 91-92 mitochondrial, in chronic alcoholism, 227-232 Pseudolupus syndrome, anti-M3 and, 132 Pyruvate carboxylase, deficiency, 32 Pyruvate dehydrogenase complex, 139-141 deficiency, 30-32

215-218

R

Ρ Parkinson's disease, mitochondrial disorders, 122

Pearson's marrow /pancreas syndrome, 2 4 Pearson's syndrome mtDNA rearrangements causing, 7 7 ° H + > O H + deletions in, 8 1 - 8 5 Peripheral vascular disease, mitochondrial disorders and, 1 2 0 Phosphate for mitochondrial diseases, 1 1 9 - 1 2 0 Phospholipid, antibodies against, 1 3 1 - 1 3 2 31 Phosphorus magnetic resonance spectroscopy brain metabolism disorders, 1 1 5 - 1 1 6 mitochondrial diseases, 1 0 1 - 1 0 4 Phosphorylation potential, cellular, ethanol treatment and, 2 1 8 - 2 2 1 Phylloquinone, 1 3 Positron emission tomography brain metabolism disorders, 1 1 6 - 1 1 7 mitochondrial diseases, 1 0 4 - 1 0 5 Primary biliary cirrhosis, 1 3 5 - 1 6 4 antibody systems associated with, 1 5 2 - 1 5 5 antibody titres and disease staging in, 1 5 5 156

anti-M4 antibodies in, anti-M9 antibodies in,

247

153-154 154-155

Reactive oxygen species, in mitochondria, 4-5 Redox compounds as therapeutic agents, 1314 Redox effects of ethanol metabolism, 206-208 Reduced CoQ-cytochrome c oxidoreductase, defects of, 39-41 Renal failure, mitochondrial disorders and, 120-121 Reperfusion, see also Ischemia-reperfusion injury by, 176-177 mitochondrial "stunning" and, 189-191 Respiratory chain defects of, 35-50 combined, 45-50 in complex I, 39 in complex II, 39 in complex III, 39-41 in complex IV, 41-44 in complex V, 44-45 enzymes of, properties of, 36 mitochondrial, 2-3 Respiratory failure, mitochondrial disorders and, 120 p- mutations, in degenerative diseases, 10 Riboflavin, 13 for mitochondrial diseases, 119

antimitochondrial antibodies in pathogenesis of,

159-160

antimitochondrial antibody production in, stimulus for, 1 5 6 - 1 5 9 M 2 antigen in, 1 3 6 - 1 5 2

S Sarcosine dehydrogenase, antibodies against, 130-131

248

INDEX

Skeletal muscle, mitochondrial diseases exercise, 110-113 recovery from exercise, 106-110 resting state, 113-115 Spectroscopy, magnetic resonance brain metabolism disorders, 115-116 of mitochondrial diseases, 101-104 Subacute necrotizing encephalomyelopathy, 43 Substrate transport, defects, 26-30 Substrate utilization defects, mitochondrial myopathies from, 30-33 Succinate, 13 Succinate-coenzyme Q oxidoreductase, defect of, 39 Superoxides, production by mitochondrion, 177-180 syn- mutations, in degenerative diseases, 9 Syphilis, anti-Mi ΑΜΑ in, 129-130 Systemic lupus erythematosus, anti-M5 IN, 131-132

Τ TCA cycle enzymes, loss in ischemiareperfusion, 187-188 Τ cells in pathogenesis of primary biliary cirrhosis, 160-162 Thiol groups, in antigenicity, 150-151 Tissue injury, mitochondrial energy metabolism and, in chronic alcoholism, 234-235 Tomography, positron emission brain metabolism disorders, 116-117 mitochondrial diseases, 104-105

V Venocuran, anti-M3 and, 132 Vitamin C, 13 Vitamin K,, 13 Vitamin K 3, 13 for mitochondrial diseases, 117-118

Contents of Previous Volumes

Volume 1

Energy-Linked Reactions of Plant Mitochondria /. B. Hanson and T. K. Hodges

Kinetics and Intermediates of the Oxygen Evolution Step in Photosynthesis Bassel Kok and George M. Cheniae

1 8

0 and Related Exchanges in Enzymic Formation and Utilization of Nucleoside Triphosphates P. D. Boyer

