Mitochondrial Dysfunction in Neurodegenerative Disorders
Amy Katherine Reeve • Kim Jennifer Krishnan Michael R. Duchen • Doug M. Turnbull Editors
Mitochondrial Dysfunction in Neurodegenerative Disorders
Editors Amy Katherine Reeve, B.Sc (Hons) Ph.D. Centre for Brain Ageing and Vitality Mitochondrial Research Group Institute for Ageing and Health Newcastle University Newcastle upon Tyne, Tyne and Wear UK
Michael R. Duchen, B.A., M.B., B.S. MRCP, Ph.D Department of Cell and Developmental Biology, Consortium for Mitochondrial Research University College London London UK
Kim Jennifer Krishnan, B.Sc.(Hons), Ph.D. Centre for Brain Ageing and Vitality Institute for Ageing and Health Newcastle University Newcastle upon Tyne, Tyne and Wear UK
Doug M. Turnbull, M.B.B.S.(Hons), M.D. Ph.D., FRCP Institute for Ageing and Health Newcastle University Newcastle upon Tyne UK
ISBN 978-0-85729-700-6 e-ISBN 978-0-85729-701-3 DOI 10.1007/978-0-85729-701-3 Springer London Dordrecht Heidelberg New York British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Library of Congress Control Number: 2011941274 © Springer-Verlag London Limited 2012 Whilst we have made considerable efforts to contact all holders of copyright material contained in this book, we may have failed to locate some of them. Should holders wish to contact the Publisher, we will be happy to come to some arrangement with them. Apart from any fair dealing for the purposes of research or private study, or criticism or review, as permitted under the Copyright, Designs and Patents Act 1988, this publication may only be reproduced, stored or transmitted, in any form or by any means, with the prior permission in writing of the publishers, or in the case of reprographic reproduction in accordance with the terms of licenses issued by the Copyright Licensing Agency. Enquiries concerning reproduction outside those terms should be sent to the publishers. The use of registered names, trademarks, etc., in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant laws and regulations and therefore free for general use. Product liability: The publisher can give no guarantee for information about drug dosage and application thereof contained in this book. In every individual case the respective user must check its accuracy by consulting other pharmaceutical literature. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)
This book is dedicated to the memory of Dr. Denise Kirby.
Preface
The loss of neurons at any stage of life is a catastrophic event. These post-mitotic cells lack an ability to regenerate, and therefore their loss later in life will inevitably lead to functional defects. The most widely known diseases in which neuronal cell loss occurs are related to advancing age, for example, Parkinson’s disease and Alzheimer’s disease, but there are several neurodegenerative diseases which tend to occur at a much younger age, for example, multiple sclerosis and motor neuron disease. Proposed roles for mitochondria in the pathogenesis of these diseases are appearing at a rapid rate, especially since discoveries that familial forms of Parkinson’s disease are caused by mutations in genes encoding proteins associated with mitochondria. In a world where the aging population is increasing, these diseases will become more prevalent and their impact on society is set to expand to epidemic proportions. With increasing numbers of papers being published in the field of “mitochondria and neurodegenerative disease” we wanted to bring together a collection of articles, from leaders in the field, to illustrate how varied and important this area of research is. It is remarkable how a number of distinct mechanisms of mitochondrial dysfunction can lead to neurodegeneration, while there is also a remarkable degree to which common mechanisms are recapitulated between various disease models. We hope the reader will gain insight into the various ways in which mitochondrial dysfunction contributes to the pathogenesis of these diseases. In this book, we aim to lead the reader through the basic functions of mitochondria and the mechanisms that lead to their dysfunction, to the consequences of this dysfunction on neuronal function before finishing with the modeling of these disorders and our hopes for the future. Our hopes for the future are high, especially since as we uncover the mechanisms which lead from mitochondrial dysfunction to neurodegeneration we stumble upon new potential therapeutic targets. This expanding field of research focusing on mitochondria in relation to these diseases forms a small but increasingly important part of neurodegeneration research. Amy Katherine Reeve Kim Jennifer Krishnan Michael R. Duchen Doug M. Turnbull
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Acknowledgements
The Editors would like to acknowledge the following funding bodies without whose support work in the field of mitochondria and neurodegeneration would not be possible – The Wellcome Trust, The Medical Research Council, The Muscular Dystrophy Campaign and Parkinson’s UK. We also acknowledge the support of the Biotechnology and Biological Sciences Research Council, Engineering and Physical Sciences Research Council, Economic and Social Research Council and Medical Research Council as part of the Lifelong Health and Wellbeing Initiative and UK NIHR Biomedical Research Centre for Ageing and Age-Related Disease.
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Contents
Part I 1
Mitochondrial Function and Genetics
An Introduction to Mitochondria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Amy Katherine Reeve and R.N. Lightowlers
Part II
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Mitochondrial Dysfunction: Causes and Effects?
2
Neurodegeneration in Primary Mitochondrial Disorders . . . . . . . . . Nichola Lax and Evelyn Jaros
3
Mutations in Nuclear Genes That Affect Mitochondrial Function in Parkinson’s Disease . . . . . . . . . . . . . . . . . Gavin Charlesworth and Nicholas W. Wood
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Common mtDNA Polymorphisms and Neurodegenerative Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . Joanna L. Elson and David C. Samuels
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4
5
mtDNA Mutations in Brain Aging and Neurodegeneration . . . . . . . Kim Jennifer Krishnan and Amy Katherine Reeve
Part III 6
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Functional Consequences of Mitochondrial Dysfunction
Mitochondrial Dysfunction in Neurodegenerative Disease: Protein Aggregation, Autophagy, and Oxidative Stress . . . . . . . . . . . Jianhua Zhang and Victor Darley-Usmar
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7
Mitochondria, Sodium, and Calcium in Neuronal Dysfunction . . . . David G. Nicholls
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8
Mitochondrial Membrane Potential and Dynamics . . . . . . . . . . . . . . Jorge M.A. Oliveira
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9
Dysfunction of the Ubiquitin/Proteasome System and Mitochondria in Neurodegenerative Disease . . . . . . . . . . . . . . . . Matthew Y. Tang and Douglas A. Gray
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Contents
10 Neuronal Mitochondrial Transport and Dysfunction. . . . . . . . . . . . . Talia A. Atkin, Andrew F. MacAskill, and Josef T. Kittler 11 Mitochondrial Changes Associated with Demyelination: Consequences for Axonal Integrity . . . . . . . . . . . . . . . . . . . . . . . . . . . Graham Robert Campbell, Kenneth J. Smith, and Don J. Mahad Part IV
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Modeling Mitochondrial Contribution to Neurodegenerative Disease
12 Modeling Mitochondrial Dysfunction in Neurodegenerative Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Johannes C. Komen and David R. Thorburn Part V
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The Future
13 Potential Therapies for Mitochondrial Dysfunction. . . . . . . . . . . . . . Claire Henchcliffe and M. Flint Beal
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Summary and Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Amy Katherine Reeve
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Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Contributors
Talia A. Atkin, B.A. Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK M. Flint Beal, M.D. Neurology and Neuroscience, New York Presbyterian Hospital, Weill Cornell Medical Center, New York, NY, USA Graham Robert Campbell, B.Sc.(Hons), M.Res., Ph.D. Mitochondrial Research Group, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, Tyne and Wear, UK Gavin Charlesworth, B.Sc.(Hons), M.B.B.S., MRCP Department of Molecular Neuroscience, Neurogenetics, UCL Institute of Neurology, London, UK Victor Darley-Usmar, Ph.D. Department of Pathology and Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL, USA Joanna L. Elson, Ph.D. Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, Tyne and Wear, UK Douglas A. Gray, B.Sc.(Hons), Ph.D. Centre for Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, Tyne and Wear, UK Claire Henchcliffe, M.D., DPHIL Neurology and Neuroscience, Movement Disorders Division, Weill Medical College of Cornell University, New York, NY, USA Evelyn Jaros, Ph.D. Neuropathology/Cellular Pathology, Newcastle upon Tyne Hospitals, NHS Foundation Trust, Royal Victoria Infirmary, Newcastle upon Tyne, Tyne and Wear, UK Institute for Ageing and Health/UK NIHR Biomedical Research Centre for Ageing and Age-related diseases, Newcastle University, Newcastle upon Tyne, Tyne and Wear, UK
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Contributors
Josef T. Kittler, Ph.D. Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK Johannes C. Komen, Ph.D. Department of Paediatrics, Murdoch Childrens Research Institute, Royal Children’s Hospital, Melbourne, VIC, Australia Kim Jennifer Krishnan, B.Sc.(Hons), Ph.D. Centre for Brain Ageing and Vitality, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, Tyne and Wear, UK Nichola Lax, M.Res., B.Sc.(Hons) Mitochondrial Research Group, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, Tyne and Wear, UK Institute for Ageing and Health/UK NTHR Biomedical Research Centre for Ageing and Age-Related diseases, Newcastle University, Newcastle upon Tyne, Tyne and Wear, UK R.N. Lightowlers, Ph.D. Mitochondrial Research Group, Institute for Ageing and Health, Newcastle University Medical School, Newcastle upon Tyne, Tyne and Wear, UK Andrew F. MacAskill, Ph.D. Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK Don J. Mahad, M.B.Ch.B., Ph.D. Mitochondrial Research Group, Institute of Ageing and Health, Newcastle University, Newcastle upon Tyne, Tyne and Wear, UK David G. Nicholls, M.A., Ph.D. Beck Institute for Age Research, Novato, CA, USA Jorge M.A. Oliveira, Ph.D. REQUIMTE, Department of Drug Sciences, Faculty of Pharmacy, University of Porto, Porto, Portugal Amy Katherine Reeve, Ph.D. Centre for Brain Ageing and Vitality, Mitochondrial Research Group, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, Tyne and Wear, UK David C. Samuels, Ph.D. Department of Molecular Physiology and Biophysics, Center for Human Genetics Research, Vanderbilt University Medical Center, Nashville, TN, USA Kenneth J. Smith, B.Sc., Ph.D. Department of Neuroinflammation, Institute of Neurology, University College London, London, UK Matthew Y. Tang, B.Sc., M.Sc. Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada Centre for Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada
Contributors
David R. Thorburn, Ph.D. Department of Paediatrics, Murdoch Childrens Research Institute, Royal Children’s Hospital, Melbourne, VIC, Australia Department of Paediatrics, University of Melbourne, Melbourne, VIC, Australia Nicholas W. Wood, Ph.D., FRCP, FMedSci Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK Jianhua Zhang, Ph.D. Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA
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General Introduction
While some neurodegenerative diseases have been recognized for centuries and research into their causes has been taking place for decades, one fundamental question which still remains to be answered is: why do neurons die in these diseases? While studying these diseases has taught us an extraordinary amount about neuronal function, protein metabolism and cell signaling, we often cannot answer this most simple of questions. Understanding these diseases, however, is not even as simple as understanding why neurons die; we need to also understand how these cells function, how they respond to changes in their local environment, what happens when one process within them is disrupted, and how they degrade dysfunctional organelles. In this book we have focused on the energy generating mitochondrion as a potential central player in the loss or dysfunction of neurons in a large number of neurodegenerative diseases. We examine the impact of mitochondrial dysfunction on a wide range of neuronal processes and speculate how this will impact on cell survival and function. In order to do this we have brought together articles by an array of international experts in the field of mitochondrial biology to discuss the implications of mitochondrial dysfunction on mitochondrial function and ultimately cell survival, and also the potential therapeutic hopes of preventing the disruption of mitochondrial dysfunction in common neurodegenerative diseases (Chap. 13).
The Mitochondrion Sometimes described as the “batteries” of the cell, mitochondria are essential for cellular survival since they generate the vast majority of the cell’s energy in the form of ATP (Chap. 1). In cells which demand a lot of energy, which includes neurons, these organelles are particularly important, and changes or defects within mitochondria have major consequences for cellular function. However it is not only their function as energy producers which necessarily make mitochondria the potential “bad guys” in neurodegenerative disease but also their ability to buffer calcium concentrations within the cell (Chap. 7), their generation of damaging reactive oxygen radical species (Chap. 6), and their dynamic nature (Chaps. 8 and 10). Mitochondria are derived ultimately from free living bacterial organisms; their residence within eukaryotic cells began with an endosymbiotic relationship between xvii
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a bacterium and a heterotrophic anaerobe. The benefit of this symbiosis was protection for the bacterium while for the anaerobe it was the ability to use oxygen to generate energy, thus leading to the evolution of aerobic species and all eukaryotic cells (Chap. 1). Over the course of evolutionary time, the bacterium has lost all of its functions as a separate entity and become the mitochondrion. While the vast majority of the DNA content of the mitochondrion has been donated to the nuclear genome. However, ~16Kb of circular DNA remains encoding a few essential electron transport chain proteins, as well as a full complement of tRNAs and rRNAs, essential for the synthesis of mitochondrial encoded proteins (Chap. 1). All proteins which are not directly synthesized within the mitochondria are encoded in the nucleus, created within the cytoplasm and actively transported into the mitochondria. Mitochondria have a double membrane, surrounding a central space called the matrix containing tubular structures called cristae (Chap. 1). The electron transport chain resides on the inner mitochondrial membrane and the flow of electrons through four respiratory complexes of the chain generates a proton gradient, also expressed as a membrane potential (DYm) across the membrane which is then utilized by the fifth complex, the F0F1 ATP synthase, to generate energy, in the form of ATP. Although the textbook image of a mitochondrion is that of an ovoid shaped organelle, in reality these are highly dynamic organelles which constantly undergo the processes of fission and fusion, thus creating a constantly changing reticular network (Chap. 8). The fusion of mitochondria, which requires protein interactions of both the inner and outer membranes, allows the mixing of the contents of these organelles, and it is thought that these processes are related to the mitochondrial membrane potential (Chap. 8). The loss of DYm leads to a loss of essential fusion proteins and may target mitochondria for degradation through the process of autophagy (Chap. 6). Autophagy is a dynamic process through which long lived proteins and intact organelles can be degraded, and their component amino acids recycled by the cell. This process can degrade proteins in response to different cellular stresses (including starvation) and environmental conditions (including hypoxia). Mounting evidence suggest that changes in this process are important in a number of different diseases and also in normal aging. The relationship of this process to other protein degradation pathways is also providing information for disease pathways (Chaps. 6 and 9). Yet little is known about the rate at which these organelles are turned over, in terms of both their generation and degradation. It is thought that in post-mitotic cells, such as neurons and muscle fibers, turnover occurs at a much slower rate than in replicative tissues such as blood and colon. A slower rate of turnover in neurons may provide clues as to the accumulation of mitochondrial dysfunction in these tissues with aging and in disease. Mitochondria perform a number of important functions within the cell apart from simply energy generation; they buffer calcium, are involved in the synthesis of iron sulfur cluster proteins, and are important for apoptosis (Chap. 1). Although responsible for these essential functions within the cell, mitochondria are also significant generators of reactive oxygen species (Chap. 6). These species are generated as a normal by-product of oxidative phosphorylation, and, when generated in excess by damaged mitochondria, may damage many cellular components including DNA,
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membranes, and proteins. This damage has been proposed to be important in both aging and in many degenerative diseases, especially those associated with protein aggregation. Recent data do show that these species are not always damaging and are actually also important for cell signaling pathways.
Neurodegenerative Disease and Mitochondria Neurodegenerative diseases such as Parkinson’s disease (PD) and Alzheimer’s disease (AD) have been characterized for many years, but we still do not fully understand their pathogenesis or why they target specific individuals and specific neuronal populations. The only thing we definitively know is that for many of them the main risk factor is advancing age, and yet not all elderly individuals develop the symptoms of these diseases. Due to advances in medical science and in light of the modern world in which we all reside, the world population as a whole is aging. It is predicted that by 2030, 1 in 8 of us will be over 65. This equates to one billion people worldwide. As such, age related diseases will increase in prevalence and as such will impact more significantly on medical resources. Since the vast majority of the diseases we discuss in this book are age related and advancing age is the primary risk factor for developing them, it is hugely important that we, as scientists, understand the mechanisms of cell loss in these diseases so that we can devise therapies to improve “healthspans” as lifespan increases. Mitochondria have been proposed as contributors to a number of neurodegenerative disorders including Alzheimer’s disease (AD) (see Chaps. 4 and 5), Parkinson’s disease (PD) (see Chaps. 3, 4 and 5), multiple sclerosis (MS) (see Chap. 11), and Huntington’s disease (HD) (see Chap. 8). While defects in mitochondria caused by mitochondrial DNA mutations are known to directly cause a number of neurological diseases including Kearns Sayre syndrome and mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) (Chap. 2). Perhaps one of the first neurodegenerative diseases to be described in detail, along with its pathology, was Parkinson’s disease. This disease first described in 1817 in an article entitled “An Essay on the Shaking Palsy” is the second most common age related neurodegenerative disease and is characterized by a number of motor symptoms including tremor and bradykinesia. The first published description of patients with this disorder was provided by James Parkinson, but the term “Parkinson’s Disease” was introduced by Charcot who was the first to describe amyotrophic lateral sclerosis (motor neuron disease, in 1869) and multiple sclerosis (in 1868). Alzheimer’s disease, now the most prevalent neurodegenerative disease, was first described in 1906 by Alois Alzheimer, who described this “presenile dementia.” These two disorders are the most common degenerative diseases, but there are many other forms of neurodegenerative disease in which mitochondria have been implicated, for example, Huntington’s disease and multiple sclerosis (Chaps. 8 and 11). Of these diseases, perhaps the most clearly associated with mitochondrial dysfunction is Parkinson’s disease. In the 1980s it was found that a compound known as MPTP caused Parkinson’s disease like symptoms when a number of Californian
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drug users developed these symptoms after using MPTP contaminated drugs. We now know that it is a metabolite of this drug MPP+ which causes the above symptoms by inhibiting complex I of the mitochondrial electron transport chain. The use of such inhibitors in the lab have also been reported to cause PD like symptoms in animal models, and a complex I defect has been described in human samples. Most recently however genetic studies investigating familial, early onset forms of PD have found that a number of genes responsible for these rare forms of PD are actually important for mitochondrial function as well, for example, the proteins pink1 and parkin have effects on mitochondrial fission and fusion and also mitochondrial turnover (Chap. 3). Mutations within the mitochondrial genome are known to cause a group of degenerative disorders, with predominant neurological symptoms and significant muscular involvement. Degeneration occurs in several brain regions, predominantly in the cerebellum, and is associated with loss of mitochondrial proteins and reductions in protein activities. The neuropathological changes seen in these diseases can vary with the type of mitochondrial DNA mutation, but all mitochondrial disorders involve some degree of neurodegenerative change (Chap. 2). These mitochondrial protein changes are also seen in more common neurodegenerative diseases such as PD and AD, but unlike these more common diseases there seems to be no accumulation of protein into inclusions or plaques in mitochondrial disease patients.
Mitochondrial Dysfunction in Neurodegenerative Disease: Format of the Book Part I Mitochondrial Function and Genetics The first section of this book introduces the mitochondrion; we discuss its origins, various functions, and its genome. We give an introduction to mitochondrial DNA genetics, from mitochondrial DNA translation and transcription to maternal inheritance and heteroplasmy. It is intended to give the reader a good introduction to mitochondria to allow them to understand following chapters.
Part II Mitochondrial Dysfunction: Causes and Effects The second section of this book (Chaps. 2, 3, 4 and 5) will introduce the basics of mitochondrial function and genetics. The chapters in Part II discuss the neuropathology of mitochondrial DNA disorders, the intricacies of mitochondrial DNA in various diseases and recent evidence that nuclear mutations which cause familial Parkinson’s disease impact on mitochondrial function.
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Part III Functional Consequences of Mitochondrial Dysfunction Part III (Chaps. 6, 7, 8, 9, 10 and 11) considers the functional consequences of mitochondrial dysfunction within neurons by considering a number of essential neuronal processes which may be affected. Here we consider reactive oxygen species damage, effects of mitochondrial dysfunction of calcium handling, neuronal transport, and myelination. We also examine the effects of loss of mitochondrial membrane potential and how protein turnover may be affected by mitochondrial dysfunction and how this may be related to the neuropathology of several neurodegenerative diseases.
Parts IV and V “Modeling Mitochondrial Contribution to Neurodegenerative Disease” and “The Future” Part IV of this book will consider what we have learned about the implications of mitochondrial dysfunction, for neurons and the brain, through the use of animal models. In this section we review a vast number of models and reflect on their contributions to our understanding of neurodegenerative diseases. In our final section we look to the future and discuss whether by understanding these roles of mitochondria in neurodegenerative diseases we may be able to consider and design therapeutic measures. After all, the overall aim of investigating and understanding the pathogenesis of these diseases is to be able to create treatments to ultimately prevent the loss of neurons and other cells, to offer hope to the thousands of sufferers of these disorders. We end this book with a reflection of the importance of mitochondria and mitochondrial dysfunction in neurodegenerative diseases beyond those caused directly by mutations within the mitochondrial genome. Amy Katherine Reeve Kim Jennifer Krishnan Michael R. Duchen Doug M. Turnbull
Part I Mitochondrial Function and Genetics
1
An Introduction to Mitochondria Amy Katherine Reeve and R.N. Lightowlers
Abstract
Mitochondria are essential organelles for all nucleated eukaryotic cells, responsible for numerous key cellular functions such as energy transduction, buffering cellular calcium, apoptosis, and the synthesis of Fe-S clusters. In this chapter we give an introduction into these fascinating organelles from their evolution to some of their functions. We introduce a number of concepts which are crucial for later chapters as well as giving a brief history of mitochondrial research. Keywords
Mitochondria • Mitochondrial function • Mitochondrial DNA • mtDNA genetics
1.1
Introduction
1.1.1
What Are Mitochondria and Where Did They Come From?
Due to the pioneering work of Rudolf Albert von Kölliker we have known about the presence of mitochondria or “threadlike granules” in the cytoplasm of eukaryotic cells for more than 150 years, but what exactly are these organelles, where did they come from and what do they do?
A.K. Reeve Centre for Brain Ageing and Vitality, Mitochondrial Research Group, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, Tyne and Wear, UK e-mail:
[email protected] R.N. Lightowlers (*) Mitochondrial Research Group, Institute for Ageing and Health, Newcastle University Medical School, Newcastle upon Tyne, Tyne and Wear, UK e-mail:
[email protected] A.K. Reeve et al. (eds.), Mitochondrial Dysfunction in Neurodegenerative Disorders, DOI 10.1007/978-0-85729-701-3_1, © Springer-Verlag London Limited 2012
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Mitochondria, or the related hydrogenosomes and mitosomes are believed to be present in all nucleated cells of the eukarya [1], often comprising more than 20% of the total cell volume. There is a general consensus that mitochondria have evolved from an alpha eubacterial ancestor, which became engulfed by a host cell in a process referred to as endosymbiosis [2]. The origin of the host cell, however, remains contentious (see [3]). Following seminal work on electron microscopy of the cell, Palade and colleagues in the early 1950s revealed that mitochondria contained an outer membrane encasing an inner, often highly invaginated, membrane [4]. These folds have been termed cristae and massively expand the surface area of the inner membrane available for biochemical reactions. It rapidly became apparent that the form and complexity of these infoldings varied enormously between tissue type and even within type, dependent on the developmental stage or physiological condition. Thus the classic textbook vision of a mitochondrion was borne with the two soluble compartments, the internal matrix and the intermembrane space being encased by an outer membrane and separated by the inner membrane. These early ultrastructural studies were impressive, but exactly what form of invagination constituted the cristae has only recently been resolved. Electron tomography is a method for reconstituting three-dimensional structures from serial sections. Frey, Manella, and colleagues applied EM tomography to serial sections of rat liver mitochondria and were able to show that the ultrastructure of the inner membrane had discrete subdomains: an inner boundary membrane, which is closely opposed to the outer membrane, and the tubular cristae membranes, which are separated by distinct cristae junctions [5]. Various studies have now shown an enrichment of specific mitochondrial proteins and complexes in these subcompartments [6]. EM tomography has increased our understanding of mitochondrial ultrastructure, but is unable to give substantial information about mitochondrial dynamics. It had been well established that under certain physiological conditions mitochondria could generate large reticular networks in a variety of species. The study of mitochondrial dynamics has however been revolutionized by use of time-lapse fluorescence microscopy. The mitochondrion is not static. It is connected to the cytoskeleton and is capable of fusing and budding. These processes of fission and fusion are thought to be key for a number of cellular and mitochondrial processes and require a plethora of protein interactions. Mutations in genes encoding these essential proteins have been shown to cause a number of degenerative diseases, such as hereditary optic atrophy and Charcot Marie Tooth disease type 2A [7, 8]. A detailed discussion of the molecular mechanisms underlying fission and fusion is beyond the scope of this chapter and readers are recommended the following reviews and Chap. 8 for future reference [9, 10].