Fluorescence Yield in Photosynthetic Systems and Its Relations to Electron Transport Warren L. Butler

On the Role of Ubiquinone A. Kröger and M. Klingenberg

Uncoupling and Energy Transfer Inhibition in Photophosphorylation Norman Good, Seikichi Izawa, and Geoffrey Hind

Energy-Linked Reactions in Chemoautotrophic Organisms Lutz A. Kiesow

The Chemistry of Bioluminescence /. W. Hastings

Respiration and Adenosine Triphosphate Synthesis in Nuclei Thomas E. Conover

Structure and Function of the Contractile Protein Myosin A. Stracher and P. Dreizen

The Oscillation of Insect Flight Muscle R. Τ Tregear

Energized Calcium Transport and Relaxing Factors Annemarie Weber

Contractile Mechanisms in Cilia and Flagella Michael Holwill

Ion Transport to Mitochondria E. J. Harris, J. D. Judah, and K. Ahme da

Genesis of the Cochlear Endolymphatic Potential Brian M. Johnstone

AUTHOR INDEX-SUBJECT INDEX

AUTHOR INDEX-SUBJECT INDEX

Volume 2

Volume 3

Mechanism of Photoinduced Electron Transport in Isolated Chloroplasts Mordhay Auron

A Scrutiny of Mitchell's Chemiosmotic Hypothesis of Respiratory Chain and Photosynthetic Phosphorylation G. D. Greville

The Energized Movement of Ions and Water by Chloroplasts Lester Packer and Antony R. Crofts

Electron Transfer and Energy Conservation Robert J. P. Williams 249

250

CONTENTS OF PREVIOUS VOLUMES

Translocations in Bimolecular Lipid Membranes: Their Role in Dissipative and Conservative Bioenergy Transductions Paul Mueller and Donald O. Rudin Energy Relationships and the Active Transport of Ions Peter C. Caldwell Energy Utilization and Oxidative Recovery Metabolism in Skeletal Muscle Frans F. Jobsts The Mechanism of the Visual Process Sjoerd L. Bonting Energy Transduction in Algal Phototaxis Gordon Tollin AUTHOR INDEX-SUBJECT INDEX

Volume 4 Nonequilibrium Thermodynamics and Its Application to Bioenergetics S. Roy Caplan The Design and Use of Fluorescent Probes for Membrane Studies G. K. Radda Energy Transformations in the Respiratory Chain V. P. Skulachev

Sodium and Potassium Transport /. C. Skou AUTHOR INDEX-SUBJECT INDEX

Volume 5 X-Ray Diffraction Studies on Biological Membranes C. R. Worthington Chlorophyll and Light Energy Transduction in Photosynthesis Joseph J. Katz and James R. Norris, Jr. Chemically and Physically Induced Luminescence as a Probe of Photosynthetic Mechanisms Darrell E. Fleischman and Berger C. Mayne The Reducing Side of Photosystem I James Siedow, Charles F. Yocum, and Anthony San Pietro The Chemistry of Vertebrate and Invertebrate Visual Photoreceptors Edwin W. Abrahamson and Roger S. Fager Mechanism of Actomyosin ATPase and the Problem of Muscle Contraction Edwin T. Taylor

Profiles of Mitochondrial Monovalent Ion Transport Cyril L. Moore

Energy-Transducing Components in Mitochondrial Respiration David F. Wilson, P. Leslie Dutton, and Michal Wagner

Coupling of Ion and Electron Transport in Chloroplasts Richard A. Dilley

Kinetics of Cytochromes b Maria Erecinska, Michal Wagner, and Britton Chance

Energy Conversion Reactions in Bacterial Photosynthesis Herrick Baltscheffsky, Margareta Baltscheffsky, and Anders Thore

Mitochondrial Coupling Factors R. Brian Beechy and Kenneth J. Cattel

Electrogenic Ion Pumping in Nervous Tissue J. Murdoch Ritchie Sequence of Steps in the (Na + K)Activated Enzyme System in Relation to

AUTHOR INDEX-SUBJECT INDEX

Volume 6 Energy Balance in Muscle Contraction: A Biochemical Approach Martin J. Kushmerick