1.1.2
What Are the Functions of These Essential Organelles?
Mitochondria have been known to house the central metabolic pathways for oxidation of food stuffs for well over 50 years. One of their major and perhaps most
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An Introduction to Mitochondria
5
Complex I Complex III Complex V Complex II Complex IV H+ Caspase 3
Cell death Cyt c
Caspase 9
Apaf-1
H+ e-
H+ e-
H+ ADP ATP
Bcl2 Cytochrome c
Mitochodria are required for the formation of iron sulphur clusters. These clusters are essential for the function of a number of proteins.
Mitochondria are essential for maintaining Ca2+ homeostasis. this is achieved through the permeability transition pore as well as through calcium and sodium transporters.
Fig. 1.1 The many functions of the mitochondrion. The mammalian mitochondrion houses specific enzymes involved in the biosynthesis of various essential cellular components, as well as the metabolic pathways for the oxidation of fats and sugars. Reduced cofactors that are produced from these processes pass electrons into the respiratory chain, a set of four enzyme complexes housed in the mitochondrial inner membrane. Protons that are concomitantly passed across the membrane generate a proton electrochemical gradient, which is harnessed by the FOF1 ATP synthase (complex V) to produce ATP. This process is referred to as oxidative phosphorylation (upper right). One of the major pathways of apoptosis is mediated by proteins from within the mitochondrion such as cytochrome c. On release of this protein from the intermembrane space, it associates with Apaf-1 to produce the apoptosome that activates various cysteine proteases as part of a signaling pathway, which eventually leads to cell death (upper left panel – see text for details). Ca2+ buffering is another important function of the mitochondrion. Finally, the only function that has been retained in the mitochondrion and its evolutionary relatives, the hydrogenosome and mitosome, is the production of Fe-S clusters, essential cofactors that are found in a plethora of enzymes throughout the cell
celebrated functions is to couple cellular respiration to the production of adenosine triphosphate in a process referred to as oxidative phosphorylation (OXPHOS). Determining how the transfer of electrons through a complex series of intramembranous enzymes could result in the formation of ATP was a highly emotive issue in the 1950s and 1960s. The explanation lay in how several of these enzymes pumped protons across the impermeable inner membrane concomitantly on electron transfer, generating a proton electrochemical gradient which was harnessed by a fifth enzymatic complex, the FOF1 ATP synthase to produce ATP, which was subsequently transported to the cytosol by the adenine nucleotide translocase. This chemiosmotic hypothesis of oxidative phosphorylation [11] was recognized by award of the Nobel Prize for Chemistry to Peter Mitchell in 1978. In more recent years, it has become apparent just how mitochondria are essential players not only in metabolism and energy transduction, but also in apoptosis, Ca2+ homoeostasis and intracellular signaling (Fig. 1.1), which are each detailed in other chapters.
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Strikingly, the only process that has been retained in the organelle from all species during reductive evolution is the ability to form Fe-S clusters (ISCs). These important cofactors can be exported into the cytosol where they are incorporated into numerous enzymes which are essential for cell function. Detailed analysis of the many components involved in this crucial process has now been documented, as described in [12]. Although the complete set of ISC-containing proteins has yet to be described for any cell, it was recently shown that several proteins involved in genome maintenance contain ISCs [13]. Yeast cells, which had a reduced mitochondrial membrane potential due to a lack of OXPHOS capacity, were subject to loss of heterozygosity a condition indicative of genome instability. Transcriptomic experiments indicated that the reduction in membrane potential resulted in a profile indicative of iron starvation in cells. Further, it was demonstrated that ISC formation in the mitochondrion was severely impaired. These data are consistent with decreased mitochondrial membrane potential leading to genomic instability as a consequence of the lack of ISC formation which is required for the function of several proteins involved in genomic maintenance. Mitochondrial also contain their own DNA, a remnant of their bacterial origins, this genome encodes a number of proteins essential for OXPHOS. The genetics of this mitochondrial genome and its characteristics are discussed below.
1.2
Oxidative Phosphorylation
The production of ATP through the process of oxidative phosphorylation (OXPHOS) is the main and most well-known function of mitochondria and has led to these organelles often being described as the “powerhouses” or “batteries” of the cell. The number or mass of mitochondria per cell does generally reflect the energy demands of that cell, for example, energy-demanding tissues such as muscle and neurons tend to have more mitochondria than less energy-demanding cells. This energy transduction occurs through the transport of electrons through a chain of proteins. These protein complexes lie within the inner mitochondrial membrane and have components encoded by both the mitochondrial and nuclear genomes. The donation of electrons to this chain of protein complexes is possible through the enzymatic reactions of glycolysis and the tricarboxylic acid cycle, these reactions oxidize carbon to generate electrons. These electrons are then passed from reduced cofactors to complex I (NADH:CoQ oxidoreudctase) or complex II (succinate:ubiquinone oxidoreductase) and flow through additional complexes before being finally transferred to molecular oxygen. The sequential passage of electrons along this chain is linked to the production of a proton gradient across the inner mitochondrial membrane, which is utilized by the FOF1 ATP synthase to form ATP. The first complex of this chain is by far the largest complex and is composed of over 40 subunits with a combined molecular weight of 980 kDa [14], for review see [15]. Seven of these subunits are encoded by the mitochondrial genome (MT-ND1 to 6 and MT-ND4L), with the remainder encoded by nuclear DNA. This complex has a characteristic L-shape, and it resides within the inner mitochondrial membrane with
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one arm protruding into the mitochondrial matrix. All of the subunits encoded by the mitochondrial genome are highly hydrophobic, contain transmembrane helices and are all essential functional subunits. Complex I (NADH:Ubiquinone Oxidoreductase) catalyzes the oxidation of NADH, derived from the citric acid cycle, which yields two electrons. These electrons pass to a flavin mononucleotide and through a series of iron sulfur clusters to ubiquinone reducing it to ubiquinol. The transfer of these two electrons allows the translocation of four protons across the inner mitochondrial membrane. The exact structure of the mammalian complex I has yet to be resolved, but the recent report of the structure of the ortholog found in Thermus thermophilus is a tour de force [15]. This beautiful structure shows how a large helical domain that lies parallel to the membrane is able to move in a piston-like manner as a consequence of electron transfer, facilitating the translocation of protons across the membrane. Succinate:ubiquinone oxidoreductase (complex II), is also an essential component of the citric acid cycle, where it is responsible for oxidizing succinate to fumarate. Electrons from this oxidation are then donated to the electron transport chain (ETC) through the reduction of ubiquinone. Complex II is the smallest complex of the ETC, is the only complex which is entirely encoded by the nuclear genome, and electron flow through this complex is not associated with proton translocation. Succinate dehydrogenase is composed of only four subunits. The two hydrophilic groups A and B are a flavoprotein and an iron sulfur protein, respectively, while the two hydrophobic subunits are integral membrane proteins which contain heme groups (for review see [16]). Following the reduction of ubiquinone by complexes I and II, its reoxidation is catalyzed by complex III, ubiqinol:cytochrome c oxidoreductase. This third complex is composed of 11 subunits, which with the exception of cytochrome b are entirely nuclear encoded. Three of the subunits of this complex contain redox groups which are cytochrome b which contains two heme groups, cytochrome c1 and the Rieske protein (or the iron sulfur cluster) [17]. The oxidation of ubiquinol releases the two electrons transferred from complexes I and II. The transfer of these two electrons from ubiquinol to cytochrome c occurs in two steps. First, one of the electrons is transferred to the iron sulfur cluster and then to cytochrome c via cytochrome c1 while the second one is recycled to ubiquinol in a reaction called the Q-cycle. In this cycle two ubiquinols are oxidized, two electrons are transferred and one ubiquinone reduced. For each electron transferred two protons are translocated across the inner mitochondrial membrane. Electrons are finally transferred to complex IV (cytochrome c oxidase), of which three subunits are encoded by the mitochondrial genome (COXI, II, III). The substrate for this complex is cytochrome c, which transfers the electrons between complexes III and IV. This hemoprotein donates electrons to the intermembrane space side of complex IV whose structure in bovine mitochondria has been resolved to 2.8 Å [18]. Electrons are donated one by one to the CuA site; these single electrons are then sequentially transferred via a heme group to the catalytic site which contains another Cu (CuB) and heme a, a3. Cytochrome c is only capable of donating single electrons but a total of four are required and so this protein must shuttle between complexes III and IV. The CuB site is reduced following an addition of two
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electrons; this is followed by the reduction of a heme site, which can now bind oxygen. Upon binding of oxygen, the heme site is in the peroxy state, following the additions of electrons three and four this site passes through the ferryl and oxidized states, respectively. Each of these transitions results in the translocation of a proton across the membrane. As electrons are passed down this chain to molecular oxygen, a total of eight protons are translocated across the inner mitochondrial membrane. This proton gradient is then utilized by the final complex of the oxidative phosphorylation machinery to generate ATP. Complex V, FOF1 ATP synthase is a large multi-subunit complex containing two subunits encoded by the mitochondrial genome (ATPase 6 and 8). This complex is composed of two main domains: FO which embedded in the inner membrane is composed of 9–12 subunit c molecules and the ab2 complex which acts to link FO to F1, and F1. F1, the catalytic domain, forms the head and stalk of the complex and is located on the matrix side of the inner membrane. F1 is composed of five different subunits a, b, g, d, and e [19]. Subunits a and b form a cylinder around subunit g, with the b subunits possessing catalytic activity and subunit e forms a stalk and interacts with FO subunit C9–12 [20]. The current structural model of ATP synthase suggests that subunits C9–12 are connected to a complex formed by subunits g and e forming the rotor. The stator arm of the enzyme is then formed from the ab2 complex and subunit d [21]. b subunits can possess three conformations, open, loose, and tight. In these conformations, the catalytic sites are empty, contain bound ADP + Pi or tightly bound ATP, respectively. Any of the b subunits may be in any of these states at any time, and conformation is determined by altering the contact with the g subunit. The movement of protons across the inner membrane from the intermembrane space side promotes rotation of the F1, causing the b subunits to transfer between the three conformational states. The energy harnessed during this rotation is used to synthesize ATP which can then be released on the next rotation. Three protons are required for each molecule of ATP since one proton is required for each b subunit [20, 21]. The generation of ATP through oxidative phosphorylation is an extremely efficient method of aerobic glucose metabolism, far more efficient than through the substrate level phosphorylation of anaerobic glucose metabolism merely by glycolysis. Therefore, it is easy to comprehend how any defect which affects the ability of mitochondria to carry out this essential process will greatly affect the production of ATP which will be detrimental to cellular function. The consequences of mitochondrial dysfunction are considered in the remaining sections of this book, in particular in relation to the central nervous system.
1.3
Apoptosis
Apoptosis is a critical process throughout life, it is responsible for development (e.g., in limb modeling and neurodevelopment), it is also important for maintaining tissue homeostasis and intervening in this process could lead to treatments for cancer and also degenerative diseases. This process was first observed in terms of cellular morphology in the 1970s. Kerr et al. observed the phenomena of membrane
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blebbing and chromatin condensation in cells undergoing apoptosis [22]. Apoptosis therefore can be easily characterized and is a highly regulated form of cell death. There are two main apoptosis pathways which occur in mammalian cells, the death receptor (intrinsic) pathway and the mitochondrial (extrinsic) pathway. The death receptor pathway is triggered by members of the death-receptor superfamily, for example, CD95, which is a component of the death-inducing signaling complex which is capable of recruiting other molecules such as caspase-8 leading to apoptosis. Both this pathway and the mitochondrial pathway involve caspases and converge at the mitochondrion [23]. The mitochondrial apoptosis pathway has the ability to respond to both intracellular and extracellular cues, as exemplified by DNA damage. The most potent signaling molecule in this apoptosis pathway is cytochrome c which, as mentioned above, normally shuttles electrons between complexes III and IV of the electron transport chain. The release of cytochrome c occurs following the loss of mitochondrial membrane potential following mitochondrial outer membrane permeabilization. On release of cytochrome c from the mitochondrial intermembrane space to the cytosol, it activates caspase-9, which in turn forms a complex with Apaf-1 in the presence of cytochrome c, increasing the proteolytic activity of caspase 9, which can then activate caspase-3 leading to apoptosis [24, 25]. For this to occur, however, cytochrome c must move from the mitochondrion to the cytosol. This movement is regulated by the Bcl-2 family of proteins (including Bax and Bak) [26, 27]. These proteins are localized to the outer mitochondrial membrane and may be pro- or antiapoptotic. It is thought that these proteins permeabilize the mitochondrial outer membrane either through interactions with the voltage-dependent anion channel (porin) (reviewed in [28]) or with these Bcl-2 proteins forming a channel in the outer membrane themselves. These proteins may also allow the movement of DIABLO (an inhibitor of apoptosis protein) and AIF (apoptosis-inducing factor) from the mitochondrion into the cytosol (reviewed in [29, 30]). Recent evidence has also suggested that the Bcl-2 family of proteins may also be important in regulating the mitochondrial network during apoptosis but also in healthy cells. Mitochondrial fragmentation occurs during apoptosis and it has been proposed that this fragmentation may be crucial for the release of cytochrome c (reviewed in [31]). The relationship of the mitochondrion to neuronal cell loss and death will be considered in a number of other chapters.
1.4
The Human Mitochondrial Genome
A unique attribute for any organelle in a mammalian cell outside the nucleus is that the human mitochondrion contains expressed nucleic acid. This compact circular genome (mtDNA) comprises 16,569 bp and encodes 13 polypeptides, all of which are components of the oxidative phosphorylation machinery (see above, Fig. 1.2a). The additional 34 genes encode the complete set of mitochondrial tRNAs required for the intramitochondrial synthesis of these 13 proteins and 2 ribosomal RNAs that are essential for assembly of the 2 mitochondrial ribosomal subunits [32]. Any further proteins required for mitochondrial function are synthesized in the cytosol and
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D-Loop (i) OH
Light strand Heavy strand
(ii)
OL
(iii)
a
b The Synchronous model of mitochondrial DNA replication. OH OH OH
The Asynchronous model of mitochondrial DNA replication. OH OH OH
OL
OL
OL
OL
OL
OL OH
OH OH
OH
OH
OH OH
OL
OL
OH
OL
OL OL
OL
OL OL
c
d
Fig. 1.2 Mitochondrial genetics and models of mitochondrial genome replication. (a) The mitochondrial genome encodes components of the oxidative phosphorylation machinery. Genes encoding components of complex I are shown in yellow and those encoding members of complex III, IV and V shown in pink, blue, and green, respectively. Genes encoding the two ribosomal RNAs are shown in purple and tRNAs are denoted in black. (b) Due to the multicopy nature of mtDNA, a unique genetic situation can arise where all mtDNA molecules within the mitochondria are the same, a situation termed homoplasmy (i). However, a mutation event may occur in a few genomes (heteroplasmy), but its effect will not be felt as substantial wild-type levels are present and the vast majority of mutations are recessive; (ii), however a given mutation may expand over time until it is represented in the majority of the genomes, eventually causing mitochondrial dysfunction (iii). (c) There are several theories for how mtDNA replicates, but perhaps two models are the current leaders. First, is the Clayton model of asynchronous replication where replication commences from the origin of heavy strand replication OH and the light strand is displaced. Replication proceeds around the genome until the origin of light strand replication, OL, is reached and exposed. Initiation is depicted in a counter clockwise direction. In this model there is a delay between the replication of the two strands. (d) Shows Holts proposed model of synchronous replication. In this model replication commences from a zone around the origin of heavy strand replication. Initiation of light strand replication commences, with replication of this “lagging” strand being the result of the generation of short Okazaki fragments. Replication then proceeds bidirectionally around the genome. A third model, RITOLS, invokes a replication intermediate whereby RNA is incorporated throughout the lagging strand [45]
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are then actively transported into the mitochondrion through transport proteins on the outer and inner mitochondrial membranes, the TOM and TIM complexes, respectively. The mitochondrial genome is dwarfed by its nuclear counterpart (approximately 3.4 billion base pairs per haploid genome) but can still constitute almost 1% of the total amount of cellular DNA in mature oocytes. It is present in thousands of copies in most cell types and up to millions of copies in mature oocytes or early zygotes [33]. As mtDNA is tethered to the inner side of the inner mitochondrial membrane, it is close to the components of the electron transfer chain, the major site of reactive oxygen species generation in the cell. Exposure to ROS is known to damage DNA and the mitochondrial genome is no exception. Although the organelle contains an impressive array of DNA damage repair enzymes [34], it is highly likely that the toxic oxidative environment overwhelms these systems leading to the eventual generation of mtDNA mutations. The production of a second, genetically distinct, mtDNA molecule produces a situation referred to as heteroplasmy (Fig. 1.2b). Originally, it was believed that all mtDNA molecules in a cell or tissue were invariant. Next generation sequencing of mtDNA sequences has now shown this to be a rather naïve supposition, with allelic variants being extremely common in tissues [35], with a small fraction of the variation being inherited from the mother and the majority occurring early in development. The complexities of human mitochondrial genetics and disease will be covered in detail in Chap. 4.
1.4.1
How Is mtDNA Maintained, Replicated, and Inherited?
A massive amount of supportive data shows that mtDNA is invariably inherited down the maternal lineage. Consistent with almost every law in the life sciences, there is always one exception that proves the rule, and maternal transmission of mtDNA is no different. One case of the paternal transmission of pathogenic mtDNA has been recorded [36], but this is a unique report. Not only is sperm mtDNA massively outnumbered in the fertilized zygote, but it is highly likely that there is also an active process for degradation of male-derived mtDNA. Such processes have been shown in mouse intra-species crosses [37]. Further, it has been proposed that during fertilization of mammalian oocytes, mitochondrial proteins from the sperm mid piece, which accesses the oocyte, presumably on the outer membrane, are selectively ubiquitinated leading to degradation within the fertilized egg [38]. Our understanding of how the levels and integrity of human mtDNA is maintained has been greatly enhanced by the study of patients with mitochondrial disease (see Chap. 2). It has been known for over 20 years that defects in mtDNA can cause a wide variety of disorders, which often (but not solely) affect muscle and the central nervous system (recently reviewed in [39]). More recently, however, it has become apparent that many combined disorders of OXPHOS complexes are also caused by mutations in nuclear genes whose products affect mtDNA maintenance, causing multiple deletions (e.g., POLG1, POLG2, ANT1, PEO1, OPA1, TYMP) or indeed mtDNA depletion (DGUOK, SUCLA2, SUCLG1, RRM2B, MPV17, TK2) (reviewed
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in [40]). Mutations are found in proteins that function directly in mtDNA replication, such as the mitochondrial DNA polymerase g (POLG), its accessory unit and the critical DNA helicase, twinkle, or in the homeostasis of mitochondrial nucleotide pools. All these proteins are essential for maintaining mtDNA, but exactly how does this molecule replicate? Based on a series of published observations, Clayton and colleagues reported a model where mtDNA replicated in a strand asynchronous mechanism, priming from the light strand promoter within the major noncoding region of the genome (Fig. 1.2c) [41]. This RNA primer was then extended by the mitochondrial DNA polymerase, copying the entire Heavy strand. As the polymerase passed approximately twothirds of the entire length of the L-strand template, a region of the H-strand was revealed. This region folded to produce a stable stem-loop structure that acted as the promoter of L-strand synthesis. RNA was laid down by an unknown “primase” and was in turn fully extended by the DNA polymerase. This model was neat but unusual, with no obvious similar precedents known in the DNA replication field. Holt and colleagues challenged the assumption that strand asynchronous replication was the universal method for mtDNA replication (Fig. 1.2d). Researchers working in the field of DNA replication commonly visualized replication intermediates by the use of native 2D gel electrophoresis [42]. When applied to mtDNA, replication intermediates were more consistent with a strand-coupled then strand-asynchronous model [43]. This work also highlighted the previously established and surprising observation that ribonucleotides are incorporated into the genome [44]. Taken to extremes, this has also led to a third replication model, RITOLS (RNA incorporation throughout the lagging strand), whereby ribonucleotides are present throughout the entire lagging strand [45]. Many questions still remain and it is currently unclear exactly how mtDNA is replicated. It is possible that mtDNA may be replicated by different modes at different stages of development or in different tissues. However, it is of interest to note a third approach to tackling this difficult problem. Falkenberg and colleagues have been attempting to reconstitute mtDNA replication in vitro [46]. Intriguingly, they have been able to show that the region referred to as OriL by Clayton et al. can indeed form a structure which can be primed by the mitochondrial RNA polymerase (POLRMT) [47]. This primer is then faithfully extended by the mitochondrial DNA polymerase gamma. Native gel analysis was unable to assign a replication initiation site at this region, but did show this site to be an important replication pause site. Deletion break point analysis of mtDNA molecules capable of in vivo replication has shown a marked lack of deletion of OriL, arguing strongly in favor of the importance of this site in mtDNA replication.
1.5
From DNA to RNA: Mitochondrial Transcription, Processing, Maturation, and RNA Turnover
Mitochondrial transcription is unusual in that the core transcription complex containing the mtRNA polymerase and the two characterized transcription factors TFAM and TFB2 facilitate transcription of almost the entire genome from promoters borne
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on both strands. Detailed analysis of mitochondrial transcription and its regulation is beyond the scope of this chapter, but can be found in the excellent recent review by Larsson and colleagues [48]. These large polycistronic units are essentially punctuated by mitochondrial tRNA structures, which are thought to spontaneously fold and are subsequently liberated by the mt-tRNA processing enzyme activities RNase P [49] and RNase Z. This tRNA punctuation model appears to be correct in general, but it cannot explain how certain mRNA species are processed, as they are not surrounded by tRNA or antisense tRNA (RNA14, MT-CO3, MT-ND5, MT-ND6) [50]. All the resultant mitochondrial mRNA and tRNAs are matured, either by the 3¢ terminal addition of a CCA trinucleotide extension ([CCA] nucleotidyl transferase [51]) to all mt-tRNAs or polyadenylation of mt-mRNAs of approximately 35–60 nucleotides by a mitochondrial poly(A) polymerase [52]. It must be noted, however, that at steady state a subpopulation of mt-mRNAs carries a shorter oligoadenylated extension that is not due to 3¢ 5¢ exonucleolytic degradation. These oligoadenylated species are the predominant form of mt-mRNAs in patients with defective mtPAP [53] or under conditions of mtPAP depletion [52, 54], leading to the suggestion that a second poly(A) polymerase activity must be present in the mitochondrial matrix. Why are mt-mRNAs polyadenylated? The answer is still frustratingly unclear, although addition of at least one or two adenines is essential to complete a termination codon for 7 of the 13 open reading frames. Poly/oligoadenylation of RNAs is a well-established phenomenon in all domains of life [55]. It can lead to RNA instability in prokaryotes, chloroplasts, or the eukaryote nucleus, yet it promotes both RNA stability and translation in the eukaryote cytosol. In protist mitochondria, it can lead to decay or to stability, dependent on the editing state of the molecules [56]. The situation in human mitochondria is complex. Removal of the poly(A) tail can lead to transcript decay, increased stability, or be unaffected dependent on the mtmRNA species [57]. Indirect evidence also suggests that polyadenylation increases the translatability of the transcript [57]. Although much is known about mitochondrial transcription, RNA processing and maturation, our current understanding of mitochondrial RNA turnover is negligible. To our knowledge, there is no report of any mitochondrial ribonuclease of any form that is involved in RNA turnover and the mitochondrial proteome fails to identify any obvious candidates. The impressive algorithms that have been established over the past few years to predict mitochondrial proteins also fail to identify any credible mitochondrial-specific matrix ribonucleases. It is remarkable that we are still unaware of the identity of proteins that perform such a fundamental role as mitochondrial RNA turnover.
1.6
The Mitochondrial Ribosome and Translation of Mature Mitochondrial mRNA
Pioneering studies by, amongst others, O’Brien, Watanabe, and Spremulli have been somewhat hampered by the absence of a faithful in vitro reconstituted system for mammalian mitochondrial protein synthesis. Consequently, although we are aware
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of numerous proteins that are crucial in each of the four stages of protein synthesis, initiation (If2mt, If3mt) elongation (mtEFG1, mtEFTu, mtEFTs), termination (mtRF1a, mtEFG2) and recycling (mtRRF), functional assays have had to rely on heterologous systems (for a recent review see [58]). Highly purified bovine mitoribosomes have been subjected to proteomic analyses, and cryoEM studies have successfully visualized the structure to almost 10 Å [59]. This structure has at least 83 polypeptides and only 2 ribosomal RNAs. Thus, unlike all other ribosomes to date, the mitoribosome is mostly comprised of protein (approximately 70%) over RNA (30%) where the norm is more 65%:35% RNA to protein. Consequently, the mitoribosome is less dense (55S particle), comprising a 39S large and 28S small subunit. A 5S RNA has been found in mitochondria and import has been demonstrated [60, 61]. Although it is not present in the highly purified mitoribosome, it is formally possible that it has been lost during purification or that it has no role in mitochondrial protein synthesis. Another striking absence from the cryoEM structure is an Exit site for the mitochondrial tRNA. To date, an E site is present in all other ribosomal structures. The mitoribosome has an exquisite and essential interaction with the mitochondrial membrane, possibly reflecting the highly hydrophobic nature of all 13 mitochondrial gene products. This may also partly explain why it has been so difficult to reconstitute a faithful system in vitro. This critical association has been highlighted by studies of patients with mitochondrial OXPHOS disorders. As described above, two important subsets of patients with mitochondrial disease are caused either by direct mutations in the mitochondrial genome or by nuclear mutations in proteins that are involved in mtDNA maintenance. Recently, a third subset of disorders has been described. These patients have defects in mitochondrial protein synthesis without involvement of mtDNA quality or quantity. Patients have combined OXPHOS disorders, with a wide spectrum of clinical presentation (recently reviewed in [62]). Patients with a form of hereditary spastic paraplegia, HSP7, have been shown to harbor mutations in the protein paraplegin [63]. This protein is a member of the heteromultimeric mAAA protease that is responsible for cleaving membrane-bound proteins. Langer and colleagues were able to show that the reason for the severe mitochondrial protein synthesis defect in cells lacking normal mAAA protease activity was due to the loss of maturation of the mitoribosomal protein MRPL32. Further, when this protein is not cleaved, the mitoribosome is unable to associate with the inner membrane. Intriguingly, mutations in the second of the heteroligomeric components, AFG3L2, also causes severe combined OXPHOS disorder, but clinical presentation is predominantly a spinocerebellar ataxia [64].
1.6.1
How Does the Mitochondrion Control the Quality of Its Synthesized Proteins?
The process of protein synthesis is notoriously complex, with problems often occurring early in the process leading to stalling of the ribosome and loss of the nascent peptide [65]. Stalling can also occur when mRNAs lack termination codons. Once the ribosome has stalled, there must be mechanisms to remove the peptidyl-tRNA
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from the complex and dissociate the ribosomal subunits. A well-described process for rescuing stalled ribosomes is found in eubacteria, where a novel RNA, tmRNA encoded by the SsrA gene, is able to access the ribosomal A site and promote its own translation, leading to the addition of a short oligopeptide to the C-terminus of the nascent protein which acts as a substrate for ubiquitination and subsequent degradation [66, 67]. This tmRNA also contains a termination codon, facilitating the hydrolysis of the complete peptidyl-tRNA and recycling of the ribosome. In the absence of a tmRNA ortholog in mitochondria, how are stalled ribosomes recognized? Human genome analysis identifies four members of a mitochondrial peptidyl-tRNA release factor family. Following the observation that the rare codons AGA and AGG promote ribosome frameshifting at the terminus of two mitochondrial open reading frames [68], we now know that only one of the release factor family, mtRF1a, is capable of terminating translation of all 13 open reading frames [69]. It is therefore highly likely that other members of this family will be intimately involved in rescuing stalled ribosomes. Indeed, one of these members, ICT1, has been shown to have become integrated into the mitochondrial ribosome [70]. Although its exact function is currently unknown, it is highly likely to be involved in ribosome rescue, particularly as its peptidyl tRNA hydrolase activity is codon independent, potentially allowing it to release peptides stalled at any or indeed no A site codon. The roles of the remaining two factors are unknown although defects of one member, C12orf65, have been shown to cause mitochondrial disease [71]. Although tmRNA is a mechanism used widely by eubacteria for rescuing stalled ribosomes, it is not essential for viability, and recently other proteins have been also shown to promote such rescue, one of which, YaeJ, is believed to be the ortholog of ICT1 [72]. Such observations make it highly likely that the other members of this family will also be important in quality control of the translation system in mammalian mitochondria.
1.7
Conclusions
In summary, the mammalian mitochondrion houses perhaps as many as 1,500 different types of protein which are involved in multiple functions critical to the cell. Although we have made substantial progress in unraveling many of these molecular mechanisms, as is well documented in the chapters that follow. However, we are still surprisingly unsure of the function of a substantial subpopulation of these proteins. It is envisioned that the next generation of scientists will have great success in describing their roles and elucidating the crucial processes of how this organelle monitors its own function and how it is able to communicate with the host cell.
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32. Anderson S, Bankier AT, Barrell BG, et al. Sequence and organization of the human mitochondrial genome. Nature. 1981;290:457–65. 33. Craven L, Tuppen HA, Greggains GD, et al. Pronuclear transfer in human embryos to prevent transmission of mitochondrial DNA disease. Nature. 2010;465(7294):82–5. 34. Boesch P, Weber-Lotfi F, Ibrahim N, et al. DNA repair in organelles: pathways, organization, regulation, relevance in disease and aging. Biochim Biophys Acta. 2011;1813(1):186–200. Epub Oct 13, 2010. 35. He Y, Wu J, Dressman DC, et al. Heteroplasmic mitochondrial DNA mutations in normal and tumour cells. Nature. 2010;464(7288):610–4. 36. Schwartz M, Vissing J. Paternal inheritance of mitochondrial DNA. N Engl J Med. 2002; 347(8):576–80. 37. Kaneda H, Hayashi J, Takahama S, et al. Elimination of paternal mitochondrial DNA in intraspecific crosses during early mouse embryogenesis. Proc Natl Acad Sci USA. 1995;92(10): 4542–6. 38. Sutovsky P, Moreno RD, Ramalho-Santos J, et al. Ubiquitinated sperm mitochondria, selective proteolysis, and the regulation of mitochondrial inheritance in mammalian embryos. Biol Reprod. 2000;63(2):582–90. 39. Tuppen HA, Blakely EL, Turnbull DM, et al. Mitochondrial DNA mutations and human disease. Biochim Biophys Acta. 2010;1797(2):113–28. 40. Copeland WC. Inherited mitochondrial diseases of DNA replication. Annu Rev Med. 2008;59:131–46. 41. Clayton DA. Replication and transcription of the vertebrate mitochondria DNA. Annu Rev Cell Biol. 1991;7:453–78. 42. Brewer BJ, Fangman WL. A replication fork barrier at the 3¢ end of yeast ribosomal RNA genes. Cell. 1988;55(4):637–43. 43. Holt IJ, Lorimer HE, Jacobs HT. Coupled leading- and lagging-strand synthesis of mammalian mitochondrial DNA. Cell. 2000;100(5):515–24. 44. Grossman LI, Watson R, Vinograd J. The presence of ribonucleotides in mature closed-circular mitochondrial DNA. Proc Natl Acad Sci USA. 1973;70(12):3339–43. 45. Yasukawa T, Reyes A, Cluett TJ, et al. Replication of vertebrate mitochondrial DNA entails transient ribonucleotide incorporation throughout the lagging strand. EMBO J. 2006;25(22): 5358–71. 46. Korhonen JA, Pham XH, Pellegrini M, et al. Reconstitution of a minimal mtDNA replisome in vitro. EMBO J. 2004;23(12):2423–9. 47. Fuste JM, Wanrooij S, Jemt E, et al. Mitochondrial RNA polymerase is needed for activation of the origin of light-strand DNA replication. Mol Cell. 2010;37(1):67–78. 48. Falkenberg M, Larsson NG, Gustafsson CM. DNA replication and transcription in mammalian mitochondria. Annu Rev Biochem. 2007;76:679–99. 49. Holzmann J, Frank P, Loffler E, et al. RNase P without RNA: identification and functional reconstitution of the human mitochondrial tRNA processing enzyme. Cell. 2008;135(3):462–74. 50. Temperley RJ, Wydro M, Lightowlers RN, et al. Human mitochondrial mRNAs-like members of all families, similar but different. Biochim Biophys Acta. 2010;1797(6–7):1081–5. 51. Nagaike T, Suzuki T, Tomari Y, et al. Identification and characterization of mammalian mitochondrial tRNA nucleotidyltransferases. J Biol Chem. 2001;276(43):40041–9. 52. Tomecki R, Dmochowska A, Gewartowski K, et al. Identification of a novel human nuclearencoded mitochondrial poly(A) polymerase. Nucleic Acids Res. 2004;32(20):6001–14. 53. Crosby AH, Patel H, Chioza BA, et al. Defective mitochondrial mRNA maturation is associated with spastic ataxia. Am J Hum Genet. 2010;87(5):655–60. 54. Slomovic S, Schuster G. Stable PNPase RNAi silencing: its effect on the processing and adenylation of human mitochondrial RNA. RNA. 2008;14(2):310–23. 55. Gagliardi D, Stepien PP, Temperley RJ, et al. Messenger RNA stability in mitochondria: different means to an end. Trends Genet. 2004;20(6):260–7. 56. Kao CY, Read LK. Opposing effects of polyadenylation on the stability of edited and unedited mitochondrial RNAs in Trypanosoma brucei. Mol Cell Biol. 2005;25(5):1634–44.
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57. Wydro M, Bobrowicz A, Temperley RJ, et al. Targeting of the cytosolic poly(A) binding protein PABPC1 to mitochondria causes mitochondrial translation inhibition. Nucleic Acids Res. 2010;38(11):3732–42. 58. Koc EC, Haque E, Spremulli LL. Current views of the structure of the mammalian mitochondria. Isr J Chem. 2010;50(1):45–59. 59. Sharma MR, Koc EC, Datta PP, et al. Structure of the mammalian mitochondrial ribosome reveals an expanded functional role for its component proteins. Cell. 2003;115(1):97–108. 60. Magalhaes PJ, Andreu AL, Schon EA. Evidence for the presence of 5S rRNA in mammalian mitochondria. Mol Biol Cell. 1998;9(9):2375–82. 61. Smirnov A, Tarassov I, Mager-Heckel AM, et al. Two distinct structural elements of 5S rRNA are needed for its import into human mitochondria. RNA. 2008;14(4):749–59. 62. Chrzanowska-Lightowlers ZM, Horvath R, Lightowlers RN. 175th ENMC International Workshop: mitochondrial protein synthesis in health and disease. Neuromuscul Disord. 2011;21(2):142–7. Epub Dec 15, 2010. 63. Nolden M, Ehses S, Koppen M, et al. The m-AAA protease defective in hereditary spastic paraplegia controls ribosome assembly in mitochondria. Cell. 2005;123(2):277–89. 64. Di Bella D, Lazzaro F, Brusco A, et al. Mutations in the mitochondrial protease gene AFG3L2 cause dominant hereditary ataxia SCA28. Nat Genet. 2010;42(4):313–21. 65. Das G, Varshney U. Peptidyl-tRNA hydrolase and its critical role in protein biosynthesis. Microbiology. 2006;152(Pt 8):2191–5. 66. Keiler KC, Waller PRH, Sauer RT. Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA. Science. 1996;271:990–3. 67. Muto A, Ushida C, Himeno H. A bacterial RNA that functions as both a tRNA and an mRNA. Trends Biochem Sci. 1998;23(1):25–9. 68. Temperley R, Richter R, Dennerlein S, et al. Hungry codons promote frameshifting in human mitochondrial ribosomes. Science. 2010;327(5963):301. 69. Soleimanpour-Lichaei HR, Kuhl I, Gaisne M, et al. mtRF1a is a human mitochondrial translation release factor decoding the major termination codons UAA and UAG. Mol Cell. 2007;27(5):745–57. 70. Richter R, Rorbach J, Pajak A, et al. A functional peptidyl-tRNA hydrolase, ICT1, has been recruited into the human mitochondrial ribosome. EMBO J. 2010;29(6):1116–25. 71. Antonicka H, Ostergaard E, Sasarman F, et al. Mutations in C12orf65 in patients with encephalomyopathy and a mitochondrial translation defect. Am J Hum Genet. 2010;87(1):115–22. 72. Handa Y, Inaho N, Nameki N. YaeJ is a novel ribosome-associated protein in Escherichia coli that can hydrolyze peptidyl-tRNA on stalled ribosomes. Nucleic Acids Res. 2011;39(5): 1739–48. Epub Nov 3, 2010.
Part II Mitochondrial Dysfunction: Causes and Effects?
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Neurodegeneration in Primary Mitochondrial Disorders Nichola Lax and Evelyn Jaros
Abstract
Mitochondria are critically responsible for the generation of energy in the form of ATP through the electron transport chain (ETC). The central nervous system (CNS) performs highly energy-intensive tasks and is therefore particularly dependent on ATP. Defects residing within the complexes comprising the ETC can affect the synthesis of ATP and consequently severely compromise neuronal function. It is unsurprising then that primary mitochondrial DNA mutations are an important cause of neurological disease. The clinical presentation is often heterogeneous in terms of age of onset and different neurological signs and symptoms which might include ataxia, seizures, cognitive decline, blindness, and stroke. The clinical course can vary considerably but in many patients there is progressive neurological decline and marked neurodegeneration. Our understanding of the mechanisms underpinning neurodegenerative changes due to mitochondrial DNA mutations is limited due to the availability of appropriate animal models of disease. However, studies on human postmortem CNS tissues have provided an invaluable insight into the distribution and severity of neuronal degeneration in patients harboring mitochondrial DNA defects. In this chapter, we describe the neuropathological changes occurring in the CNS associated with N. Lax (*) Mitochondrial Research Group, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, Tyne and Wear, UK Institute for Ageing and Health/UK NTHR Biomedical Research Centre for Ageing and Age-Related diseases, Newcastle University, Newcastle upon Tyne, Tyne and Wear, UK e-mail:
[email protected] E. Jaros Neuropathology/Cellular Pathology, Newcastle upon Tyne Hospitals, NHS Foundation Trust, Royal Victoria Infirmary, Newcastle upon Tyne, Tyne and Wear, UK Institute for Ageing and Health/UK NTHR Biomedical Research Centre for Ageing and Age-Related diseases, Newcastle University, Newcastle upon Tyne, Tyne and Wear, UK e-mail:
[email protected] A.K. Reeve et al. (eds.), Mitochondrial Dysfunction in Neurodegenerative Disorders, DOI 10.1007/978-0-85729-701-3_2, © Springer-Verlag London Limited 2012
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different mutations of the mitochondrial genome and discuss the mechanisms which might contribute to neural dysfunction and cell death. Keywords
Mitochondria • Mitochondrial DNA • Neurodegeneration
2.1
Introduction
Mitochondrial disease was first described in 1962, when Luft and colleagues described a patient with non-thyroidal hypermetabolism [1]. Since this discovery, major advances in our understanding of mitochondrial biology and genetics have permitted the recognition of a number of mitochondrial disorders. Mitochondrial diseases are a heterogeneous group of disorders that arise from defects in either the mitochondrial or nuclear genome. As has been discussed elsewhere in this book (Chap. 1), mitochondria contain their own circular double-stranded DNA encoding for 13 polypeptides of the electron transport chain (ETC), 22 transfer RNAs (tRNA), and 2 ribosomal RNAs (rRNA) [2]. This genome is under dual genetic control, with many mitochondrial maintenance genes encoded by the nuclear genome. Mitochondrial diseases present an important social and economic burden with the estimated prevalence of 1 in 10,000 of people clinically manifesting the disease and a further 1 in 6,000 who are at risk of developing mitochondrial disease [3]. In mitochondrial disease, the central nervous system (CNS) can be particularly vulnerable to neuronal cell loss. Firstly, the brain is highly metabolically active and therefore particularly susceptible to bioenergetic failure. Secondly, it has considerably fewer antioxidant defenses than other tissues, so is less able to protect against excessive reactive oxygen species (ROS) production. Thirdly, with the exception of subventricular zone, olfactory epithelium, and the hippocampus, most neurons within the brain are postmitotic, and therefore irreplaceable. Consequently, any particular neuronal insult will prove fatal if not alleviated in some way. In addition, there is increasing evidence linking mitochondrial dysfunction to neuronal cell loss in age-related neurodegenerative disorders, including Alzheimer’s disease and Parkinson’s disease.
2.2
Neurological Features and Genetics of Mitochondrial Diseases
Patients with mitochondrial diseases present with a wide variety of neurological manifestations, in particular optic atrophy, ataxia, seizures, dementia, stroke-like episodes, extrapyramidal features, and neuropathy. Whilst it is recognized that some of the symptoms are associated with particular patterns of CNS pathology and with certain mitochondrial DNA (mtDNA) mutations, little is understood about the mechanisms that underlie these associations.
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2.2.1
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Kearns–Sayre Syndrome
Kearns–Sayre syndrome (KSS) presents with a characteristic triad of symptoms including development of retinitis pigmentosa, progressive external ophthalmoplegia, and an onset before 20 years of age [4]. Additional neurological features include cerebellar ataxia, raised cerebrospinal fluid (CSF) protein levels, subclinical neuropathy, cognitive impairment (defined by defects in visuospatial attention and executive function), and deafness. As this is a multisystem disorder, other clinical manifestations such as proximal myopathy, complete heart block, cardiomyopathy, endocrinopathies, short stature, and dysphagia are particularly prominent [5]. This is predominantly a sporadic condition caused by a single large-scale deletion or complex rearrangements of mtDNA [6]. There is no specific deletion responsible for the disease as all deletions will remove multiple genes encoding ETC proteins and tRNAs. However, the majority of mtDNA deletions are located within the major arc between the two proposed origins of replication (OH and OL). Often mtDNA deletions may be flanked by short direct repeats. Approximately, one-third of these patients harbor a 4,977 bp deletion, known as the common deletion which has a 13 bp repeat sequence [7].
2.2.2
Chronic Progressive External Ophthalmoplegia
Chronic progressive external ophthalmoplegia (CPEO) is defined by a slowly progressive ophthalmoplegia and ptosis. The age of onset is variable, with some patients first noticing ptosis in the third or fourth decade of life. As the disease progresses, associated proximal muscle weakness and fatigue commonly occur but are rarely debilitating. The genetic defect in CPEO can vary and can be caused by mtDNA rearrangements [8], single nucleotide substitutions in specific tRNA genes [9–11], or mutations in nuclear-encoded mitochondrial maintenance genes which cause multiple mtDNA deletions [12].
2.2.3
Mitochondrial Encephalopathy Lactic Acidosis and Stroke-Like Episodes
Mitochondrial encephalopathy lactic acidosis and stroke-like episodes (MELAS) presents with a triad of lactic acidosis, seizures, and stroke-like episodes which remain central to diagnosis and pathology. Although the m.3243A>G mutation was the first and most frequently described mutation in association with MELAS, a number of other mutations including m.3253A>G [13], m.3271T>C [14], and m.3291T>C [15] have subsequently been identified. Furthermore, m.1642G>A in mt-tRNAVal [16], mutations in complex IV of the ETC, cytochrome c oxidase (COX) subunit III (such as m.9957T>C) [17], and mutations within the mtDNA-encoded complex I (MTND) subunit genes have been described [18–22]. It is also important to note that the m.3243A>G mutation can lead to other distinct clinical phenotypes, such as maternally inherited diabetes and deafness [23] and CPEO [9].
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Myoclonic Epilepsy Ragged Red Fibers
Myoclonic epilepsy ragged red fibers (MERRF) is a severe neurodegenerative disorder which presents during childhood or early adulthood. Clinically, myoclonus is often the first presenting feature which becomes progressive. Other neurological symptoms comprise focal and generalized epilepsy, cerebellar ataxia, dementia, pyramidal signs, neuropathy, sensorineural hearing loss, and optic atrophy. Nonneurological symptoms include muscle weakness and wasting, hypertrophic cardiomyopathy, multiple lipomas, and Wolff–Parkinson–White syndrome [24]. Initially, MERRF was described in association with m.8344A>G point mutation in the MTTK gene encoding for tRNALys. Whilst other mutations in the same tRNA have been reported, the m.8344A>G change remains the most common [25].
2.2.5
Leigh Syndrome
Leigh syndrome is a progressive neurodegenerative disorder which has an onset in infancy and childhood. This disease is characterized by symmetric necrotic lesions distributed along the brainstem, diencephalon, and basal ganglia which have been documented in both neuroimaging and postmortem studies. Common neurological features include ataxia, dystonia, and developmental delay. Other clinical features include respiratory abnormalities, nystagmus, and hypotonia [26]. A severe failure of oxidative metabolism within the brain of infants with Leigh syndrome has been described in association with a variety of nuclear and mtDNA mutations.
2.2.6
Leber’s Hereditary Optic Neuropathy
Leber’s hereditary optic neuropathy (LHON) was first described by Theodore Leber in 1871 [27]. The salient clinical feature of LHON is the almost exclusive involvement of the optic nerve. Patients typically present with a subacute or acute, painless, loss of central vision usually between the ages of 20 and 40 years with a strong predilection for males. The first mtDNA point mutation identified was m.11778G>A [28], however additional point mutations m.3460G>A and m.14484T>C have subsequently been identified. It has been shown that over 95% of LHON pedigrees harbor one of these mtDNA point mutations, all involving genes encoding complex I subunits of the ETC [29].
2.2.7
Mitochondrial Diseases Associated with Primary Mutations in POLG Gene
Mitochondrial DNA polymerase g is the only polymerase responsible for the synthesis and repair of mtDNA in mammalian cells. The human mtDNA polymerase is a 195 kDa heterotrimer consisting of a 140 kDa catalytic subunit (POLG1) and two identical 55 kDa accessory units (POLG2). The C-terminus of the catalytic
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subunit of POLG1 is responsible for the polymerase function whilst the N-terminus is responsible for exonuclease activity and proofreading of mtDNA. The linker mediates a focal contact with the dimeric accessory subunit. POLG1 is encoded by the nuclear POLG gene. Primary mutations in the POLG gene represent one of the mechanisms that cause secondary mutations in the mtDNA, including depletion of mtDNA, multiple mtDNA deletions, or multiple point mutations in mtDNA [30]. Recently, numerous mutations have been described in POLG in association with a spectrum of clinical/neurodegenerative phenotypes, including autosomal dominant and autosomal recessive progressive external ophthalmoplegia (PEO) [31–33], autosomal recessive sensory ataxic neuropathy with dysarthria and ophthalmoplegia (SANDO) [34], mitochondrial inherited recessive ataxia syndrome without ophthalmoplegia (MIRAS) [35], Parkinsonism [36], and Alpers syndrome [37]. In a large cohort of patients with POLG mutations, childhood presentation has been associated with the Alpers-Huttenlocher syndrome, characterized by fatal hepatoencephalopathy with refractory epilepsy, stroke-like episodes, cortical blindness, and the development of acute cerebral lesions with a propensity for the striate cortex. Presentation in teenage years or adulthood has been associated with ptosis, PEO, and unexplained multisystem neurological disorder or isolated neurological disorder with family history [38]. The reasons for the large variability in the clinical/ neurodegenerative phenotype in relation to the POLG genotype, and in turn to the secondary mtDNA mutations, are poorly understood. Involvement of some, as yet unidentified, epigenetic factors has been suggested by a report on two siblings with identical compound heterozygous POLG mutations, one of whom presented with Alpers syndrome and the other with SANDO [39].
2.3
Neurodegeneration in Mitochondrial Diseases
2.3.1
Brain Atrophy
Postmortem brain atrophy is a common observation in patients with mitochondrial diseases, though quantitative data are sparse. In a Newcastle series of 15 brains dissected at postmortem from patients with genetically diverse mitochondrial disease, evidence of atrophy was present in 12 (Professor Robert Perry, personal communication). The average fresh weight was reduced by about 19% (range: 6–37%; Table 2.1) compared to brains from normal individuals matched for age and gender [40]. Of the 12 patients with atrophic brains, six were aged between 20 and 60 years, received MELAS diagnosis and harbored m.3243A>G, three patients were aged between 18 and 34 years and harbored mutations in POLG, one patient was aged 42 years with MERRF diagnosis and m.8344A>G, one patient was aged 40 years with KSS diagnosis and a single, large-scale mtDNA deletion, and the final patient was aged 55 years and harbored homoplasmic m.14709T>C. Three of the 15 brains had the fresh weight within normal limits. This included a brain from a patient, aged 34 years, with neuropathological features of Leigh/MELAS and m.13094T>C, and two brains from patients aged 59 and 71 years, who had mutations in POLG.