CONTENTS OF PREVIOUS VOLUMES

251

Transport in Membrane Vesicles Isolated from the Mammalian Kidney and Intestine Bertram Sacktor

Volume 8 Photosynthesis: Part Β

Membranes and Energy Transduction in Bacteria Franklin M. Harold

Alternate Fates of the Photochemical Reducing Power Generated in Photosynthesis: Hydrogen Production and Nitrogen Fixation Norman I. Bishop and Larry W. Jones

Proton Translocation in Chloroplasts G. Hauska and A. Trebst The Use of Ionophores and Channel Formers in the Study of the Function of Biological Membranes A. Gomez-Puyou and C. Gomez-Lojero Mitochondrial Calcium Transport Fyfe L. Bygrave SUBJECT INDEX

Volume 7 Photosynthesis: Part A Photochemistry of Chlorophyll in Solution: Modeling Photosystem II G. R. Seely Picosecond Events and Their Measurement Michael Seibert The Primary Electron Acceptors in GreenPlant Photosystem I and Photosynthetic Bacteria Bacon Ke The Primary Reaction of Chloroplast Photosystem II David B. Knaff and Richard Malkin Photosynthetic Electron-Transport Chains of Plants and Bacteria and Their Role as Proton Pumps A. R. Crofts and P. M. Wood

Chlorophyll-Protein Complexes and Structure of Mature and Developing Chloroplasts Ν. K. Boardman, Jan M. Anderson, and D. J. Goodchild Dynamic Structural Features of Chloroplast Lamellae Charles J. Arntzen Structure and Development of the Membrane System of Photosynthetic Bacteria Gerhart Drews Genetic Control of Chloroplast Proteins N. W. Gilham, J. E. Boynton, and N-H. Chua Mutations and Genetic Manipulations as Probes of Bacterial Photosynthesis Barry L. Marrs SUBJECT INDEX

Volume 9 Irreversible Thermodynamic Description of Energy Transduction in Biomembranes H. V. Westerhoffand Κ. Van Dam Intracellular pH: Methods and Applications R. J. Gillies and D. W. Dreamer Mitochondrial ATPases Richard S. Criddle, Richard F. Johnston, and Robert J. Stack

The ATPase Complex of Chloroplasts and Chromatophers Richard E. McCarty

Ionophores and Ion Transport Across Natural Membranes Adil E. Shamoo and Thomas J. Murphy

SUBJECT INDEX

Reaction Mechanisms for ATP Hydrolysis

252

CONTENTS OF PREVIOUS VOLUMES

and Synthesis in the Sarcoplasmic Reticulum Taibo Yamamoto, Haruhiko Takisawa, and Yuji Tonomura Flavoproteins, Iron Proteins, and Hemoproteins as Electron-Transfer Components of the Sulfate-Reducing Bacteria Jean LeGall, Daniel V. DerVartanian, and Harry D. Peck, Jr. Applications of the Photoaffinity Technique to the Study of Active Sites for Energy Transduction Richard John Guillory

Biochemistry and Genetics of Bacterial + H -Translocating ATPases Robert H. Fillingame Proton-Linked Transport in Chromaffin Granules David Njus, Jane Knoth, and Michael Zallakian Regulation of the Synthesis and Hydrolysis of ATP in Biological Systems: Role of + Peptide Inhibitors of H -ATPases Peter L. Pedersen, Klaus Schwerzmann, and Nitza Cintron

SUBJECT INDEX

Structure and Mechanism of the (Ha, K)ATPase Lewis C. Cantley

Volume 10

Actomyosin ATPase and Muscle Contraction J. A. Sleep and S. J. Smith

Application of Fluctuation Spectroscopy to Muscle Contractility Julian Borejdo +

Respiration-Linked H Translocation in Mitochondria: Stoichiometry and Mechanism Marten Wikström and Klaas Krab Uptake and Release of Bivalent Cations in Mitochondria Nils-Erik Saris and Karl E. O. Akerman Role of Subunits in Proton-Translocating ATPase (F 0-F,) Masamitsu Futai and Hiroshi Kanazawa Control of Mitochondrial Substrate Oxidation Richard G. Hansford Electrochemistry of Nitrogenase and the Role of ATP Robert V. Hageman and R. H. Burris INDEX