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Table 2.1 Postmortem brain weights Mitochondrial cases Wt Age Sex Genetic defect Wt (g) (% normal) 18 F POLG 1,098 81.9 20 F m.3243A>G 829 63.3 24 F POLG 1,081 83.2 30 M m.3243A>G 1,174 81.5 34 F m.13094T>C 1,260 97.7 34 F POLG 973 75.4 36 F m.3243A>G 930 72.1 40 F Single mtDNA 1,040 80.6 deletion 42 F m.3243A>G 1,070 82.9 42 F m.8344A>G 1,075 83.3 55 M m.14709T>C 1,325 94 57 F m.3243A>G 1,150 92 59 M POLG 1,480 108 60 F m.3243A>G 984 78.7 71 M POLG 1,340 99.3
2.3.2
Wt (% reduction) 18.1 36.7 16.8 18.5 0 24.6 27.9 19.4
Normal cases (Dekaban [40]) Age Wt (g) mean ± range Sex SD 16–18 F 1,340 ± 40 19–21 F 1,310 ± 50 22–30 F 1,300 ± 10 22–30 M 1,440 ± 20 31–40 F 1,290 ± 30 31–40 F 1,290 ± 30 31–40 F 1,290 ± 30 31–40 F 1,290 ± 30
17.1 16.7 6 8 0 21.3 0
41–50 41–50 51–55 56–60 56–60 56–60 71–75
F F M F M F M
1,290 ± 20 1,290 ± 20 1,410 +/− 10 1,250 ± 20 1,370 ± 20 1,250 ± 20 1,350 ± 20
Neuroradiological Imaging
Neuroradiological imaging in patients with mitochondrial diseases often demonstrates focal lesions in cerebral cortex, white matter, basal ganglia and brainstem and, particularly in patients with longer disease duration, generalized cerebral and cerebellar atrophy, all suggestive of CNS neuron loss. Enlargement of the fourth ventricles is often profound in younger patients and could be a predictor of cerebellar atrophy [41, 42]. Most of the imaging findings can be diverse and nonspecific with little correlation with genotype or biochemical phenotype but in some of the mitochondrial diseases the lesions show predilection for particular anatomical areas of the brain. For instance, in MELAS patients there is often prominent dilation of occipital horns with focal stroke-like cortical lesions (SLCL) in a nonvascular distribution generally in the posterior of the brain consistent with stroke-like episodes (SLEs). In early stages of the disease, the SLCL show features of vasogenic edema, rather than ischemic-like lesions [43] and often exhibit rapid resolution associated with clinical improvement. However, presence of cerebral atrophy in long-standing disease suggests association with gradual attrition of neuronal cells [41, 42]. In MERRF patients harboring the m.8344A>G mutation, neuroradiological imaging most often shows atrophy in cerebral cortex, cerebellum, superior cerebellar peduncle and brainstem [44, 45]. More recently, MRI showed evidence of thalamic demyelination in a young male harboring m.8344A>G but without the typical clinical presentation [46]. In patients with Leigh syndrome necrotic-like lesions distributed along the brainstem,
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thalamus, and basal ganglia are considered characteristic [41]. Patients harboring POLG mutations demonstrate a combination of thalamic and cortical lesions without basal ganglia involvement but with lesions in deep cerebellar nuclei and inferior olivary nuclei which can be indicative of mitochondrial spinocerebellar ataxia and epilepsy. In contrast, the main finding in patients with POLG mutations and Alpers disease with pediatric onset are stroke-like lesions in occipital cortex [47], although thalamic changes have also been described, particularly in patients presenting as young adults [48, 49]. In some mitochondrial diseases imaging shows predominantly white matter lesions. In KSS patients symmetrical lesions in the white matter of the cerebrum, cerebellum, globus pallidus, dorsal midbrain, and thalamus are consistently reported [41]. In mitochondrial neurogastrointestinal encephalopathy (MNGIE) patients there are diffuse white matter lesions in cerebral hemispheres, brainstem, and cerebellar peduncle in addition to demyelinating peripheral neuropathy [50]. Intracerebral calcification has been commonly reported in MELAS, KSS, and Leigh syndrome. A neuroimaging study from 1998 revealed that basal ganglia (BG) calcification, involving the caudate, putamen, thalamus, and global pallidus, was the most common finding in 54% of MELAS patients harboring the m.3243A>G mutation [42]. A similar distribution pattern is seen in ageing suggesting that BG calcification in mitochondrial disease, and particularly in MELAS, may occur due to accelerated ageing [51]. This particular feature is also seen in a range of insults to the brain including anoxia [52], exposure to radiation [53], and parathyroid [54], and hypothyroid disorders [55] suggesting that this brain region responds to damage by laying down minerals. Although BG calcification in mitochondrial diseases is often severe, it has not been reported in association with BG atrophy. It is also unclear if there are any functional symptoms related to this pathology in the patients with mitochondrial diseases, and whilst there is evidence of BG calcification in other diseases, there does appear to be a lack of correlation between the degree of pathology and emergence of clinical symptoms [56, 57]. It is intriguing that in the very old and also in younger patients with psychiatric illnesses psychotic symptoms are strongly associated with basal ganglia calcification [51].
2.4
Neuropathology of Mitochondrial Diseases
When postmortem brains from patients with mitochondrial diseases are examined microscopically there is profound neuron loss, often with microcystic change and astrogliosis and/or axon and glial loss from the anatomical areas of focal lesions seen by neuroradiological imaging. There is also more subtle microscopic pathology, not detectable by neuroradiology. These include variable reductions in neuronal density and in levels of mitochondrial ETC proteins detectable within neurons and cerebral vasculature using histochemistry and immunohistochemistry. Such microscopic changes are often reported in the cerebellar cortex, cerebellar dentate nucleus, medullary inferior olives, and other brain areas of many mitochondrial diseases but quantitative data are sparse (Fig. 2.1).
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a
b
d
e
g
h
c
f
i
j
Fig. 2.1 Cerebellar pathology in patients harboring primary mtDNA mutations. (a) Infarct-like lesion involving the molecular layer, Purkinje cells, granular cell layer, and the white matter of the cerebellar cortex in a patient harboring the m.3243A>G mutation (Cresyl fast violet stain, scale bar = 100 mm). (b) Gross atrophy of the cerebellar cortex in a patient harboring a m.3243A>G mutation (Cresyl fast violet stain, scale bar = 100 mm). (c) Foci of mild spongiform degeneration and moderate myelin loss in the cerebellar deep white matter from a patient with KSS harboring a single large-scale mtDNA deletion (Loyez myelin stain). (d) Formation of an axonal torpedo (arrow) in the granular cell layer in a patient with KSS harboring a single large-scale mtDNA deletion (Antiphosphorylated neurofilament, scale bar = 100 mm). (e) Multiple axonal torpedos (arrows) evident in the granular cell layer of a patient harboring m.14709T>C mutation (Bielschowsky’s silver stain, scale bar = 100 mm). (f) Trapped mitochondria within swollen Purkinje cell dendrites in the molecular layer in a patient with KSS due to a single, large-scale deletion of mtDNA (Anti-porin, scale bar = 100 mm). (g) Porin immunohistochemistry in the dentate nucleus of a patient harboring the m.3243A>G mutation reveals abundant neuronal mitochondria (scale bar = 100 mm). (h) Complex I 30 kDa deficient neurons (arrowhead) are evident in the dentate nucleus of a patient harboring the m.3243A>G mutation (scale bar = 100 mm). (i) Intact complex II 70 kDa proteins levels in the dentate nucleus of a patient harboring the m.3243A>G mutation (scale bar = 100 mm). (j) Marked reduction in complex IV subunit I protein expression in the neuronal population (arrowhead) of the dentate nucleus of a patient harboring m.3243A>G (scale bar = 100 mm)
2.4.1
MELAS
MELAS is neuropathologically characterized by the presence of multiple “infarctlike” lesions in the occipital, parietal, and temporal lobes of the brain and less in the cerebellar cortex (Fig. 2.1a). The lesions may be asymmetric, and when severe may be observed externally on the surface of the brain. Although microscopically they resemble true infarcts, commonly involving considerable neuronal cell loss, neuronal eosinophilia, astrogliosis, and microvacuolation, unlike infarcts, they
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have cortical laminar distribution and are not be restricted to particular vascular territories of specific cerebral arteries or watershed zones [58]. These “infarct-like” lesions represent the chronic stage of the disease. They are seen as SLCL by neuroradiological imaging and may be responsible for the SLE in MELAS. The pathogenic mechanism underlying the preferentially posterior distribution of the neuron loss in MELAS is not fully understood. There appears to be no correlation between distribution of the lesions, COX (cytochrome c oxidase)-deficient neurons and intraneuronal levels of m.3243A>G. Based on findings of profound deficiency of COX in the walls of leptomeningeal and cortical vessels, it has been proposed that coupling of vascular mitochondrial dysfunction with cortical spreading depression originating in the occipital lobe are involved [59]. Microscopic examination of areas of basal ganglia calcification commonly seen on neuroimaging of MELAS patients show, often prominent, mineralization of the vessel walls within the basal ganglia. However, within the basal ganglia, there are no intraneuronal calcium deposits, the neuronal populations are usually spared, and there is no evidence of infarct-like lesions [42].
2.4.2
MERRF
Microscopically, MERRF is characterized by severe neuronal cell loss and astrogliosis within the cerebellar dentate nucleus, cerebellar cortex, including Purkinje cells, in the inferior olivary nuclei, red nucleus and substantia nigra [60]. Within these regions, abnormally enlarged mitochondria containing inclusion bodies have been observed [61]. Additionally, neuron loss has been detected within the gracile and cuneate nuclei, Clarke’s column within the spinal cord, the sensory neurons within the dorsal root ganglia, and less in the basal ganglia and thalamus. Myelin loss, likely secondary to neuronal/axonal loss, is an additional degenerative feature documented in the superior cerebellar peduncle and posterior spinal columns [62]. The selective vulnerability to degeneration of specific neuronal populations in MERRF remains under investigation. An immunohistochemical investigation showed selective reduced expression of COX II within remaining neurons of the cerebellar dentate nucleus, inferior olivary nuclei, and frontal cortex [63]. Also, electron chain abnormalities of complex I, III, and IV have been reported in MERRF [64]. However, a microdissection study conducted on a patient harboring the m.8344A>G mutation demonstrated an apparent lack of correlation between extent of pathology and mutation load within individual CNS neurons [65]. This suggests that in MERRF there are important factors other than proportion of mutant mtDNA that determine the degree of mitochondrial dysfunction and the fate of neurons.
2.4.3
KSS
White matter abnormalities are often described as being the neuropathological hallmark of KSS, however gray matter involvement, in particular of the cerebellar cortex and brainstem nuclei, has also been documented (Fig. 2.1c). The defining characteristic
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is the presence of widespread spongiform degeneration of white matter tracts (spongiform leukoencephalopathy) in the cerebrum, brain stem, spinal cord, cerebellum, thalamus, and basal ganglia [62]. The degree of spongiform degeneration can vary from slight myelin pallor at early stages through mild spongy changes to coarse vacuolation, resulting in a “sieve-like appearance.” The selective involvement of white matter pathology observed in KSS is proposed to be a consequence of a specific vulnerability of the myelin-producing glia, oligodendrocytes [66]. Although white matter abnormalities are the predominant feature in KSS, gray matter pathology has also been documented, particularly within the cerebellum and brainstem. Evidence of Purkinje cell loss, formation of axonal torpedos (Fig. 2.1d), and abnormal dendrites (Fig. 2.1e) have all been described in the cerebellar cortex of patients with KSS. The deep cerebellar nuclei, including the dentate nucleus, show intact neuronal cell density despite evidence of reduced ETC protein expression, a loss of synaptic contact from Purkinje cells and spongy changes [67].
2.4.4
Leigh Syndrome
The symmetrical lesions identifiable by MRI in the brainstem, basal ganglia, and thalamus of patients with Leigh syndrome are microscopically characterized by “vasculonecrotic” microcystic degeneration with neuron loss, astrogliosis, myelin loss, and vascular proliferation [26]. In addition, more subtle microscopic lesions are widespread, commonly involving the dorsal pons, inferior olives in the medulla, roof nuclei in the cerebellum, and posterior columns and anterior horns in cervical spinal cord [58, 68]. In the gray matter nuclei, the main features are neuronal ischemic-type change and neuronal loss, whilst in the white matter there is both myelin and axonal loss. Subacute lesions show neuropil edema and swollen cell processes. The cerebral cortex and cerebral white matter are generally only mildly affected. Given that Leigh syndrome has been associated with mutations in a multitude of nuclear and mtDNA genes encoding mitochondrial enzymes, including pyruvate dehydrogenase, and respiratory complexes I, II, IV, and V the molecular mechanisms involved in the pathogenesis of these lesions may vary, depending on the underlying mutation.
2.4.5
LHON
In general, neuropathology in LHON is limited to the optic system, in particular to neurodegeneration of the retinal ganglion cell (RGC) layer, with sparing of the retinal pigment epithelium and photoreceptors. Within the RGC layer, there is pronounced cell body and axonal degeneration, with associated myelin loss and atrophy observed from the optic nerves to the lateral geniculate bodies [69]. There is evidence of impaired glutamate transport, increased mitochondrial reactive oxygen species (ROS), and apoptosis within the RGCs [70]. Rare LHON patients have been described with additional distinct spinal cord pathologies: an inflammatory MS-like disorder – “classic” Harding disease [71] or a subacute metabolic axonopathy of posterior columns with associated features of Leigh-like syndrome; the
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a
b
c
d
e
f
Fig. 2.2 Evidence of prominent deficiencies of ETC proteins within the sensory neurons of the dorsal root ganglia in a patient harboring two primary heterozygous mutations, p.A467T and p.X1240C, in POLG1 and secondary multiple deletions of the mtDNA. (a) Unusually reduced mitochondrial density in one neuron (arrow) and uneven cellular distribution within sensory neurons (anti-porin immunohistochemistry). (b) Profound neuronal deficiency of the 15 kDa subunit of complex I with focal cytoplasmic clumping of immunoreactivity. (c) Reduced expression of the 70 kDa subunit of complex II within the neurons of the dorsal root ganglia, similar to the distribution observed with porin. (d) Dual COX/SDH histochemistry reveals a high level of complex IV (COX) deficiency in some neurons (blue), whilst in other neurons the COX activity is relatively unaffected (brown). (e) Markedly reduced expression and focal cytoplasmic clumping of subunit I of complex IV in all neurons. (f) Reduced expression and focal cytoplasmic clumping of subunit IV of complex IV in some neurons (arrow). Scale bar represents 100 mm
pathogenesis of the subacute selective axonopathy is unclear as the patient was homoplasmic for m.3460G>A in all tissues [72].
2.4.6
Mitochondrial Diseases Associated with Multiple mtDNA Deletions
The neuropathology in a patient harboring two recessive heterozygous mutations, p.A467T and a novel p.X1240T in POLG with secondary multiple mtDNA deletions, was initially described in 2000 [73]. In this patient, the inferior medullary olives were the most severely affected region, suffering massive neuronal loss. Moderate to extensive neuronal loss was observed throughout the cerebellum and to a lesser extent in the dentate nucleus and the red nuclei, while the pons remained unaffected. This pattern is indicative of cerebello-olivary atrophy and probably underlies the cerebellar ataxia observed in patients with multiple deletions. The spinal cord tissues demonstrated evidence of severe myelin and axonal loss within the posterior columns and in dorsal spinal roots. Severe neuronal loss and ETC abnormalities were also present in dorsal root ganglia and in paraspinal sympathetic ganglia (Fig. 2.2). Ventral and lateral spinal tracts, motor roots, and motor neurons were intact. There was also severe depletion of neurons from the substantia nigra without
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Lewy body formation. Throughout the brain of this patient there was lack of correlation between the distribution of COX-deficient neurons and the degree of neuropathological damage, similar to the findings in patients with MELAS and MERRF. More recently, a study on a patient harboring multiple deletions due to two heterozygous POLG mutations revealed loss of pigmented neurons in the substantia nigra (SN) with alpha-synuclein-reactive Lewy body formation. In addition, unlike in patients with Lewy body Parkinson’s disease (PD), but similar to patients with some mitochondrial diseases, there was loss of Purkinje cells, loss of neurons from the cerebellar dentate nucleus, profound loss of myelin and axons from posterior columns of spinal cord, and loss of neurons from gracile nucleus. In the patient with the POLG mutations, the remaining SN neurons had high levels of mtDNA deletions (~65%) associated with COX-deficiency affecting 21% of the neurons. This was in contrast to the percentage of COX-deficiency observed in SN neurons in PD and ageing at <3% and <1%, respectively [74]. Multiple mtDNA deletions and/or mtDNA depletion also underlie severe degeneration of the cerebellum, brainstem, and spinal cord that occur in mitochondrial inherited recessive ataxia syndrome (MIRAS) and in infantile-onset spinocerebellar ataxia with sensory neuropathy (IOSCA) [75]. In both of these diseases, neuronal complex I protein expression is either absent or dramatically reduced in the cerebellum and frontal cortex. In MIRAS, the brain shows multiple deletions of mtDNA and mtDNA depletion due to a primary mutation in POLG, whilst in IOSCA, caused by a primary mutation in C10orf2 gene that encodes the mitochondrial DNA helicase Twinkle, the brain shows only mtDNA depletion but no multiple deletions of mtDNA [35].
2.5
Mechanisms of Neurodegeneration in Mitochondrial Diseases
Understanding molecular processes that lead to neuronal loss in the various mitochondrial diseases remains elusive. In particular, it is unclear why particular neuronal types/systems show greater susceptibility to neurodegeneration in association with certain mtDNA mutations. There appears to be little correlation between the degree of neuropathological damage, intraneuronal level of deficit in mtDNAencoded enzymes, and intraneuronal levels of mutated mtDNA (heteroplasmy) in patients with many mitochondrial diseases, including multiple mtDNA deletions, MELAS, and MERRF. This suggests involvement of additional factors. There is evidence for mitochondrial ETC protein deficiency and dysfunction in cerebral vasculature and possibly regional dysfunction of astroglia in MELAS patients. In addition, existence of differences in the patterns of neuronal activity/physiological properties, and neurotransmitter metabolism between neurons in different brain anatomical areas is well documented. It is likely that particular patterns of neurodegeneration can be accounted for by interactions between energy demand and variable types of deficiencies in the mitochondrial ETC proteins, back-up mechanism, and calcium handling at the levels of individual neurons, supporting astroglia and cerebral vasculature. The complexities of these interactions are yet to be unraveled.
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33
Mitochondrial Homeostasis
There is evidence for a mitochondrial cause of neurodegeneration in patients harboring mtDNA mutations or defects in nuclear-encoded components involved in mitochondrial homeostasis. Although mitochondria are essential for ATP generation through the ETC, mitochondria also play additional key roles in cellular homeostasis, including production of iron-sulfur clusters, calcium handling, regulation of apoptosis (Chap. 2), and oxidative stress (Chap. 6). To some extent, all of these processes have been implicated in neurodegenerative disease.
2.5.2
Mitochondria and Calcium Handling
Mitochondria are well-recognized intracellular calcium buffers with increased calcium uptake during oxidative phosphorylation [76] mediated by activation of calciumsensitive dehydrogenases from the Krebs cycle and pyruvate dehydrogenase [77]. Calcium homeostasis is particularly important for neuronal function where calcium is intricately linked to neurotransmitter vesicle release, synaptic plasticity, and organelle movement (see Chap. 7). Calcium is critical for the regulation of mitochondrial distribution throughout the brain, particularly for mitochondrial movement along axons (see Chap. 10). Neuronal mitochondria transition between mobile and stationary states in response to intracellular calcium [78]. It is thought that this mechanism facilitates mitochondrial retention in presynaptic terminals and postsynaptic dendritic spines, where calcium influx is dynamic and homeostasis maintenance is heavily required. Whilst there have been a limited number of studies investigating the effect of mtDNA defects on calcium homeostasis, these have often focused upon non-neuronal cell lines where physiological differences are manifold. Fibroblasts obtained from patients harboring the m.3243A>G mutation associated with MELAS tend to reveal higher basal calcium levels and a sustained elevation of calcium in response to stimulation with potassium [79]. Furthermore, fibroblasts from patients harboring complex I deficiency due to nuclear subunit deficiencies show evidence of a prolonged increase in calcium following stimulation with bradykinin [80–82]. Clearly, there does seem to be some abnormalities of calcium homeostasis due to mtDNA mutations. Indeed, a recent study on neurons derived from mouse embryonic stem cells has revealed a progressive calcium handling deficit in those cells harboring mtDNA mutations [83].
2.5.3
Mitochondria and Cell Death
There are at least three cell death pathways which might become induced in energetically compromised neurons. These processes include: apoptosis, autophagy, and necrosis and each are morphologically distinct and are induced differentially. The process of regulated cell death takes place via the process of apoptosis, and mitochondria are intricately involved in this pathway through the intrinsic mitochondrial apoptotic pathway.
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Although the mechanism of neuronal cell death in patients with mitochondrial disease has not been investigated, there are few studies which examine the apoptosis in muscle in association with ETC defects. However, the results from these reports are inconsistent and contradictory, with some papers suggesting the presence of molecular executioners of apoptosis [84–87] whilst others do not detect any evidence of these proteins [88]. Therefore, the role of apoptosis in the demise of muscle fibers due to mtDNA defects is highly contentious. It remains to be determined how cell death might become initiated in neurons containing dysfunctional mitochondria.
2.5.4
Oxidative Stress
Mitochondrial metabolism is also responsible for the majority of the ROS production in cell. The formation of ROS occurs when unpaired electrons escape the ETC and react with molecular oxygen, generating superoxide. Complexes I and III of the ETC are the main sites implicated in superoxide generation. Although ROS can be very damaging to the cell as they can react with DNA, proteins, and lipids leading to oxidative stress, they also play an important role in intracellular signaling [89]. Excessive ROS generation is associated with both neurodegenerative diseases and ageing (see Chap. 6). The maintenance of physiological ROS levels is critical to normal cellular functioning. Exploration of ROS generation as a consequence of mtDNA defects stems mainly from animal models or cell culture. An increase in ROS production has been reported in neuronal NT2 cybrid cells that harbor a mutation causing LHON [90]. A study conducted on patient cybrid cells harboring the m.8883T>G mutation associated with neurogenic muscle weakness, ataxia, and retinitis pigmentosa revealed that antioxidant treatment conferred cellular protection. However, contrasting results have been observed in transgenic mice harboring a POLG mutation with no evidence of increased ROS [91, 92].
2.5.5
Cell Culture and Animal Models of Mitochondrial Disease
The recent development of transgenic animal models and cell culture systems has proved to be invaluable in exploring mechanisms of neurodegeneration in mitochondrial disease (see Chap. 12). Recent work conducted in differentiated neurons from mouse embryonic stem cells harboring mtDNA defects has allowed a more mechanistic approach to understanding neurodegeneration in mitochondrial disease. Four cell lines have been generated to date and these consist of: wild-type parental cells, polymorphic cells, mild complex IV mutant cells, and severe complex I mutant cells. Those neurons harboring severe complex I mutations revealed the most dramatic alterations, with a reduced ability to undergo neuronal differentiation and delayed development [93]. Despite this, differentiated neurons showed maintenance of a high mitochondrial membrane potential with a reversal of the ATP synthase and marked ROS production [94].
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Subsequent analysis following repetitive neuronal stimulation demonstrated that the ability of the neurons to respond to calcium transients was impaired [83]. Recently, the development of an animal model, the “mutator” mouse, harboring a proofreading deficiency of polymerase g has shown the accumulation of high level of mtDNA point mutations and deletions [92, 95]. The phenotype of the “mutator” mouse show evidence of accelerated ageing, curvature of the spine, weight loss, alopecia, and reduced lifespan. Whilst no neuropathological studies have been conducted in this mouse, neurological changes would be interesting to investigate.
2.5.6
Other Neurodegenerative Disorders
Deciphering the mechanisms of neurodegeneration in patients with mtDNA diseases poses a difficult challenge to neuroscientists because of the diversity in clinical presentations, different types of mtDNA mutations, and variable pattern of brain involvement. However, increased understanding of the processes underlying CNS degeneration in patients with mtDNA diseases will be incredibly informative for other neurodegenerative disorders where a mitochondrial etiology is suspected. There is some evidence that mutations within the mitochondrial genome or mitochondrial dysfunction play a role in a number of neurodegenerative disorders including PD, Huntington’s disease (HD), Alzheimer’s disease (AD), and Multiple sclerosis [96–99] (see Chap. 5). Whilst the role of these reported mitochondrial abnormalities, whether causative for the onset of neurodegeneration or arising secondary to other neurodegenerative mechanisms, it is clear that mitochondrial dysfunction would act to exacerbate disease pathogenesis. However, much like the mitochondrial encephalomyopathies, our understanding of the pathophysiological mechanisms involved in these diseases remains incomplete. Acknowledgments This work was in part supported by the UK NIHR Biomedical Research Centre for Ageing and Age-related disease award to the Newcastle upon Tyne Hospitals NHS foundation Trust, UK.
Glossary Ataxia Progressive loss of coordination and balance in hands, arms, and legs often due to dysfunction of the cerebellum. Bradykinesia An abnormal slowness of movement, often associated with the clinical symptoms seen in Parkinson’s disease. Cardiomyopathy The deterioration of heart muscle resulting in compromised heart function. Chronic progressive external ophthalmoplegia Slowly progressive paralysis of the extraocular muscle which results in bilateral, symmetrical, progressive ptosis (drooping of the eyelids) followed by ophthalmoparesis (paralysis) months to years later.
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Dysarthria A speech disorder characterized by poor articulation. Often present in patients suffering from cerebellar ataxia. Dysphagia Swallowing problems often present in conjunction with dysarthria in patients suffering from cerebellar ataxia. Dystonia Dystonia is characterized by involuntary and uncontrollable muscle spasms which can force affected parts of the body into abnormal, sometimes painful, movements or postures. Endocrinopathy A disorder in the function of the endocrine gland which can often result in hormone imbalances. Epilepsy Recurrent seizures (often described as fits). Extrapyramidal features Maybe defined as an inability to initiate movements or an inability to remain motionless. Hypotonia A state of low muscle tone resulting in reduced muscle strength. Lipoma A benign tumor consisting of brown fat cells that forms under the skin. Myoclonus Brief involuntary twitching of a muscle or a group of muscles. Nystagmus A form of uncontrolled eye movements. Peripheral neuropathy Damage to the nerves comprising the peripheral nervous system, which can result in a combination of weakness, autonomic dysfunction, and sensory abnormalities. Proximal myopathy Weakness of those muscles located closely (proximal) to the body. Ptosis Droopy eyelids. Retinitis pigmentosa Retinitis pigmentosa is a disorder of the retina which is characterized by dysfunction of the photoreceptors resulting in incurable blindness or tunnel vision. Stroke-like episodes Neurological deficits often described as resembling stroke however do not conform to a vascular territory. They are often accompanied by cortical blindness, hemianopsia, or hemiparesis. Wolff–Parkinson–White syndrome This syndrome is a heart condition caused by extra electrical activity and can lead to increased heart rate.
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74. Betts-Henderson J, Jaros E, Krishnan KJ, et al. Alpha-synuclein pathology and Parkinsonism associated with POLG1 mutations and multiple mitochondrial DNA deletions. Neuropathol Appl Neurobiol. 2009;35(1):120–4. 75. Lonnqvist T, Paetau A, Nikali K, et al. Infantile onset spinocerebellar ataxia with sensory neuropathy (IOSCA): neuropathological features. J Neurol Sci. 1998;161(1):57–65. 76. Jouaville LS, Pinton P, Bastianutto C, et al. Regulation of mitochondrial ATP synthesis by calcium: evidence for a long-term metabolic priming. Proc Natl Acad Sci USA. 1999;96(24): 13807–12. 77. Hansford RG. Dehydrogenase activation by Ca2+ in cells and tissues. J Bioenerg Biomembr. 1991;23(6):823–54. 78. Yi M, Weaver D, Hajnoczky G. Control of mitochondrial motility and distribution by the calcium signal: a homeostatic circuit. J Cell Biol. 2004;167(4):661–72. 79. Moudy AM, Handran SD, Goldberg MP, et al. Abnormal calcium homeostasis and mitochondrial polarization in a human encephalomyopathy. Proc Natl Acad Sci USA. 1995;92(3): 729–33. 80. Visch HJ, Koopman WJ, Leusink A, et al. Decreased agonist-stimulated mitochondrial ATP production caused by a pathological reduction in endoplasmic reticulum calcium content in human complex I deficiency. Biochim Biophys Acta. 2006;1762(1):115–23. 81. Visch HJ, Rutter GA, Koopman WJ, et al. Inhibition of mitochondrial Na+−Ca2+ exchange restores agonist-induced ATP production and Ca2+ handling in human complex I deficiency. J Biol Chem. 2004;279(39):40328–36. 82. Willems PH, Valsecchi F, Distelmaier F, et al. Mitochondrial Ca2+ homeostasis in human NADH:ubiquinone oxidoreductase deficiency. Cell Calcium. 2008;44(1):123–33. 83. Trevelyan AJ, Kirby DM, Smulders-Srinivasan TK, et al. Mitochondrial DNA mutations affect calcium handling in differentiated neurons. Brain. 2010;133(Pt 3):787–96. 84. Aure K, Fayet G, Leroy JP, et al. Apoptosis in mitochondrial myopathies is linked to mitochondrial proliferation. Brain. 2006;129(Pt 5):1249–59. 85. Formichi P, Battisti C, Bianchi S, et al. Evidence of apoptosis via TUNEL staining in muscle biopsy from patients with mitochondrial encephaloneuromyopathies. J Submicrosc Cytol Pathol. 2003;35(1):29–34. 86. Ikezoe K, Nakagawa M, Yan C, et al. Apoptosis is suspended in muscle of mitochondrial encephalomyopathies. Acta Neuropathol. 2002;103(6):531–40. 87. Mirabella M, Di Giovanni S, Silvestri G, et al. Apoptosis in mitochondrial encephalomyopathies with mitochondrial DNA mutations: a potential pathogenic mechanism. Brain. 2000;123(Pt 1):93–104. 88. Sciacco M, Fagiolari G, Lamperti C, et al. Lack of apoptosis in mitochondrial encephalomyopathies. Neurology. 2001;56(8):1070–4. 89. Camello-Almaraz C, Gomez-Pinilla PJ, Pozo MJ, et al. Mitochondrial reactive oxygen species and Ca2+ signaling. Am J Physiol Cell Physiol. 2006;291(5):C1082–8. 90. Wong A, Cavelier L, Collins-Schramm HE, et al. Differentiation-specific effects of LHON mutations introduced into neuronal NT2 cells. Hum Mol Genet. 2002;11(4):431–8. 91. Kujoth GC, Hiona A, Pugh TD, et al. Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging. Science. 2005;309(5733):481–4. 92. Trifunovic A, Hansson A, Wredenberg A, et al. Somatic mtDNA mutations cause aging phenotypes without affecting reactive oxygen species production. Proc Natl Acad Sci USA. 2005;102(50):17993–8. 93. Kirby D, Smulders-Srinivasan T, Rennie K, et al. Transmitochondrial embryonic stem cells containing pathogenic mtDNA mutations are compromised in neuronal differentiation. Cell Prolif. 2009;42(4):413–24. Epub Jun 23, 2009. 94. Abramov AY, Smulders-Srinivasan TK, Kirby DM, et al. Mechanism of neurodegeneration of neurons with mitochondrial DNA mutations. Brain. 2010;133(Pt 3):797–807. 95. Trifunovic A, Wredenberg A, Falkenberg M, et al. Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature. 2004;429(6990):417–23.
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96. Bender A, Krishnan KJ, Morris CM, et al. High levels of mitochondrial DNA deletions in substantia nigra neurons in aging and Parkinson disease. Nat Genet. 2006;38(5):515–7. 97. Campbell G, Ziabreva I, Reeve AK, et al. Mitochondrial DNA deletions and neurodegeneration in multiple sclerosis. Ann Neurol. 2011;69(3):481–92. 98. Horton TM, Graham BH, Corral-Debrinski M, et al. Marked increase in mitochondrial DNA deletion levels in the cerebral cortex of Huntington’s disease patients. Neurology. 1995;45(10):1879–83. 99. Lin MT, Simon DK, Ahn CH, et al. High aggregate burden of somatic mtDNA point mutations in aging and Alzheimer’s disease brain. Hum Mol Genet. 2002;11(2):133–45.
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Mutations in Nuclear Genes That Affect Mitochondrial Function in Parkinson’s Disease Gavin Charlesworth and Nicholas W. Wood
Abstract
Over the last two decades, mutations in a growing number of nuclear genes have been shown to cause parkinsonism. Although these mutations account for only a small percentage of the number of cases of Parkinson’s disease, their discovery has provided a means of identifying and modeling the molecular pathways thought to be dysfunctional in the disorder. There is now growing evidence that several of these pathways converge on the mitochondrion. PINK1 and Parkin have been implicated in a single pathway functioning to maintain mitochondrial homeostasis and remove irreparably damaged organelles, while DJ-1 emerges as an “on-demand” mitigator of oxidative stress. Alpha-synuclein appears to directly damage mitochondria in its aggregated, protofibrillar form, while only limited evidence exists to suggest that mutant LRRK2 may also adversely affect mitochondrial function. These discoveries raise the possibility that measures aimed at redressing the balance in mitochondrial homeostasis might delay or prevent neuronal cell death in Parkinson’s disease, thus presenting a viable target for the disease-modifying therapies that are currently lacking. Keywords
Mitochondria • PINK1 • Parkin • Alpha synuclein • LRRK2 • Autophagy • Fission • Fusion
G. Charlesworth Department of Molecular Neuroscience, Neurogenetics, UCL Institute of Neurology, London, UK e-mail:
[email protected] N.W. Wood (*) Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK e-mail:
[email protected] A.K. Reeve et al. (eds.), Mitochondrial Dysfunction in Neurodegenerative Disorders, DOI 10.1007/978-0-85729-701-3_3, © Springer-Verlag London Limited 2012
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Table 3.1 Some genes known to cause prominent parkinsonism with clinical phenotypes, neuropathological inclusion types, and likelihood of mitochondria involvement Gene Dominant SNCA LRRK2 Ataxin-2
Locus
Clinical
Pathology
Mitochondrial involvement
PARK1/4 Typical PD with dementia PARK8 Typical PD SCA2 Mostly ataxia; sometimes parkinsonism, especially in Asians SCA3 Mostly ataxia; sometimes parkinsonism, especially in Africans FTDP-17 Mostly dementia; sometimes parkinsonism PARK13 Relevance uncertain – parkinsonism in German study
Lewy bodies Lewy bodies Polyglutamine inclusions Polyglutamine inclusions Tau pathology
Yes Possibly – two reports Not known
Not known
Yes
PARK2
Young onset PD, slow progression
Yes
PARK6
Young onset PD, slow progression
DJ-1 PARK7 ATP13A2 PARK9
Young onset PD, slow progression Complex dystonia-parkinsonism, often with pyramidal features Complex dystonia-parkinsonism, often with pyramidal features Usually spastic paraparesis but sometimes complex parkinsonism with pyramidal features
Usually not Lewy body One report only – Lewy bodies Not known Not known Lewy bodies
Not known
Not known
Not known
Ataxin-3 MAPT Omi/ HtrA2 Recessive Parkin PINK1
PLA2G6
PARK14
Spatacsin
SPG11
Possibly – two reports Possibly – one report
Yes Yes Not known
Adapted from Hardy [140]
3.1
Introduction
Before embarking on a discussion of the evidence linking nuclear genes to mitochondrial dysfunction in Parkinson’s disease, it is necessary to give some consideration what we understand by the term Parkinson’s disease (PD). Unlike some other neurodegenerative disorders, such as Alzheimer’s disease, where the condition is defined neuropathologically, PD has traditionally been defined clinically. Moreover, parkinsonism (which is not synonymous with PD but shares many of the core clinical features) may be a prominent feature of many other conditions, a subsection of which will have a genetic cause (see Table 3.1). This has led to a situation that is confused in two respects. Firstly, some genes that can be associated with prominent parkinsonism are not considered PARK loci (e.g., Ataxin2, Ataxin3, Spatacsin, MAPT), while some PARK loci are linked to genes that cause clinically distinct, complex parkinsonian syndromes that differ considerably from sporadic PD (e.g., ATP13A2, PLA2G6, FBXO7) [1–5]. Secondly, even among the most established of the PARK loci, where the clinical syndrome most resembles that of sporadic PD, mutations in SCNA, LRRK2, and probably PINK1 lead to disease that is characterized
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Mutations in Nuclear Genes That Affect Mitochondrial Function in Parkinson’s Disease 45
neuropathologically by typical Lewy body inclusions, for example, while mutations in Parkin associated with relatively pure nigrostriatal degeneration with little or no evidence of Lewy body pathology in nearly all cases [6, 7]. This latter distinction is important as it may indicate that there are at least two distinct pathological processes capable of producing a “Parkinson’s disease” phenotype. For the purpose of this chapter, I have chosen to concentrate on those genes that have been consistently associated with a clinical phenotype most resembling idiopathic PD, with the possible exception of age of onset, namely, SNCA, LRRK2, PINK1, Parkin, and DJ-1. Mention will be made of other genes where appropriate, but they will not be discussed in detail.
3.2
PINK1
The role of mitochondria in PD had been the subject of rather inconclusive speculation since the first description of parkinsonism as a result of exposure to the complex I inhibitor, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) [8]. However, the identification of mutations in PINK1, a putative mitochondrial protein kinase, as the cause of PARK6-linked, autosomal recessive PD served as the spark that reignited scientific interest in this area. There is now a growing consensus that mitochondrial dysfunction is a major contributor to the pathogenesis of PD [9, 10]. PINK1 encodes a protein of 581 amino acids in length, containing a domain highly homologous to the serine/threonine protein kinases of the calcium/calmodulin family, a theoretical transmembrane domain and a canonical n-terminal mitochondrial leading sequence [10, 11]. It is widely expressed throughout the brain and in many other human tissues [12, 13]. Within the cell, both wild-type and mutant PINK1 have been conclusively shown to localize to the mitochondria, but are also present within the cytosol in both processed and full-length forms where they may fulfil different physiological roles [12, 14]. It is currently believed that PINK1 may function to protect neurons from death secondary to oxidative stress. This view is supported by work on toxin models, in which knockdown of PINK1 by RNA interference (RNAi) increased levels of cell death after exposure to mitochondrial toxins [10, 15, 16]. Conversely, both transient and stable overexpression of wild-type, but not mutant PINK1, has been shown to protect against a variety of toxic insults, most notably the complex I inhibitor MPTP and the proteasome inhibitor MG-132 [10, 15, 17–19]. The importance of the mitochondria to this neuroprotective effect is suggested by the fact that PINK1 deficient cells display reduced mitochondrial membrane potential and increased production of reactive oxygen species (ROS), while reduced activity of complex I has been observed in the striatum of PINK1 knockout mice as well as in the fibroblasts and immortalized lymphoblasts of individuals carrying homozygous PINK1 mutations [16, 20–23]. These phenotypes may be partly explained by recent work implicating PINK1 in the control of mitochondrial calcium homeostasis by regulating ion flux through the Na+/Ca2+ exchanger [20]. PINK1 deficiency was shown to result in accumulation of calcium in the mitochondria, which stimulates production of ROS
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that interfere with the activity of the glucose transporter. The outcome is reduced substrate delivery, impaired respiratory complex functions, and a lowered mitochondrial membrane potential, all of which can be reversed by specifically blocking mitochondrial calcium uptake [20, 24]. Recent attention has been drawn to the role of mitochondrial trafficking as any reduction in mitochondrial mobility would be expected to be particularly detrimental to neurons, which rely heavily on organelle transport to meet the high energy requirements of different regions of the cell over time [25]. PINK1 has been shown to be capable of forming a multiprotein complex with Miro and Milton, which, along with kinesin-1, are necessary for the anterograde microtubule-based transport of these organelles [26]. Moreover, elevated levels of calcium are known to arrest microtubular transport of mitochondria [27]. This may be of some relevance, given PINK1’s role in the regulation of calcium flux in the mitochondria [20, 27], but further experimental evidence will be required before any solid conclusions can be drawn. Generally, one needs mutations on both alleles of PINK1 (i.e., autosomal recessive inheritance) to develop PD. Furthermore, some of the mutant alleles are nonsense with clear loss of function. Other mutations appear to interfere with PINK1 functionality by at least two different mechanisms. The majority of disease-causing mutations in PINK1 are found within the kinase domain, including one that resides within the adenosine triphosphate (ATP) binding pocket. This indicates that loss of PINK1 kinase activity is a key step in disease initiation and suggests a search for its physiological substrates and an understanding of their roles (see next section) would shed further light on the pathways underlying PD [28]. Another group of mutations are clustered around the N-terminal region of PINK1 and recent work has shown that they act by reducing cleavage of full length PINK1 at residue A103 [29]. Cleavage occurs in the mitochondria and involves the Rhomboid protease PARL [11, 29]. Moreover, SH-SY5Y cells expressing these mutations exhibit a mitochondrial phenotype similar to that seen in PINK1 knock-out cells, with a reduced mitochondrial membrane potential, increased production of ROS, and an altered mitochondrial network [29]. These findings suggest that the PINK1 cleavage (predominantly to DN-PINK1) is essential to its neuroprotective functions.
3.3
Putative PINK1 Substrates Link It to Oxidative Stress and the Mitochondria
Attempts to find physiological substrates have been difficult, but the mitochondrial chaperone TRAP1 and the E3 ligase Parkin likely fulfil this role, while the serine protease Omi/HtrA2 (also linked to PD as PARK13) and the heat-shock proteins Hsp90/Cdc37 are possible mitochondrial-binding partners [19, 30–32]. Phosphorylated TRAP1 has been shown to prevent cytochrome c release and H2O2-induced apoptosis, although the mechanism remains unclear. Interestingly, overexpression of wild-type PINK1 is no longer capable of safeguarding cells from stress-induced apoptosis in the absence of TRAP1, suggesting that this molecule is a prerequisite to PINK1’s neuroprotective effects [19].
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Omi/HtrA2 is a member of the conserved high-temperature requirement family of oligomeric serine proteases [33]. In bacteria, these proteins have been implicated in stress tolerance and pathogenicity [34]. In humans, Omi/HtrA2 is expressed as a 49 kDa proenzyme that is targeted to the intermembrane space of the mitochondria, where it undergoes processing by removal of the first 133 amino acids, possibly by autoproteolysis [35, 36]. Although initially identified as a pro-apoptotic factor, the protein probably functions within the mitochondria as a protease and chaperone, helping to degrade or refold damaged proteins. Levels of the Omi/HtrA2 have been shown to increase several-fold in response to protein misfolding triggered by tunicamycin or heat shock [37]. The relevance of the Omi/HtrA2 gene to Parkinson’s disease remains a topic of debate. Initially, a large screen of 518 German PD patients detected one missense mutation (G399S) and one rare polymorphism (A141S) that was over represented in the case cohort [38]. In vitro data demonstrating functional consequences for these changes appeared to support a pathogenic role [38]. However, a subsequent replication study in the North American population of 644 PD patients and 826 normal controls not only failed to confirm the association with A141S, but also found the G399S substitution in more controls than in PD patients [39]. Conversely, a mutational analysis in 266 Belgian patients revealed nine patient-specific heterozygous variants (including the A141S polymorphism) and one novel missense mutation (A404T), only five amino acids downstream of the previously reported G399S variant that was found to decrease protease activity [40]. However, none of these polymorphisms proved positive in a subsequent association analysis [40]. Despite their uncertain relevance, it is interesting to note that the A141T and G399S amino acid substitutions are immediately adjacent to two serine residues that are putative phosphorylation sites for proline-directed serine/threonine kinases [41]. Moreover, PINK1 appears to be important in influencing phosphorylation status, at least at serine 142 [31, 41]. Phosphorylation increases Omi/HtrA2’s proteolytic activity, which may provide enhanced protection against stress-induced accumulation of misfolded proteins. Indeed, phosphorylation of Omi/HtrA2 was virtually abolished in brain tissue originating from individuals with PD-associated PINK1 mutations [41]. The importance of HtrA2 in vivo is demonstrated by mice that are either homozygous for an S276C mutation that greatly reduces it functionality as a protease or have undergone a targeted deletion of the Omi/HtrA2 gene [36, 42]. Both sets of animals develop a parkinsonian neurodegenerative phenotype, with decreased mobility, tremor, reduced coordination, and premature death. Taken as a whole, this work suggests that failure of phosphorylation of Omi/HtrA2 either due to mutations in Omi/HtrA2 itself or in its binding partner PINK1 may represent another pathway to neuronal cell death, most likely mediated by increased vulnerability to mitochondrial stress.
3.3.1
Parkin
Parkin encodes an ubiquitin E3 ligase involved in ubiquitin-dependent proteosomal degradation pathways [43]. Mutations in the Parkin gene cause PARK2-linked
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autosomal recessive PD [44]. Numerous substrates of Parkin have been identified, including synaptic proteins such as a-synuclein and synphilin-1, and one hypothesis holds that mutations disrupting Parkin’s E3 ubiquitin ligase function may lead to toxic accumulation of its physiological targets [45, 46]. Parkin overexpression also appears to be protective against a number of cellular stressors, including a-synuclein toxicity, but the relevance of these cell line experiments to Parkin’s in vivo functions at normal physiological levels has been called in to question [45, 47–50]. Initial evidence to suggest that Parkin may have a role in mitochondrial function, and that this role might involve interaction with PINK1, came from transgenic animal models. Drosophila engineered to carry Parkin loss-of-function mutations display a severe phenotype, which includes swollen mitochondria with fragmented cristae in muscle and germline tissues, dopaminergic cell loss and increased susceptibility to oxidative stress [51, 52]. Similar results were obtained with mutant Drosophila expressing the pathogenic Parkin mutation R275W [53]. Collectively, these observations implied a role for Parkin in mitochondrial maintenance, but since Parkin is not specifically a mitochondrial protein it was suggested that it may be part of a larger pathway involving other proteins. Clues to the identity of at least one these other proteins came with the subsequent creation of Drosophila that either expressed loss-of-function mutations in PINK1 or were PINK1 deficient. The phenotype of these animals was almost identical to that seen in the Parkin mutant Drosophila, with similar aberrations in muscle mitochondrial morphology, dopaminergic degeneration and increased sensitivity to oxidative stress [54–56]. In view of this resemblance, investigators went on to test the hypotheses that overexpression of Parkin would alter the PINK1 mutant phenotype and vice versa. In fact, Parkin overexpression completely rescues the phenotype of PINK1 mutant flies, while PINK1 overexpression has no effect on the phenotype of Parkin mutants [54– 56]. This implicated both PINK1 and Parkin in a single pathway-mediating mitochondrial maintenance and places PINK1 upstream of Parkin. In comparison to Drosophila, PINK1 and Parkin mutant mice have a much milder phenotype: neither exhibit dopaminergic neuron loss, and the presence of any significant behavioral abnormalities is disputed [21, 57–62]. Closer examination, however, did reveal some subtle deficits in dopaminergic nigrostriatal neurotransmission [58, 61]. More interestingly, both knockouts also show mitochondrial abnormalities [21, 59]. In PINK1 knockout mice, higher numbers of larger mitochondria were observed with evidence of respiratory chain dysfunction and an increased sensitivity to H2O2 [21]. Parkin knockout mice also showed evidence of respiratory chain dysfunction, though their mitochondria appeared morphologically normal [59]. Although the very mild and nonprogressive phenotype of these animals has limited their use as a model of PD, these observations do at least suggest an early and central role for mitochondrial dysfunction in the disease caused by mutations in PINK1 and Parkin. Over the last few years, experimental evidence has begun to gather that suggests two possible mechanisms by which these proteins might contribute to mitochondrial quality control, namely [1]: by regulation of targeted mitophagy; and [2] by regulation of mitochondrial fission and fusion.