INDEX

Volume 12 On the Structure and Genetics of the Proteolipid Subunit of the ATP Synthase Complex W. Sebald and J. Hoppe Biochemistry of Bacterial Bioluminescence Miriam M. Ziegler and Thomas O. Baldwin The Electron Transport System and Hydrogenase of Paracoccus dentrificans Paulette M. Vignais, Michèle-France Henry, Edith Sim, and Douglas B. Kell Electron Transfer, Proton Translocation, and ATP Synthesis in Bacterial Chromatophores A. Baccarini-Melandri, R. Casadio, and B. A. Melandri INDEX

Volume 11 Proton-ATPase of Chloroplasts Nathan Nelson

Volume 13 Determination of the Proton

CONTENTS OF PREVIOUS VOLUMES Electrochemical Gradient across Biological Membranes Giovanni Felice Λ ζ zone, Daniela Pietrobon, and Mario Zoratti Application of Electron Paramagnetic Resonance in the Study of Iron-Sulfur Clusters in Energy-Transducing Membranes S. P. J. Albracht Recent Developments in Spin Label EPR Methodology for Biomembrane Studies Leslie W. -M Fung and Michael E. Johnson Probing Structure and Motion of the Mitochondrial Cytochromes B. P. Sudha, N. Dixit and Jane M. Vanderkooi Enzyme-Catalyzed Oxygen Exchange Reactions and Their Implications for Energy Coupling Robert A. Mitchell Use of Immunological Techniques to Study Membrane Proteins Howard Riezman INDEX

Volume 14 Structure of Cytochrome Oxidase Redox Centers in Native and Modified Forms: An EXAFS Study B. Chance and L. Powers X-Ray and Neutron Diffraction for Probing the Interactions of Small Molecules with Membrane Structures Leo G. Herbette

Circular Dichroism Studies of ElectronTransport Components Yash P. Myer INDEX

Volume 15 Structure of NADH-Ubiquinone Reductase (Complex I) C. Ian Ragan Structure of the Succinate-Ubiquinone Oxidoreductase (Complex II) Tomoko Ohnishi Structure of Mitochondrial UbiqinolCytochrome-c Reductase (Complex III) Hanns Weiss Structure of Cytochrome-c Oxidase Roderick A. Capaldi, Shinzaburo Takamiya, Yu-Zhong Zhang, Diego Gonzalez-Halphen, and Wayne Yanamura Evolution of a Regulatory Enzyme: Cytochrome-c Oxidase (Complex IV) Bernhard Kadenbach, Lucia KuhnNentwig, and Ursula Büge The Assembly of F^Q-ATPase in Escherichia coli Graeme B. Cox and Frank Gibson Biogenesis of Mitochondrial Energy Transducing Complexes Nikolaus Pfanner and Walter Neupert Biogenesis of Mammalian Mitochondria B. Dean Nelson Structure and Biogenesis of Chloroplast Coupling Factor CFQCFJ-ATPase Carlo M. Nalin and Nathan Nelson

19

F NMR Investigations of Membranes Chien Ho, Susan R. Dowd, and Jan F. M. Post

Metal Ion NMR: Application to Biological Systems William H. Braunlin, Torbjörn Drakenberg, and Sture Forsén

253

Mitochondrial Gene Products Anne Chomyn and Giuseppe Attardi Overview: Bioenergetics between Chemistry, Genetics, and Physics L. Kovac INDEX

254

CONTENTS OF PREVIOUS VOLUMES

Volume 16 Photosystem II: Molecular Organization, Function, and Acclimation Bertil Andersson and Stenbjörn Sty ring Photosystem I John H. Golbeck and Donald A. Bryant Electron Transport between Photosystem II and Photosystem I W. A. Cramer, P. N. Furbacher, A. Szczepaniak, and G. -S. Tae Chloroplast and Plant Mitochondrial ATP Synthases Elzbieta Glaser and Birgitta Nörting

Energy Coupling in Chloroplasts: A Calcium-Gated Switch Controls Proton Fluxes between Localized and Delocalized Proton Gradients Richard A. Dilley The Reaction Center Protein from Purple Bacteria: Structure and Function M. R. Gunner Energetics of and Sources of Energy for Biological Nitrogen Fixation Paul W. Ludden INDEX