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Mutations in Nuclear Genes That Affect Mitochondrial Function in Parkinson’s Disease 49
3.3.2
PINK1, Parkin and Mitophagy
Autophagy is the process by which cellular components are broken down, involving their sequestration within a double membrane structure known as the autophagosome, which subsequently fuses with the lysosome [63, 64]. Under nutrient-rich conditions autophagy serves a critical role in removal of damaged organelles and protein aggregates that might otherwise be toxic to the cell [65]. In this situation, the process is carefully targeted, with mitophagy being the term used when mitochondria are selectively ingested [66]. It has been shown that Parkin translocates from the cytosol to the mitochondria in response to stress induction by the mitochondrial uncoupler CCCP (as well as other mitochondrial poisons) in both neurons and HeLa cells [67]. Moreover, when only a subset of mitochondria within the cell were depolarized, Parkin was observed to selectively accumulate on these damaged mitochondria alone [67]. On continued incubation of the same cells for 24 h with mitochondrial uncouplers, Parkin-targeted mitochondria were selectively removed by mitophagy. Working on the hypothesis that PINK1 and Parkin are part of a single pathway, it was subsequently shown that both the translocation of Parkin to damaged mitochondria and the subsequent induction of mitophagy require the activity of PINK1 [68–71]. But how does PINK1 detect damaged mitochondria in the first instance when it seems to be imported into all mitochondria? One hypothesis is that the normally rapid proteolytic turnover of PINK1 in healthy mitochondria may be inhibited by membrane depolarization, effectively allowing PINK1 to accumulate “selectively” in these organelles, to which it subsequently recruits Parkin [68, 69]. The exact means by which PINK1 recruits Parkin from the cytosol remains uncertain, but direct phosphorylation of Parkin at threonine 175 may play a role [32]. Whatever the means, data suggest that PINK1 activates the E3 ligase activity of Parkin, which induces mitophagy by ubiquination of multiple mitochondrial substrates, as well as causing the degradation of key proteins such as mitofusin 1 and 2, which might otherwise allow these terminally damaged mitochondria to re-fuse with their healthy counterparts [72, 73].
3.3.3
PINK1 and Parkin in Mitochondrial Fusion and Fission
Apart from their transport and degradation, optimal functioning of the mitochondrial network is also dependent on a carefully calibrated balance of fission and fusion (see also Chap. 8). Mitochondrial fusion appears to confer resistance to many forms of cellular injury, possibly by allowing enhanced respiration, increased capacity for calcium buffering, and stabilization of mitochondrial DNA [74–76]. Mitochondrial fission, on the other hand, is important for proper cell division and mitochondrial redistribution, including the separation of damaged organelles destined for destruction [77]. An investigation of the effect of PINK1 and Parkin on these processes was thus a natural next step. Unfortunately, the results have been contradictory. In Drosophila models, knockout of dPINK1 promotes fusion and aggregation of mitochondria that can be abrogated by overexpression of Drp1 or knockdown of
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Opa1/Mfn, both of which tip the balance toward fission [78–81]. This suggested that under normal physiological conditions the PINK1/Parkin pathway would act to promote fission and/or inhibit fusion. However, the waters were quickly muddied by data from mammalian and human cell lines that demonstrated increased mitochondrial fragmentation in PINK1 and Parkin RNA interference or knockdown models [24, 79]. Similar results were obtained from primary cells cultured from patients carrying mutations in PINK1 [16, 24]. These observations were taken to indicate an increase in mitochondrial fission, suggesting that under normal physiological conditions in mammalian cells the PINK1/Parkin pathway would promote fusion and/or inhibit fission – the exact opposite of Drosophila. At present, it is not clear how to square these apparently contradictory results. Of course, it remains possible that the effect of PINK1/Parkin deficiency on mitochondrial morphology may merely be a secondary phenomenon related to more direct effects on distinct process such increased production of ROS, decreased membrane potential, and abnormalities in calcium flux. Some support for this hypothesis is provided by the observation that expression of dominant negative DRP-1 in PINK1 knockdown cells could prevent the mitochondrial morphological phenotype but did not attenuate the increase in production of ROS, which must therefore be an upstream event [82].
3.3.4
DJ-1
DJ-1 encodes a 189-amino acid protein (also known as DJ-1) that is highly conserved across species and belongs to DJ-1/Thi/PfpI protein superfamily [83]. It is expressed in a wide variety of tissues including the nervous system, where it is particularly prominent in astrocytes and astrocyte processes [84, 85]. Mutations in DJ1 cause PARK7-linked, autosomal recessive PD. In vivo, it exists as a dimer and the location of some (but not all) mutations suggests that they probably produce their effect by disruption of this structure. The phenotype of patients with DJ-1 mutations has much in common with that of patients carrying PINK1 and Parkin mutations: there is an early onset of disease with slow progression, good response to levodopa and, occasionally, psychiatric disturbance [86]. Furthermore, reports came to light of two young sisters with young-onset Parkinson’s disease who were found to be compound digenic heterozygotes for a single DJ1 and a single PINK1 pathogenic mutant allele [87]. Both of these observations hint at the possibility that DJ-1 mutations may share a pathogenic mechanism with PINK1 and Parkin. The protein appears to have multiple functions, but most notable and most consistent was the link to the oxidative stress [86]. In response to oxidative stress, there is an isoelectric point to shift toward more acidic values while the sulfhydryl group of a conserved cysteine residue can react with ROS to form cysteine sulfinic acid [88– 92]. This has led to the concept of DJ-1 as a redox sensor that is activated “on demand” by an oxidative cytoplasmic microenvironment. Furthermore, DJ-1 appears to be upregulated and redistributed to the mitochondria during cellular stress, suggesting
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Mutations in Nuclear Genes That Affect Mitochondrial Function in Parkinson’s Disease 51
the possibility that this organelle might be the main benefactor of its effects [88, 93, 94]. More recently, DJ-1 was shown to bind to NADH dehydrogenase 1 a-subcomplex 4 and ND1, nuclear and mitochondrial DNA encoded subunits of complex I, thus providing firmer evidence of DJ-1’s involvement in mitochondrial activity [95]. As with PINK1 and Parkin, knockdown of DJ-1 by RNA interference in SH-SY5Y cells increased the susceptibility of these cells to apoptosis in response to various oxidative stressors including MPTP, H2O2, and 6-OHDA [92]. Overexpression of DJ-1, on the other hand, resulted in increased resistance to stress-induced apoptosis and reduced intracellular ROS levels [94]. Morphologically, mitochondria in DJ-1 deficient cells are similar to those seen in PINK1 knockdown, with loss of membrane potential and an increased tendency to fragment. This phenotype appears to be due to increased levels of ROS, with secondary effects including reduced mitochondrial fusion and a compensatory increase in mitophagy [96]. As DJ-1 is known to be capable of binding the RNA of glutathione metabolism genes and glutathione levels can be influenced by modulating expression of DJ-1, it is interesting to note that the exposure of DJ-1-deficient cells to a cell permeable precursor of glutathione was able recover the mitochondrial membrane potential as well as improve mitochondrial morphology and mobility [96–98]. This confirms the centrality of oxidative stress to the mitochondrial defects observed in these cells and suggests one possible mechanism by which DJ-1 might act to mitigate it. Loss of DJ-1 also confers susceptibility to oxidative stress in animal models. Knockdown of the Drosophila homolog DJ-1b resulted in animals with mild locomotive impairment, which worsened significantly with exposure to levels of paraquat that produced no change in wild-type flies [99]. The fact that Drosophila lacking any DJ-1 activity were viable, active, fertile creatures with normal numbers of dopaminergic neurons, but showed increased sensitivity to mitochondrial poisons again suggests an on-demand role for DJ-1 in mitigation of oxidative stress [99, 100]. DJ-1 knockout mice are also viable and fertile, and show no gross anatomical or neuronal abnormalities, including normal numbers of dopaminergic neurons [101, 102]. Baseline motoric behavior appeared normal, although a decreased response to amphetamines, which cause dopamine release, was noted, suggesting a possible mild deficit unmasked by stress [101]. More strikingly, DJ-1 knockout mice displayed increased susceptibility to MPTP-induced striatal fiber and nigral neuronal loss, while overexpression of DJ-1 in wild-type mice blocks MPTPinduced neuronal loss in these same areas [101]. Recent work has focused on trying to elucidate the relation of DJ-1 to the PINK1/ Parkin pathway. Transient overexpression of both PINK1 and Parkin has been shown to be able to independently rescue the mitochondrial phenotype in DJ-1deficient M17 and mouse embryonic fibroblast cells [96]. This might be taken to suggest DJ-1 acts upstream of PINK1 and Parkin. However, DJ-1 was still able to rescue this mitochondrial phenotype in rotenone-treated cells in the absence of PINK1, suggesting that it may work in parallel, rather than upstream to the PINK1/ Parkin pathway [96].
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a-Synuclein/SNCA
SNCA encodes a-synuclein, a small, 140 amino acid protein that is found in abundance throughout the nervous system. It is particularly concentrated in neuronal axon terminals and there is significant evidence that it participates in neurotransmitter release and synaptic plasticity [103, 104]. It appears that it may act as a molecular chaperone by assisting cysteine string protein-a to ensure proper SNARE complex assembly [105]. Its hydrophobic, non-b amyloid domain confers on it a tendency to aggregate and it is known to form the major structural component of Lewy bodies, the neuropathological hallmark of sporadic PD, as well as acting as the precursor protein for amyloid plaques in Alzheimer’s disease [106, 107]. Both point mutations and duplications/triplications of the SCNA gene cause autosomal dominant Parkinson’s disease and have been assigned to the PARK1 and PARK4 loci, respectively [108–112]. An association between a-synuclein and the mitochondria has been reported by several studies. Both in vitro and in vivo, a proportion of a-synuclein appears to localize to the mitochondria [113–115]. A sequence in the N-terminal of the human protein contains a noncanonical mitochondrial-targeting sequence that allows a-synuclein to be imported into the mitochondria in an energy-dependent manner [113]. Once within the mitochondria, a-synuclein has been shown to associate with the inner mitochondrial membrane, where it leads to complex I impairment, increased production of ROS, increased protein tyrosine nitration, and a decreased mitochondrial membrane potential [113, 115]. This effect is exacerbated by the pathogenic A53T point mutation [113]. The physiological relevance of this observation is attested by the observation that mitochondria isolated from the substantia nigra and striatum, but not cerebellum, of PD patients show a significant accumulation of a-synuclein, mostly in association with the inner membrane, when compared to normal individuals [113]. Aggregated, but not nonaggregated, a-synuclein can also interact with the mitochondria in isolated rat brain to induce the release of cytochrome c, which activates cell death pathways [115]. The means by which it accomplishes this release are uncertain, but may involve the formation of protofibrillar pores capable of puncturing the mitochondrial membrane [116–118]. Finally, expression of mutant a-synuclein has also been shown to enhance cell death in response to dopamine oxidation products [119, 120]. Mutant drosophila expressing either mutated human a-synuclein (A53T and A30P) have been employed with some success as a model of PD. These a-synuclein expressing flies replicate several features of human PD, including locomotor dysfunction, Lewy body-like inclusion formation, and age-dependent loss of dopaminergic neurons [121]. One consistent finding to arise from this model is the contribution of oxidative stress to a-synuclein-induced neuropathology. The dopaminergic neurons of these transgenic flies have been shown to be hypersensitive to hypoxia. Reduction of dopamine levels by RNA interference directed against the tyrosine hydroxylase gene reduced neurotoxicity, while niacin, an antioxidant from grape
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extracts and measures to increase glutathione activity all improved both dopaminergic neuron survival as well as motor function and lifespan [122–126]. Interestingly, overexpression of either PINK1 or Parkin in these animals was able to ameliorate their motoric dysfunction and Parkin was shown to directly reduce dopaminergic neurodegeneration [127, 128]. Though a direct interaction remains controversial, these data do suggest the possibility that compensatory interventions to improve mitochondrial homeostasis might be effective in preventing protein toxicity. Attempts to use transgenic mice to model the pathological effects of mutant a-synuclein, however, provide both an interesting and cautionary tale. Mice expressing the human mutant A53T a-synuclein die early, after developing a severe adultonset movement disorder that proceeds from reduced motoric activity with bradykinesia, mild ataxia, and dystonia to a rapidly progressive fatal paralysis [129]. Although there is no degeneration of dopaminergic neurons in the substantia nigra pars compacta, cell death is observed in the neocortex, brainstem, and spinal cord, which is preceded by the formation of intraneuronal inclusions and mitochondrial degeneration. Their paralysis appears to result from a profound loss of neocortical and spinal motor neurons [105, 114]. On closer examination of these neurons, mitochondrial DNA damage was a frequent finding, even in the absence of nuclear DNA damage [114]. Some of the mitochondria demonstrated morphological abnormalities, including reduced size, swelling, or vacuolation, and biochemical testing revealed loss of complex IV activity [114]. Human a-synuclein appeared to be closely associated with some of these degenerating mitochondria [114]. Although this mitochondrial phenotype probably owes a good deal to oxidative stress, as evidenced by the finding that mitochondrial-associated metabolic proteins are oxidized in A30P mice, generation of nitrogen radicals may also play a role. Aggregates of nitrated human a-synuclein were found in affected neurons, raising the possibility that mitochondrial DNA may be directly damaged by genotoxic reactive nitrous species within the mitochondria [114]. The exact reason why mouse motor neurons degenerate en masse while the dopaminergic neurons of the nigra escape relatively unscathed is uncertain. It is known that A53T a-synuclein is axonopathic and the susceptibility of motor neurons may be nothing more than a function of the long length of their projections, which thus rely on oligodendrocytes and Schwann cells for support [130, 131]. If, on the other hand, credence is given to the hypothesis that nitrative stress plays a central role in causing cell death, then the fact that the mitochondria of mouse motor neurons are unusual in that they express inducible NOS may be of some importance [132]. In summary, the A53T transgenic mouse is significant as it suggests that mutant a-synuclein can cause mitochondrial dysfunction and cell death in which oxidative and possibly nitrative stress play a role. Moreover, though it fails to live up to purpose as a model of PD, the finding that expression of human mutant a-synuclein can produce a phenotype highly reminiscent of ALS is not without some intrinsic interest in itself for what it may be telling us about aspects of shared pathobiology for two common neurodegenerative conditions.
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LRRK2
LRRK2 encodes a large protein of 2,527 amino acids in length that is expressed in many tissues throughout the body [133, 134]. It has several different predicted functional domains, including a Roc (Ras in complex proteins) domain, a COR (c-terminal of Roc), a leucine-rich repeat (LRR) domain, a protein kinase catalytic domain, a WD40 domain and an ankyrin domain. Interestingly, studies in rodent brains failed to demonstrate any significant quantity of the protein in the substantia nigra while levels were found to be high in the dopamine-innervated areas of the striatum. At the cellular level, LRRK2 is mostly cytosolic but approximately 10% of the protein appears to be associated with the outer mitochondrial membrane [135, 136]. Mutations in LRRK2 have been identified as responsible for PARK8-linked, autosomal dominant PD [133, 134]. Such mutations appear to be common, accounting for 5–6% of familial PD and about 1–2% of “sporadic” PD [137]. Most (but not all) of the mutations increase kinase activity of the protein in vitro and along with the inheritance pattern, this has been taken to suggest that pathogenicity is related to a gain-of-function. There is only very limited evidence to connect LRRK2 to the mitochondria. One study showed that human SH-SY5Y as well as murine embryonic neuronal precursor cells release cytochrome c and activate caspase 3 when transfected with mutant LRRK2, leading to apoptosis [138]. Activation of caspase 3 can be prevented by removal of Apaf1, but cytochrome c was still released from the mitochondria, which indicates that initiation of the apoptotic pathway is downstream from mitochondrial damage [138]. More recently, on examination of fibroblasts cultured from patients with G2019S LRRK2 mutations, a number of mitochondrial abnormalities were reported [139]. The mitochondrial membrane potential was decreased by around 45%, with a marginal but significant reduction in ATP production despite supply of specific substrates, suggesting that the respiratory chain was compromised [139]. Morphologically, the mitochondria appeared elongated with increased interconnectivity, implying an increase in fusion in the patient cell lines, though these changes were mild [139]. Overall, in both these studies, it is difficult to know whether the observed changes are a direct result of the disruption of a physiological effect of LRRK2 at the mitochondria or whether they represent a secondary phenomenon related to a more fundamental pathogenic effect of the mutant protein elsewhere (Fig. 3.1).
3.4
Conclusion
For the “major” PD genes discussed here it is clear that most evidence exists for a direct link to the mitochondria in the cases of PINK1 and Parkin. The products of these genes appear to function in a single pathway aimed at mitochondrial quality control, in which roles for mitochondrial dynamics, calcium homeostasis, and selective mitophagy are apparent. DJ-1 and (to a lesser extent) Omi/HtrA2 also appear to be critical to the mitochondria in serving as an “on-demand” protective mechanism
3
Mutations in Nuclear Genes That Affect Mitochondrial Function in Parkinson’s Disease 55 Cytosoli recruitment
Damaged mitochondria abnormal morphology
Parkin Ubiquination PINK1 Mitophagy
ROS DJ-1
Autophagosome
HtrA2 PINK1
Misfolded proteins
n
F
n/
io
ss Fi
io us
Alpha-syn protofibrils Cytochroma c Release
Healthy mitochondrial network
Apoptosis
Alpha-syn
Fig. 3.1 Schematic representation of the involvement of PINK1, Parkin, DJ-1, HtrA2 and a-synuclein in mitochrondrial homeostasis
against oxidative stress and its consequences. It is interesting to note that the disease resulting from mutations in these genes is similar from a phenotypic viewpoint and, importantly, that it stands somewhat apart from both sporadic PD and PD caused by SNCA and LRRK2 mutations. Nonetheless, mutant a-synuclein may also damage the mitochondria directly, especially in its highly toxic protofibrillar form, which may well be capable of puncturing their membranes. It is likely, however, that the mitochondria are just one among many intracellular structures with which overload and aggregation of this protein interferes, rather than being a specific target. At present, there is only very limited evidence to connect LRRK2 to the mitochondria and what has been observed may well represent secondary change downstream from a more fundamental pathological mechanism. Where mitochondrial dysfunction has been demonstrated, it is likely – and indeed there is evidence to suggest – that this dysfunction is present in many (if not all) tissues of affected individuals that express the mutant gene. The apparent selective susceptibility of the dopaminergic neurons to degeneration must then be due to factors intrinsic to this cell population, including possibly: their long, poorly myelinated projections with extensive terminal arborizations; their high energy requirements; their relatively low mitochondrial mass; and their use of transmitters that can exacerbate oxidative stress.
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As ever, it is likely that further research in this dynamic field will shed more light on the exact mechanisms underpinning mitochondrial dysfunction in PD and, hopefully, with time, suggest a means of intervention by which the balance can be redressed.
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118. Volles MJ, Lansbury PT. Vesicle permeabilization by protofibrillar alpha-synuclein is sensitive to Parkinson’s disease-linked mutations and occurs by a pore-like mechanism. Biochemistry. 2002;41(14):4595–602. 119. Junn E, Mouradian MM. Human alpha-synuclein over-expression increases intracellular reactive oxygen species levels and susceptibility to dopamine. Neurosci Lett. 2002;320(3):146–50. 120. Tabrizi SJ, Orth M, Wilkinson JM, et al. Expression of mutant alpha-synuclein causes increased susceptibility to dopamine toxicity. Hum Mol Genet. 2000;9(18):2683–9. 121. Feany M, Bender W. A Drosophila model of Parkinson’s disease. Nature. 2000;404(6776): 394–8. 122. Jia H, Li X, Gao H, et al. High doses of nicotinamide prevent oxidative mitochondrial dysfunction in a cellular model and improve motor deficit in a Drosophila model of Parkinson’s disease. J Neurosci Res. 2008;86(9):2083–90. 123. Long J, Gao H, Sun L, et al. Grape extract protects mitochondria from oxidative damage and improves locomotor dysfunction and extends lifespan in a Drosophila Parkinson’s disease model. Rejuvenation Res. 2009;12(5):321–31. 124. Trinh K, Moore K, Wes PD, et al. Induction of the phase II detoxification pathway suppresses neuron loss in Drosophila models of Parkinson’s disease. J Neurosci. 2008;28(2):465–72. 125. Bayersdorfer F, Voigt A, Schneuwly S, et al. Dopamine-dependent neurodegeneration in Drosophila models of familial and sporadic Parkinson’s disease. Neurobiol Dis. 2010;40(1): 113–9. 126. Botella JA, Bayersdorfer F, Schneuwly S. Superoxide dismutase overexpression protects dopaminergic neurons in a Drosophila model of Parkinson’s disease. Neurobiol Dis. 2008; 30(1):65–73. 127. Haywood AFM, Staveley BE. Mutant alpha-synuclein-induced degeneration is reduced by parkin in a fly model of Parkinson’s disease. Genome. 2006;49(5):505–10. 128. Todd AM, Staveley BE. Pink1 suppresses alpha-synuclein-induced phenotypes in a Drosophila model of Parkinson’s disease. Genome. 2008;51(12):1040–6. 129. Lee MK, Stirling W, Xu Y, et al. Human alpha-synuclein-harboring familial Parkinson’s disease-linked Ala-53 -–> Thr mutation causes neurodegenerative disease with alpha-synuclein aggregation in transgenic mice. Proc Natl Acad Sci USA. 2002;99(13):8968–73. 130. Giasson BI, Duda JE, Quinn SM, et al. Neuronal alpha-synucleinopathy with severe movement disorder in mice expressing A53T human alpha-synuclein. Neuron. 2002;34(4):521–33. 131. van der Putten H, Wiederhold KH, Probst A, et al. Neuropathology in mice expressing human alpha-synuclein. J Neurosci. 2000;20(16):6021–9. 132. Chen K, Northington FJ, Martin LJ. Inducible nitric oxide synthase is present in motor neuron mitochondria and Schwann cells and contributes to disease mechanisms in ALS mice. Brain Struct Funct. 2010;214(2–3):219–34. 133. Paisán-Ruíz C, Jain S, Evans EW, et al. Cloning of the gene containing mutations that cause PARK8-linked Parkinson’s disease. Neuron. 2004;44(4):595–600. 134. Zimprich A, Biskup S, Leitner P, et al. Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology. Neuron. 2004;44(4):601–7. 135. Biskup S, Moore DJ, Celsi F, et al. Localization of LRRK2 to membranous and vesicular structures in mammalian brain. Ann Neurol. 2006;60(5):557–69. 136. West AB, Moore DJ, Biskup S, et al. Parkinson’s disease-associated mutations in leucine-rich repeat kinase 2 augment kinase activity. Proc Natl Acad Sci USA. 2005;102(46):16842–7. 137. Gilks WP, Abou-Sleiman PM, Gandhi S, et al. A common LRRK2 mutation in idiopathic Parkinson’s disease. Lancet. 2005;365(9457):415–6. 138. Iaccarino C, Crosio C, Vitale C, et al. Apoptotic mechanisms in mutant LRRK2-mediated cell death. Hum Mol Genet. 2007;16(11):1319–26. 139. Mortiboys H, Johansen KK, Aasly JO, et al. Mitochondrial impairment in patients with Parkinson disease with the G2019S mutation in LRRK2. Neurology. 2010;75(22):2017–20. 140. Hardy J. Genetic analysis of pathways to Parkinson’s disease. Neuron. 2010;68:201–6.
4
Common mtDNA Polymorphisms and Neurodegenerative Disorders Joanna L. Elson and David C. Samuels
Abstract
The prevalence of the late onset neurodegenerative disorders Alzheimer’s and Parkinson’s disease is increasing as the population ages. They impose a considerable health care cost, which will grow as the population continues to age. Both disorders have been associated with oxidative stress and mitochondrial dysfunction. It has been proposed that population variants of the DNA found inside the mitochondria have a role in the etiology of both these neurodegenerative disorders. We inherit our mitochondrial DNA (mtDNA) from our mothers and the variation seen is grouped into distinct lineages called haplogroups. As mtDNA in mammals does not undergo recombination, these haplogroups are stable and distinct blocks of SNP variation. mtDNA is located at the site of energy production and thus exposed to the free radicals associated with this process. This results in mtDNA having high variation within and between populations. The lack of recombination in mtDNA causes the variant correlations to be very complex, which makes the determination of the possible role of population variants in common disease difficult. There have been many studies trying to determine if mtDNA population variants play a role in neurodegenerative disorders. They have generated both positive and negative results, for the same phenotypes. After many years of work, the role of mtDNA variants in the development of either AD or PD still remains to
J.L. Elson (*) Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, Tyne and Wear, UK e-mail:
[email protected] D.C. Samuels Department of Molecular Physiology and Biophysics, Center for Human Genetics Research, Vanderbilt University Medical Center, Nashville, TN, USA e-mail:
[email protected] A.K. Reeve et al. (eds.), Mitochondrial Dysfunction in Neurodegenerative Disorders, DOI 10.1007/978-0-85729-701-3_4, © Springer-Verlag London Limited 2012
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be established conclusively. Here, we explore the reasons for the hypothesis that mtDNA population variants might play a role in AD and/or PD. Finally, we discuss how we might move from having an attractive hypothesis with some supporting evidence to a robust answer as to whether these variants play a role in neurodegenerative disorders. Keywords
mtDNA • Haplogroups • Neurodegenerative disorders • Oxidative stress • Mitochondrial dysfunction
4.1
Introduction
It has been shown for some time that mutations in mitochondrial DNA (mtDNA) are an important cause of disease in humans [1]. It has also been suggested that some of the frequent mtDNA variants might alter susceptibility to a number of more common complex diseases [2, 3]. Complex diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) are caused by a combination of multiple genes, environmental effects, and the interaction between both. Nuclear genetics has traditionally studied complex disease using concepts that refer to the combined effect of all genes impacting on a trait such as the heritability or sibling risk. This has been calculated by studying the recurrence risk or phenotypic correlation of relatives. Over the past decade Genome-Wide Association Studies (GWAS) have become the standard method for the dissection of heritability into effects of individual common nuclear DNA polymorphisms (SNPs) [4]. GWAS have been successful in finding a number of SNPs associated with complex traits [5–7], not found by linkage mapping over the preceding 20-year period. However, there have been questions as to why relatively little of the variation known to be heritable from classical genetics can be accounted for by the SNPs determined by GWAS studies [8, 9]. The success, and increasingly obvious limitations, of GWAS have important lessons for the study of mtDNA variations in neurodegenerative diseases. Studies to determine if mtDNA variations are associated with complex disease have also been characterized by a number of difficulties many of which are common to nuclear genetic studies [10]. This chapter focuses on a number of key elements that require consideration for the successful conduct of an mtDNA association study, to determine if common mtDNA polymorphisms play a role in neurodegenerative disorders. Haplogroup studies are the major method for doing this in the field of mitochondrial genetics. The construction of robust haplogroup studies is essential to exploring and understanding any role of mtDNA polymorphisms in neurodegenerative disorders. This chapter explores the challenges of setting up such studies and what we have learnt so far using two common neurodegenerative disorders, AD and PD. Both are late-
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Most recent common maternal ancestor (mitochondrial Eve)
Time X - Extinct lineages
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Fig. 4.1 Diagram of a phylogeny illustrating mtDNA evolution. mtDNA is characterized by the emergence of distinct lineages called mitochondrial haplotypes or haplogroups. Three major groups are shown here. Each of the major groups contains what are termed sub-haplogroups. A haplogroup or sub-haplogroup is defined by a single block of characteristic SNPs common to multiple sequences. These mutations may occur many times on the phylogeny due to the high mutation rate of mtDNA (about tenfold higher than that of the nuclear DNA). While the individual mutations might be seen more than one time, the combination of SNPs defining a haplogroup is unique
onset diseases affecting millions of people and represent a substantial and growing drain on health-care resources. Mitochondrial dysfunction has been implicated in both of these diseases (see Chap. 5) [11–13].
4.2
mtDNA Inheritance and Evolution
In mammals mtDNA is inherited maternally, so that mitochondrial lineages are uniparental. Due to maternal inheritance, the evolution of mtDNA is characterized by the emergence of distinct lineages called mitochondrial haplotypes or haplogroups (Fig. 4.1). A haplogroup is inherited as a single block of characteristic SNPs upon which new polymorphisms arise (which may or may not have occurred elsewhere on the human phylogeny) giving rise to new sub-haplogroups. Expansion of these new sub-haplogroups in the population is the only mechanism by which new haplogroups might be created as they cannot arise by recombination. Over evolutionary time some of the groups expand in number while others become extinct (see Fig. 4.1). Nine major haplogroups (H, V, T, J, U K, I, X, W) were identified by
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restriction fragment length polymorphism (RFLP) analysis in European populations [14]. There are many additional haplogroups in Asian and African populations. The African haplogroups are the oldest and have therefore accumulated the greatest levels of diversity [15, 16]. This chapter will only consider the variation in European populations as this is the population group in which those association studies on Alzheimer’s and Parkinson’s disease have been conducted. For a full understanding of the role of mitochondrial SNPs in disease, knowledge of the global phylogeny is required [17]. Steps are being taken to determine the phylogeny of other regions in the same detail as has been done for Europe [16, 18, 19], but there is still much to learn. It is worth noting that an understanding of variation globally is likely to be required for a true understanding of interaction between variation and disease. Another reason for the widespread use of human mtDNA for analyses of molecular evolution is that its overall rate of divergence is high relative to nuclear genes, providing sufficient signal for phylogenetic analysis over short time spans. However, this also leads to many changes being seen more than one time on the phylogeny. Such changes (called homoplasies, which may account for greater than 30% of the mtDNA SNPs [15]) can complicate the analysis of mtDNA variation in association with complex disease. The majority of established mtDNA haplotypes are defined by a combination of SNPs, and, by their very definition, a single homoplasy cannot be used to reliably “tag” a single mtDNA haplogroup [20].
4.3
How Might mtDNA Variation Impact Complex Disease?
Mitochondria carry out aerobic respiration producing the majority of the cell’s ATP. They are also a major source of reactive oxygen species, are important in apoptosis, and necessary for a wide range of functions including iron–sulfur cluster biogenesis, which is the only entirely conserved function of the organelle [21] (see Chap. 1). Given the wide range of functions of mitochondria in cell biology, a plausible role for mitochondrial function can be argued for a correspondingly wide range of disease phenotypes. mtDNA polymorphisms may play a role in the etiology of complex disease in two basic ways (see Fig. 4.2). 1. A disease is significantly associated with one mtDNA haplogroup, a result that suggests one or more haplogroup-associated polymorphisms modify risk for the disease. This general hypothesis is akin to the “common-disease common-variant” hypothesis, since haplogroups are defined by common polymorphisms. Homoplasy has the potential to complicate such analysis. If sample size is sufficient and the changes have occurred in different population groups homoplasy also offers a chance for independent testing of the observation [22]. However, just because a specific mtDNA variation exerts an effect on one haplogroup background it does not follow that there will be the same effect on all haplogroup backgrounds (see Fig. 4.2). 2. The next possibility is that the cumulative effects of multiple phenotypically subtle mtDNA mutations are the risk factors for the disease (the “mtDNA mutational load hypothesis”) [23]. This is a version of the “rare-variant commondisease” hypothesis (Fig. 4.2).
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I
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Fig. 4.2 Diagram of the phylogeny illustrating the two different polymorphism association hypotheses. The standard European haplogroups H, V, T, J, U, K, I, X, and W are shown. The proportions have been set to emphasize the structure of the phylogeny, not the population frequencies of the haplogroups. The stars on the phylogeny in Fig. 4.2 illustrate the two potential scenarios for the involvement of mtDNA SNPs in common disease. First, that there are few (perhaps only one) SNPs of major effect (black-and-white checker star) present once on the phylogeny. Then the situation where there are few (perhaps only one) SNPs but the change has occurred more than once on the phylogeny (grey stars). Lastly, where there are many rare SNPs (black stars) any one of which may act as a risk factor in isolation or in combination with other rare SNPs or a common SNP
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There are good population genetic reasons why we might expect to see an appreciable number of slightly deleterious mtDNA polymorphisms in populations. mtDNA SNPs have one quarter the effective population size of nuclear SNPs due to mitochondria being haploid (under the assumption that all copies of the mitochondrial chromosome are identical, homoplasy) and of uniparental inheritance. The power of natural selection to remove slightly deleterious changes that enter the population is greater in large populations than small. Where population sizes are small, random drift becomes a much more powerful determinate of the allele makeup of the population. It has been suggested that a reduction in mtDNA copy number during transmission is important for the maintenance of the mitochondrial genome, and this view has experimental support [24].
4.4
Haplogroups Can Impact the Penetrance of a Mitochondrial Disease
Leber’s hereditary optic neuropathy (LHON) was the first mitochondrial genetic disease to be identified [25, 26]. It causes blindness in young adults, with an estimated prevalence of 3.2/100,000 [27]. LHON classically presents as bilateral loss of central vision due to the focal neurodegeneration of the retinal ganglion cell layer (see Chap. 2). Over 95% of LHON cases are principally due to one of three primary mtDNA point mutations: m.3460G>A, m.11778G>A, and m.14484T>C, all of which involve genes that encode subunits of complex I of the mitochondrial respiratory chain. Less than 50% of male and 10% of female LHON carriers will develop the optic neuropathy [28, 29]. This incomplete penetrance and strong gender bias indicate that additional genetic and/or environmental factors are required for expression of the LHON phenotype. The male bias has led to the suggestion that there may be an X-linked risk factor [30]. Separate meta-analyses [28, 31] have confirmed the reported association between two of the primary LHON mutations, m.11778G>A and m.14484T>C and European haplogroup J. This is particularly strong for m.14484T>C [28]. For the purposes of this chapter, LHON can be viewed as a highly specific phenotype caused in the vast majority of cases [32] by three primary mutations in different subunits of the same protein complex that clearly is affected by the haplogroup background. Viewed in this way, LHON can be classed as a disease associated with haplogroup J, although we know that the disease is actually caused by rare variations which are not part of the definition of haplogroup J. It is quite possible that a similar process occurs for common diseases that have been associated with mitochondrial haplogroups.
4.5
Haplogroups Are Reported as a Risk Factor in the Occurrence of Stroke
Can mtDNA haplogroups affect other phenotypes without identified primary mtDNA mutations? A recent study has provided good evidence that mtDNA haplogroups modulate the risk of transient ischemic attack (TIA) and ischemic stroke
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[33]. This study was conducted using two large independent subpopulations. The use of two independent subpopulations is an important factor as conflicting results in different populations is a consistent feature of the association of mtDNA with various phenotypes [23]. The presence of mtDNA sub-haplogroup K was significantly less frequent in patients with TIA or stroke than in controls in both subpopulations separately as well as when combined. This association remained highly significant after adjustment for multiple haplogroup comparisons. Importantly, the association was also independent of known risk factors. As with many mtDNA association studies there are studies reporting different associations, one study reports mtDNA haplogroup H1 is protective for ischemic stroke in Portuguese patients [34, 35]. Differences in statistical power and diagnostic accuracy are suggested as potential reasons for the discrepancy [33]. There is good evidence to lead us to believe that mtDNA SNPs can play a role in common phenotypes. There are, however, many conflicts in the literature and this is an indication that the associations might not be simple. It might be that some haplogroup backgrounds, while not dysfunctional in themselves, are less robust and consequently rare changes occurring on these particular haplogroup backgrounds act as a “second hit” changing the susceptibility to the disease. Different haplogroups are likely to have different mutations that can act as this second hit. In the specific mitochondrial disease LHON, the rare mtDNA variants believed to be directly responsible for the phenotype were identified first through family studies [25] and the association with haplogroup J was only identified later [2]. For case–control studies of common phenotypes, such a TIA and ischemic stroke, only the haplogroup association is likely to be discovered as there may be a large number of rare changes acting in combination with the haplogroup background to change susceptibility.
4.6
Why Consider mtDNA in Alzheimer’s Disease and Parkinson’s Disease?
Evidence has been accumulating for a number of years that AD and PD are associated with mitochondrial dysfunction and increased production of free radicals (see Chap. 5). Given the role of mitochondria in apoptotic and necrotic cell-death pathways, their involvement in AD and PD seems possible but a role for mitochondria need not mean that mtDNA variations play any role in the disease as mtDNA encodes only 13 from the ~1,000 [36] proteins that constitute the mitochondrion (see Chap. 1). These 13 proteins are core components of 4 of the 5 respiratory chain complexes, with only complex II being entirely nuclear encoded.
4.6.1
Alzheimer’s Disease
Alzheimer’s disease is the most prevalent late-onset neurodegenerative disorder with an estimated 35 million sufferers worldwide [37]. It is a major and growing public health problem in light of the increasing age of Western populations with the
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incidence of AD rising rapidly over the age of 60. While there are a small number of families with autosomal dominant AD [38], more than 90% of the AD cases are classified as sporadic in origin. However, there is evidence from family studies indicating that AD has a substantial genetic component with an estimated heritability of ~60–80% [39]. A lifetime risk of AD in first-degree relatives of patients is twice that of an individual without an affected first-degree relative [40]. Other studies have been able to show that there is a maternally linked element to this risk [41] supportive of a role of mtDNA as this is passed from mother to offspring with the paternal mtDNA being degraded at fertilization. A maternal component to the genetic risk has not, however, been supported by all studies [42]. No doubt the etiology of sporadic AD is complex with a number of risk modifiers including genetic, behavioral, and environmental but are common mtDNA polymorphisms one of these factors? In addition to the characteristic late-stage amyloid plaques and neurofibrillary tangles in AD, there is evidence for abnormal mitochondrial function and oxidative stress in AD patients [11]. Parker et al. reported that platelet fractions from AD patients had lower levels of cytochrome c oxidase activity than those from agedmatched controls [43]. Several groups of investigators have subsequently reported similar results in multiple tissues from AD patients, including autopsied brain samples [44]. Additional evidence has been gathered using a histochemical staining technique called cytochrome c oxidase (COX) staining. It has also been demonstrated that COX-negative neurons occurred more frequently in AD brains than in those from controls. These COX-negative neurons are not associated with amyloid plaques, neurofibrillary tangles, or markers of apoptosis [45]. This indicates that the COX-negative neurons are unlikely to be just secondary effects of disease. When investigating the pathogenesis of novel mtDNA mutations in patients, one of the gold standards is to show that the respiratory chain defect is transmitted with the mtDNA independent of nuclear background [46]. Some investigators have reported that mitochondrial defects in AD patients could be cytoplasmically transferred to normal cell lines, indicating that the defect was due to inherited mtDNA polymorphisms [47] but no specific candidate pathogenic mtDNA mutations in such AD cybrids has been identified. Contradicting this, others have obtained no evidence for cytoplasmic transmission [48]. As such genetic epidemiological analysis [41, 42] and cybrid analysis [47, 49] have not been conclusive as to the role, if any, of mtDNA in AD and PD. Haplogroup association studies and case–control analysis of mtDNA sequence changes in the context of AD have also produced a large number of conflicting results [50]. Many AD haplogroup association studies have found associations including, but not limited to, the following: males in haplogroup U [51], individuals in the haplogroup cluster IWX [52], and individuals in the cluster HV [53] (see the clusters in Fig. 4.2). There have also been studies that have found no association with any mtDNA haplogroup and sporadic AD [54, 55]. In addition, a mutational load study found no significant evidence that AD patients had a higher mutational load of potentially mildly deleterious variations on their mtDNA [23]. In conclusion, the evidence for association of common mtDNA variants with AD is clearly mixed, with both positive and negative results. A possible explanation for these
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inconsistent results is that for pathogenicity the common mtDNA variants may require additional factors (nuclear DNA variants or rare mtDNA variants) which varied between studies.
4.6.2
Parkinson’s Disease
Parkinson’s disease (PD) is the second most common neurodegenerative disease, affecting about ~2% of individuals over the age of 65 [56]. Like AD, PD has a number of environmental and genetic risk modifiers. Autosomal recessive PD can be caused by mutations in the PARKIN gene [57], which encodes a protein which interacts with mitochondria. PINK1, which also encodes a mitochondrial protein, is another gene in which mutations cause early onset PD [58] (see Chap. 3). Most cases of PD are however sporadic. The hallmark feature of PD in either familial or sporadic cases is the loss of dopaminergic neurons from the substantia nigra and intracellular Lewy body inclusions. Defects of complex I and increased oxidative damage are consistent features of PD brains (see Chap. 5). mtDNA codes for seven polypeptides of the 45 subunits that make up complex 1. Population variants in these genes could have a subtle effect on respiratory chain function and contribute to the pathology of sporadic PD via increased oxidative stress or bioenergetic defect. As electrons pass though the electron transport chain a small portion (up to 2%) leak out of the chain, mostly at complexes I and III. These electrons then react with molecular oxygen producing the superoxide anion, which can be converted into other reactive oxygen species (ROS) such as hydrogen peroxide and the highly reactive hydroxyl radical leading to oxidative stress [59] (see Chap. 6). Chemical inhibitors of these complexes can result in increased electron leakage and thus increased oxidative stress. It is possible that non-synonymous variations in the mtDNA-encoded proteins affect either the electron leakage from the complexes, or the effect of inhibitors on the complexes, thus affecting PD susceptibility. The idea that complex I defects are an important mechanism in the development of sporadic PD through oxidative stress comes from experiments in rats where complex I inhibition using rotenone produces the PD-like symptoms tremor, rigidity, and the formation of cytoplasmic inclusions [60]. However, low concentrations of rotenone, enough to induce production of ROS, can still exert neurotoxic effects in cell culture even in the presence of an antioxidant [61]. So oxidative stress independent mechanisms might also play a role in sporadic PD linked to complex I variation. Following from this, it has also been suggested that a bioenergetic defect may itself be the central cause of PD. This is in part because antioxidant treatments have failed to benefit patients in clinical trials [62]. In addition, it is known that not all mutations in complex I lead to an increase in ROS production [63]. Decreased activity in respiratory chain complex I in platelets and substantia nigra from patients with PD [13] has been reported. Cybrid cell lines obtained from PD patients showed a complex I defect independent of the nuclear genetic background [64] but, as with AD, the finding of transfer of phenotype independent of nuclear background is not robust [65] and no candidate mtDNA SNPs have been identified from such studies.
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A study in Finland found that a relative excess of nonsynonymous mutations in complex I genes in the haplogroups clusters JT and WIX was associated with an increased risk of PD and dementia [66]. A later haplogroup association study [67] found that the UKJT haplogroup cluster was associated with a 22% reduction in population-attributable risk for Parkinson’s disease. Why the difference in result? These studies were different in size by an order of magnitude with the later study being the larger one. The former study was also carried out in an isolated population. Such populations may allow us to tease out critical details; however, there is the potential for founder effects that might simply lead to different effects when compared to larger admixed populations. How do we improve mtDNA association studies? The lack of consistent results for mtDNA haplogroup association with AD and PD after 20 years of effort is clearly disappointing. What follows is a consideration of some of the critical design issues in haplogroup studies; unless these are correctly addressed we may have more years of conflicting data.
4.7
The Power of a Study Is a Central Consideration
Almost all studies analyzing haplogroup variation in association with a complex trait have used a contingency table analysis. This creates an issue of how to deal with the low frequency haplogroups present in all populations. In European populations, the haplogroups IWX will typically have individual frequencies of less than 5%. Low frequency categories inflate the chi-square value in a contingency table analysis leading to artificially low p-values and false-positive associations [68]. This problem may be overcome by performing a Fisher’s exact test for each haplogroup in turn against the remainder of the population, with a Bonferroni correction for multiple testing. An alternative method is to group the less frequent haplogroups. Typically, these small haplogroups are collected into one category labeled “other,” but a more reasonable approach is to merge the small haplogroups with their nearest neighbor from the human mtDNA phylogenetic tree. While eliminating the low frequency problem any merging of haplogroups can mask effects associated with one of the less frequent groups. Spurious positive associations are not the limit of problems associated with small studies. Another problem is that these association studies often lack power to reject the null hypothesis.
4.8
Good Case Cohorts
Diagnosis of late onset neurodegenerative disorders such as AD and PD relies heavily on clinical data [69]. In some instances, studies are carried out on mtDNA removed from autopsy samples where diagnosis can be more certain. On the whole, specific diagnostic tests for the various dementias are rare. Autopsy case series have demonstrated a diagnostic error rate approaching ~10% for neurodegenerative
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disorders [70]. Such phenotypic misclassification reduces the power to detect a statistical association between a phenotype and common mtDNA polymorphisms.
4.9
What Are Good Controls for mtDNA Studies?
In order to detect a statistically significant difference between a disease and control cohort it is essential that the control group not only be free from disease but also well matched to the disease group. In the case of testing for association with common mtDNA SNPs “well matched” might not be as clear a concept as it would appear in the first instance. When the mtDNA variation across Europe was first defined it seemed as though the European haplogroups were evenly distributed across the continent [14]. This would mean that within Europe ethnically matching controls would be sufficient. As the number of studies increased, this assumption (perhaps not surprisingly in hindsight) was shown to be not wholly correct when examinations where made at a finer scale [71]. Torroni et al. [72] showed that in Italy there were clines in haplogroup variation between different regions supported by a study in France [73], which again highlighted the importance of regional sampling. This suggests that studies should have not just have ethnically matched controls but more stringent geographically matched controls [22, 55].
4.10
Understanding the Database Limitations
At this point it should be clear that for strong association studies large numbers of good cases and controls are required. It is however the common practice in mtDNA associations studies to mine mtDNA data from GenBank® or other genomic resources. The available sequences may or may not be suitable for use in any particular study. If banked sequences are suitable they must none-the-less be used with care as there are errors in some datasets and a number of duplicate entries are to be found on the GenBank® database. A look at some of the large mtDNA databases reveals a great number of changes that have only been observed in single individuals. In a recent survey [19] of over 5,000 human mtDNA sequences in GenBank® 36% of the reported polymorphic sequence positions were observed in only single individuals. Some of these singleton variations will be true but very rare variations, while many are likely to be sequencing errors. Singleton variations have no bearing on studies that examine haplogroup or subhaplogroup association since haplogroups are generally defined by multiple common markers. They are however the core of any study to test a mutational load hypothesis, which investigates the cumulative effects of multiple phenotypically subtle mtDNA variations. The possible complexities do not end there. As mtDNA is inherited as a single linkage block, it is possible that variants harmless or even advantageous on one haplogroup background might be deleterious on another.
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Conclusion
Properly powered haplogroup studies are essential if consistent associations are to be found. The studies must have the correct geographical matching. As the field moves from RFLP-based haplogroup analysis to whole genome analysis, the problem (and the opportunity) of analyzing subtle interactions and singleton variations must be considered carefully. The effort required will be great, but if we can gain a better understanding of the role of mtDNA variants in disease and in particular neurodegeneration, the rewards are potentially great.
Glossary Bonferroni Correction The Bonferroni correction is a method used to address the problem of multiple statistical comparisons. The correction is based on the idea that if multiple tests are carried out on a set of data, then the chance of wrongly rejecting the null hypothesis increases with the number of tests. The correction involves multiplication of the significance value by the number of tests. Common-Disease Common-Variant Hypothesis The common disease-common variant hypothesis predicts that disease-causing alleles/variants are widespread in all human populations. These variants are predicted to change the likelihood of a given disease. Each variant will have a small additive or multiplicative effect on the likelihood of developing the complex disease phenotype. Contingency Tables In statistics, a contingency table (also referred to as chi square, cross tabulation, or “cross tab”) is often used to analyze the relation between two or more categorical variables. Fisher’s Exact Test Fisher’s exact test is a test used in the analysis of contingency tables where sample sizes are small, normally in a 2 × 2 matrix. Genome-Wide Association Studies (GWAS) A GWAS is an approach involving the rapid scanning of markers across chromosomes or whole genomes, of medium to large cohorts of people to identify genetic markers associated with a particular disease. It is hoped that GWAS studies will be particularly good at detecting genetic variations that contribute to common, complex diseases affecting large numbers of the population, including diabetes, cancers, and heart disease. Human mtDNA Phylogenetic Tree/Network A phylogenetic tree (or network) of human mitochondrial DNA (mtDNA) can be based on coding and/or control region variation. Such diagrams show the relationship between haplogroups. The haplogroups themselves have a standardized nomenclature. This mtDNA trees and networks are a framework for studies in a number of areas including medical, evolutionary, and forensic genetics. Linkage Mapping Genetic linkage describes the tendency of loci/alleles to be inherited together. Linkage is tighter (that is the loci/alleles are seen together more often) when the loci/alleles are close together on the same chromosome. This is because the proximity of loci/alleles means that they are less likely to be separated by a crossover event during meiosis. A linkage map is a genetic map
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showing the position of its known genetic markers relative to each other in terms of recombination frequency. Linkage mapping has been critical for identifying the location of genes that cause genetic diseases by looking for association of markers with disease. Rare-Variant Common-Disease Hypothesis Some have suggested that rather than a small number of common variants being important in modulating susceptibility to complex diseases, the effect of many different rare variants may determine the susceptibility. This hypothesis is more difficult to test as it would involve detecting large numbers of variants that are individually present in only a small proportion of the population. SNPs A single-nucleotide polymorphism (SNP) is a DNA sequence variation occurring when a single nucleotide – A, T, C, or G – in the genome is seen to differ between members of a species (or chromosomes in an individual).
References 1. Taylor RW, Turnbull DM. Mitochondrial DNA mutations in human disease. Nat Rev Genet. 2005;6(5):389–402. 2. Reynier P, Penisson-Besnier I, Moreau C, et al. mtDNA haplogroup J: a contributing factor of optic neuritis. Eur J Hum Genet. 1999;7(3):404–6. 3. Majamaa K, Finnila S, Turkka J, et al. Mitochondrial DNA haplogroup U as a risk factor for occipital stroke in migraine. Lancet. 1998;352(9126):455–6. 4. Hirschhorn JN, Daly MJ. Genome-wide association studies for common diseases and complex traits. Nat Rev Genet. 2005;6(2):95–108. 5. International Schizophrenia Consortium, Purcell SM, Wray NR, et al. Common polygenic variation contributes to risk of schizophrenia and bipolar disorder. Nature. 2009;460(7256):748– 52. Epub Jul 1, 2009. 6. Yang J, Benyamin B, McEvoy BP, et al. Common SNPs explain a large proportion of the heritability for human height. Nat Genet. 2010;42(7):565–9. Epub Jun 20, 2010. 7. Phasukkijwatana N, Kunhapan B, Stankovich J, et al. Genome-wide linkage scan and association study of PARL to the expression of LHON families in Thailand. Hum Genet. 2010; 128(1):39–49. 8. Ertekin-Taner N. Genetics of Alzheimer disease in the pre- and post-GWAS era. Alzheimers. 2010;2(1):3. 9. Gibson G. Hints of hidden heritability in GWAS. Nat Genet. 2010;42(7):558–60. 10. Sale MM, Mychaleckyj JC, Chen WM. Planning and executing a genome wide association study (GWAS). Methods Mol Biol. 2009;590:403–18. 11. Bossy-Wetzel E, Schwarzenbacher R, Lipton SA. Molecular pathways to neurodegeneration. Nat Med. 2004;10(7):S2–9. 12. Parker WD, Filley CM, Parks JK. Cytochrome oxidase deficiency in Alzheimer’s disease. Neurology. 1990;40(8):1302–3. 13. Parker WD, Boyson SJ, Parks JK. Abnormalities of the electron-transport chain in idiopathic Parkinsons-disease. Ann Neurol. 1989;26(6):719–23. 14. Torroni A, Huoponen K, Francalacci P, et al. Classification of European mtDNAs from an analysis three European populations. Genetics. 1996;144(4):1835–50. 15. Herrnstadt C, Elson JL, Fahy E, et al. Reduced-median-network analysis of complete mitochondrial DNA coding-region sequences for the major African, Asian, and European haplogroups. Am J Hum Genet. 2002;70(5):1152–71.
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16. Salas A, Richards M, De la Fe T, et al. The making of the African mtDNA landscape. Am J Hum Genet. 2002;71(5):1082–111. 17. Kong QP, Bandelt HJ, Sun C, et al. Updating the East Asian mtDNA phylogeny: a prerequisite for the identification of pathogenic mutations. Hum Mol Genet. 2006;15(13):2076–86. 18. Palanichamy MG, Sun C, Agrawal S, et al. Phylogeny of mitochondrial DNA macrohaplogroup N in India, based on complete sequencing: implications for the peopling of South Asia. Am J Hum Genet. 2004;75(6):966–78. 19. Pereira L, Freitas F, Fernandes V, et al. The diversity present in 5140 human mitochondrial genomes. Am J Hum Genet. 2009;84(5):628–40. 20. Elson JL, Majamaa K, Howell N, et al. Associating mitochondrial DNA variation with complex traits. Am J Hum Genet. 2007;80(2):378–82. 21. Hjort K, Goldberg A, Tsaousis A, et al. Diversity and reductive evolution of mitochondria among microbial eukaryotes. Philos Trans R Soc Lond B Biol Sci. 2010;365(1541):713–27. 22. Yu X, Koczan D, Sulonen AM, et al. mtDNA nt13708A variant increases the risk of multiple sclerosis. PLoS One. 2008;3(2):e1530. 23. Elson JL, Herrnstadt C, Preston G, et al. Does the mitochondrial genome play a role in the etiology of Alzheimer’s disease? Hum Genet. 2006;119(3):241–54. 24. Stewart JB, Freyer C, Elson JL, et al. A genetic system to study mitochondrial DNA mutations and their propagation in mice. Biochim Biophys Acta Bioenerg. 2008;1777:S80. 25. Nikoskelainen EK, Savontaus ML, Wanne OP, et al. Lebers hereditary optic neuroretinopathy, a maternally inherited disease – a genealogic study in 4 pedigrees. Arch Ophthalmol. 1987;105(5):665–71. 26. Singh G, Lott MT, Wallace DC. A mitochondrial -DNA mutation as a cause of Lebers hereditary optic neuropathy. N Engl J Med. 1989;320(20):1300–5. 27. Man PY, Griffiths PG, Brown DT, Howell N, Turnbull DM, Chinnery PF. The epidemiology of Leber hereditary optic neuropathy in the North East of England. Am J Hum Genet. 2003; 72(2):333–9. Epub Jan 7, 2002. 28. Man PYW, Howell N, Mackey DA, et al. Mitochondrial DNA haplogroup distribution within Leber hereditary optic neuropathy pedigrees. J Med Genet. 2004;41(4):5. 29. Hudson G, Carelli V, Spruijt L, et al. Clinical expression of Leber hereditary optic neuropathy is affected by the mitochondrial DNA-haplogroup background. Am J Hum Genet. 2007; 81(2):228–33. 30. Bu XD, Rotter JI. X chromosome-linked and mitochondrial gene control of Leber hereditary optic neuropathy: evidence from segregation analysis for dependence on X chromosome inactivation. Proc Natl Acad Sci USA. 1991;88(18):8198–202. 31. Panahi MSS, Houshmand M, Tabassi AR. Mitochondrial D-loop variation in Leber hereditary neuropathy patients harboring primary G11778A, G3460A, T14484C mutations: J and W haplogroups as high-risk factors. Arch Med Res. 2006;37(8):1028–33. 32. Zhao FX, Guan MQ, Zhou XT, et al. Leber’s hereditary optic neuropathy is associated with mitochondrial ND6 T14502C mutation. Biochem Biophys Res Commun. 2009;389(3):466–72. 33. Chinnery PF, Elliott HR, Syed A, et al. Mitochondrial DNA haplogroups and risk of transient ischaemic attack and ischaemic stroke: a genetic association study. Lancet Neurol. 2010;9(5):498–503. 34. Rosa A, Fonseca BV, Krug T, Manso H, Gouveia L, Albergaria I, et al. Mitochondrial haplogroup H1 is protective for ischemic stroke in Portuguese patients. BMC Med Genet. 2008;9:57. 35. Benn M, Schwartz M, Nordestgaard BG, Jensen GB, Steffensen R, Tybjaerg-Hansen A. Mitochondrial haplogroups: ischemic cardiovascular disease, other diseases, mortality, and longevity in the general population. Circulation. 2008;117(19):2492–501. Epub May 5, 2008. 36. Elstner M, Andreoli C, Klopstock T, Meitinger T, Prokisch H. The mitochondrial proteome database: MitoP2. Methods Enzymol. 2009;457:3–20. 37. Ferri C, Sousa R, Albanese E, et al. World Alzheimer Report – Executive Summary. Alzheimers Dis Int. 2009;1–22. 38. Nee LE, Polinsky RJ, Eldridge R, et al. A family with histologically confirmed Alzheimersdisease. Arch Neurol. 1983;40(4):203–8.
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39. Gatz M, Reynolds CA, Fratiglioni L, et al. Role of genes and environments for explaining Alzheimer disease. Arch Gen Psychiatry. 2006;63(2):168–74. 40. Lautenschlager NT, Cupples LA, Rao VS, et al. Risk of dementia among relatives of Alzheimer’s disease patients in the MIRAGE study: what is in store for the oldest old? Neurology. 1996;46(3):641–50. 41. Edland SD, Silverman JM, Peskind ER, et al. Increased risk of dementia in mothers of Alzheimer’s disease cases: evidence for maternal inheritance. Neurology. 1996;47(1):254–6. 42. Ehrenkrantz D, Silverman JM, Smith CJ, et al. Genetic epidemiological study of maternal and paternal transmission of Alzheimer’s disease. Am J Med Genet. 1999;88(4):378–82. 43. Parker Jr WD, Filley CM, Parks JK. Cytochrome oxidase deficiency in Alzheimer’s disease. Neurology. 1990;40(8):1302–3. 44. Lin MT, Beal MF. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature. 2006;443(7113):787–95. 45. Cottrell DA, Blakely EL, Johnson MA, et al. Cytochrome c oxidase deficient cells accumulate in the hippocampus and choroid plexus with age. Neurobiol Aging. 2001;22(2):265–72. 46. McFarland R, Elson JL, Taylor RW, et al. Assigning pathogenicity to mitochondrial tRNA mutations: when ‘definitely maybe’ is not good enough. Trends Genet. 2004;20(12):591–6. 47. Khan SM, Cassarino DS, Abramova NN, et al. Alzheimer’s disease cybrids replicate betaamyloid abnormalities through cell death pathways. Ann Neurol. 2000;48(2):148–55. 48. Ito S, Ohta S, Nishimaki K, Kagawa Y, Soma R, Kuno SY, et al. Functional integrity of mitochondrial genomes in human platelets and autopsied brain tissues from elderly patients with Alzheimer’s disease. Proc Natl Acad Sci USA. 1999;96(5):2099–103. 49. Hirai K, Aliev G, Nunomura A, Fujioka H, Russell RL, Atwood CS, et al. Mitochondrial abnormalities in Alzheimer’s disease. J Neurosci. 2001;21(9):3017–23. 50. Howell N, Elson JL, Chinnery PF, et al. mtDNA mutations and common neurodegenerative disorders. Trends Genet. 2005;21(11):583–6. 51. van der Walt JM, Dementieva YA, Martin ER, et al. Analysis of European mitochondrial haplogroups with Alzheimer disease risk. Neurosci Lett. 2004;365(1):28–32. 52. Kruger J, Hinttala R, Majamaa K, et al. Mitochondrial DNA haplogroups in early-onset Alzheimer’s disease and frontotemporal lobar degeneration. Mol Neurodegener. 2010;5:6. 53. Maruszak A, Canter JA, Styczynska M, et al. Mitochondrial haplogroup H and Alzheimer’s disease – is there a connection? Neurobiol Aging. 2009;30(11):1749–55. 54. Chinnery PF, Taylor GA, Howell N, et al. Mitochondrial DNA haplogroups and susceptibility to AD and dementia with Lewy bodies. Neurology. 2000;55(2):302–4. 55. Mancuso M, Nardini M, Micheli D, et al. Lack of association between mtDNA haplogroups and Alzheimer’s disease in Tuscany. Neurol Sci. 2007;28(3):142–7. 56. Mayeux R. Epidemiology of neurodegeneration. Annu Rev Neurosci. 2003;26:81–104. 57. Darios F, Corti O, Lucking CB, et al. Parkin prevents mitochondrial swelling and cytochrome c release in mitochondria-dependent cell death. Hum Mol Genet. 2003;12(5):517–26. 58. Valente EM, Abou-Sleiman PM, Caputo V, et al. Hereditary early-onset Parkinson’s disease caused by mutations in PINK1. Science. 2004;304(5674):1158–60. 59. Balaban RS, Nemoto S, Finkel T. Mitochondria, oxidants, and aging. Cell. 2005;120(4):483–95. 60. Betarbet R, Sherer TB, MacKenzie G, et al. Chronic systemic pesticide exposure reproduces features of Parkinson’s disease. Nat Neurosci. 2000;3(12):1301–6. 61. Yadava N, Nicholls DG. Spare respiratory capacity rather than oxidative stress regulates glutamate excitotoxicity after partial respiratory inhibition of mitochondrial complex I with rotenone. J Neurosci. 2007;27(27):7310–7. 62. Storch A, Jost WH, Vieregge P, et al. Randomized, double-blind, placebo-controlled trial on symptomatic effects of coenzyme Q(10) in Parkinson disease. Arch Neurol. 2007;64(7):938–44. 63. Iuso A, Scacco S, Piccoli C, et al. Dysfunctions of cellular oxidative metabolism in patients with mutations in the NDUFS1 and NDUFS4 genes of complex I. J Biol Chem. 2006; 281(15):10374–80. 64. Swerdlow RH, Parks JK, Miller SW, et al. Origin and functional consequences of the complex I defect in Parkinson’s disease. Ann Neurol. 1996;40(4):663–71.
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65. Aomi Y, Chen CS, Nakada K, et al. Cytoplasmic transfer of platelet mtDNA from elderly patients with Parkinson’s disease to mtDNA-less HeLa cells restores complete mitochondrial respiratory function. Biochem Biophys Res Commun. 2001;280(1):265–73. 66. Autere J, Moilanen JS, Finnila S, et al. Mitochondrial DNA polymorphisms as risk factors for Parkinson’s disease and Parkinson’s disease dementia. Hum Genet. 2004;115(1):29–35. 67. Pyle A, Foltynie T, Tiangyou W, et al. Mitochondrial DNA haplogroup cluster UKJT reduces the risk of PD. Ann Neurol. 2005;57(4):564–7. 68. Roff D, Bentzen P. The statistical analysis of mitochondrial DNA polymorphisms: chi 2 and the problem of small samples. Mol Biol Evol. 1989;6(5):539–45. 69. Caslake R, Moore JN, Gordon JC, et al. Changes in diagnosis with follow-up in an incident cohort of patients with parkinsonism. J Neurol Neurosurg Psychiatry. 2008;79(11):1202–7. 70. Lansbury PT. Back to the future: the ‘old-fashioned’ way to new medications for neurodegeneration. Nat Med. 2004;10(7):S51–7. 71. Niemi AK, Hervonen A, Hurme M, et al. Mitochondrial DNA polymorphisms associated with longevity in a Finnish population. Hum Genet. 2003;112(1):29–33. 72. Torroni A, Bandelt HJ, D’Urbano L, et al. mtDNA analysis reveals a major late paleolithic population expansion from southwestern to northeastern Europe. Am J Hum Genet. 1998;62(5):1137–52. 73. Dubut V, Chollet L, Murail P, Cartault F, Béraud-Colomb E, Serre M, et al. mtDNA polymorphisms in five French groups: importance of regional sampling. Eur J Hum Genet. 2004; 12(4):293–300.
5
mtDNA Mutations in Brain Aging and Neurodegeneration Kim Jennifer Krishnan and Amy Katherine Reeve
Abstract
The cause of the majority of sporadic cases of neurodegenerative disease is unknown. An attractive hypothesis is that accumulation of mitochondrial DNA (mtDNA) mutations may contribute to the cell loss observed in these diseases. Here we describe some of the data surrounding mtDNA mutations in mitochondrial disease where the most severe symptoms are due to neurodegeneration. We also describe mtDNA mutations in aging and common neurodegenerative disorders with a focus on the consequences of mtDNA mutations and the possible contribution to neurodegeneration. Keywords
Mitochondrial DNA • mtDNA mutations • Neurodegeneration • Aging
K.J. Krishnan (*) Centre for Brain Ageing and Vitality, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, Tyne and Wear, UK e-mail:
[email protected] A.K. Reeve Centre for Brain Ageing and Vitality, Mitochondrial Research Group, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, Tyne and Wear, UK e-mail:
[email protected] A.K. Reeve et al. (eds.), Mitochondrial Dysfunction in Neurodegenerative Disorders, DOI 10.1007/978-0-85729-701-3_5, © Springer-Verlag London Limited 2012
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Mitochondrial Genetics
Mitochondria are vital organelles within a cell as they are central to the production of ATP for energy supply as well as having prominent roles in calcium storage, apoptosis, and iron sulfur cluster biogenesis. Mitochondria contain their own DNA (mtDNA) which is a 16.6 kb closed, double-stranded genome (see Chap. 1). This compact molecule codes for 37 genes, which includes 13 essential polypeptides of the oxidative phosphorylation system, 22 tRNAs, and 2 rRNAs which are required for intramitochondrial protein synthesis. Mitochondria are able to replicate independently of the nuclear genome. This allows the production of more mtDNA molecules capable of producing proteins for oxidative phosphorylation under high energy demands. There is a small, ~1 kb noncoding control region on the mitochondrial genome, but apart from this, the rest of the genome is entirely transcribed. Mitochondria are under the genetic control of both the nuclear and mitochondrial genomes. Proteins encoded by the nuclear genome are translated in the cytoplasm and imported into the mitochondria (see Chap. 1). There are multiple copies of the mitochondrial genome present in every cell, and this can vary depending on the particular cell type but is typically between 1,000 and 5,000 copies per somatic cell. mtDNA molecules are contained within nucleoids, which consist of approximately six to ten copies of the genome along with a number of proteins involved in mtDNA replication and maintenance such as Twinkle, polymerase gamma (POLG), and transcription factor A (TFAM) [1]. These nucleoids are thought to be anchored to the mitochondrial inner membrane. Mitochondria are continually fusing and dividing in a process of fission and fusion. This allows the mixing of mitochondrial genomes to allow complementation of the remaining wild-type molecules in the event of any mutations. The types of mutations that occur on mtDNA can range from single point mutations to large-scale rearrangements. As there are multiple copies of mtDNA per cell, mutant mtDNA molecules can exist among wild-type molecules in a state known as “heteroplasmy” [2]. It has been shown that the level of mutant mtDNA needs to exceed a critical threshold before a biochemical defect is seen as expressed by mitochondrial respiratory chain dysfunction within cells (Fig. 5.1). Therefore, the mechanisms influencing the accumulation of mutant mtDNA within cells are critical for maintaining mitochondrial integrity. Clonal expansion is the mechanism by which mtDNA mutations are able to accumulate within a cell. How mutant mtDNA molecules clonally expand to reach high enough levels to cause mitochondrial dysfunction of the affected tissue is still unknown. There are a number of theories of how this could occur. For example, it has been suggested that mtDNA deletions may have a selective advantage due to their smaller size [3, 4]; however, another study showed that high levels of mtDNA mutations can occur in single cells due to random genetic drift [5].
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mtDNA Mutations in Brain Aging and Neurodegeneration
Fig. 5.1 Mitochondrial dysfunction is observed in aging brain by sequential cytochrome c oxidase (COX) and succinate dehydrogenase (SDH) histochemistry. COX deficient cells (blue) occur amongst COX normal cells (brown) in the hippocampus (a) and choroid plexus (b) of an aged individual
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5.2
mtDNA Disease and Neurodegeneration
Mutations on the mitochondrial genome are an important cause of genetic disease. What is interesting is that in these patients, the most prominent and disabling features are due to neurodegeneration (see Chap. 2). The exact prevalence of mtDNA disease is difficult to assess, but in the North East of England it has been reported to be approximately 1 in 10,000 [6]. Point mutations on the mitochondrial genome can cause both inherited and sporadic mtDNA disease [2]. The most common of these are the m.3243A>G mutation associated with MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) and the m.8344A>G mutation associated with MERRF (myoclonic epilepsy with ragged red fibers). Patients with MELAS often present with
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stroke-like episodes and seizures which particularly affect the parieto-occipital region of the brain leading to visual field defects. Patients with MERRF often present with myoclonus which progresses into a mixed picture of myopathy, often with pronounced proximal muscle wasting in a limb-girdle distribution, and central neurological features of focal and generalized epilepsy, cerebellar ataxia, optic atrophy, pyramidal signs, and hearing loss. Single, large-scale deletions are generally observed in sporadic disorders, such as Pearson’s syndrome (PS) and Kearns–Sayre syndrome (KSS) [7, 8]. PS is a rare disorder of infancy characterized by anemia with pancytopenia and exocrine pancreatic failure. KSS is a multisystem disorder and patients often develop severe neurological complications including cerebellar ataxia, cognitive impairment, and deafness as well as non-neurological features. The most commonly reported mtDNA deletion is a 5 kb deletion often referred to as the “common deletion.” As mitochondria are under the dual genetic control of both the mitochondrial and nuclear genome, mutations in nuclear DNA encoding genes involved in mtDNA maintenance and mitochondrial nucleotide metabolism can also cause mtDNA disease. For example, mutations in POLG (the only mitochondrial polymerase), PEO1 (encoding the mitochondrial helicase Twinkle), and SLC25A4 (adenine nucleotide translocator) can cause multiple mtDNA deletions which are associated with the disease chronic progressive external ophthalmoplegia (CPEO) [9–11]. CPEO is characterized by a progressive paralysis of the eye muscles leading to impaired eye movement and ptosis. It is becoming increasingly apparent that strikingly similar mitochondrial abnormalities to those observed in mtDNA disease are also present in aging and patients with common neurodegenerative diseases such as Parkinson’s disease (PD) [12].
5.3
mtDNA Mutations and Aging
Over the last 20 years, mtDNA mutations, identical to those found in mtDNA disease, have been shown to accumulate with age in a number of tissues [13, 14]. In earlier studies, the levels of the mutations were found at low levels (<0.1%), which led to questions over their contribution to the aging process. However, newer technologies allowing investigation into single cells has revealed that high levels of mtDNA mutations can be found to occur focally in a small subset of cells which may compromise these cells and could contribute to the age-related decline in tissues [15] (see Fig. 5.1). Many different types of mtDNA mutations have now been described to occur with age. The first association of mtDNA deletions with age came from studies looking for the common deletion in tissues. This deletion removes a 5 kb region between the genes encoding ATPase8 and ND5 and had previously been described in patients with mitochondrial disease [16]. The common deletion was shown to be present in a number of tissues from aged individuals including skeletal muscle, cardiac muscle, brain, skin, retina, ovary, sperm, and liver [14, 17–23] but was not observed in young individuals. Since these first reports, high levels of mtDNA deletions have
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been reported to occur in single cells from aged tissues, particularly postmitotic cells such as neurons and muscle [12, 24, 25]. mtDNA point mutations have also been suggested to occur with age. Earlier studies focused on the mtDNA disease causing A3243G MELAS [26] and A8344G MERRF [27] mutations, with both showing a presence in only adult tissues. There was initially a great deal of excitement in the field when a T414G transversion was shown to be present at high levels in over 50% of human fibroblasts above 65 years of age which was not detected in young individuals [28]. However, this mutation was subsequently undetected in aged skeletal muscle [29] or brain [30] from normal controls and was suggested to be only fibroblast-specific. A fascinating example of mtDNA point mutations occurring with age is observed in the human colon. By the age of 80 years approximately 15% of all colonic crypts are respiratory chain deficient with clonally expanded mtDNA point mutations shown to account for this respiratory chain deficiency [31]. mtDNA deletions were not observed in the colon which suggests different mechanisms for mutation generation, and clonal expansion occurs particularly between postmitotic and mitotic tissues. More recently, mtDNA point mutations have been detected in other stem cell populations such as the gut [32], stomach [33], and liver [34] in association with respiratory chain deficiency. Does the presence of mtDNA mutations in aged cells have any effect on the agerelated decline of tissues? A boost to this hypothesis was given by the creation of transgenic mice with knock in mutations in Polg [35, 36]. The mutation in Polg resulted in the mice having increased mtDNA mutations in all tissues and interestingly these mice developed features characteristic of premature aging such as weight loss, reduced subcutaneous fat, alopecia, and kyphonia. The lifespan of the mice was ~46 weeks, with all of them dying by 61 weeks. This was the first direct link to show that mtDNA mutations could contribute to aging.
5.4
mtDNA Mutations and Parkinson’s Disease
Parkinson’s disease (PD) is a progressive movement disease that affects the dopamineproducing neurons of the substantia nigra. In the early 1980s, a direct relationship for a mitochondrial involvement in PD was established. This occurred after heroin drug users injected 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) and subsequently developed Parkinsonian features [37]. MPTP is metabolized into MPP+ by monoamine oxidase B. MPP+ is a toxin that inhibits complex I of the mitochondrial respiratory chain. This finding has since been confirmed in models where MPTP has been injected into animals that also developed Parkinsonian symptoms with selective loss of the dopaminergic neurons of the substantia nigra [38]. Lewy bodies are the characteristic hallmark of PD and have been shown to consist largely of alpha-synuclein along with a number of other proteins but also interestingly mitochondria [39, 40]. MPTP treatment in overexpressing alpha-synuclein mice produces abnormal and enlarged mitochondria [41]. It is intriguing that the damaging effects shown by MPTP treatment in mice is absent in alpha-synuclein
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null-containing mice; however, rotenone could still inhibit complex I [42]. The authors suggested that the inhibition of complex I by rotenone may lie upstream of that of MPTP. Studies using double-mutant alpha-synuclein knock-in mice produced abnormal mitochondria in neuronal somata together with reduced complex I activity in the substantia nigra [43]. Recent in vitro studies have shown that alpha synuclein can interact in a much more intimate fashion with mitochondria and that this association coincides with increasing mitochondrial dysfunction [44, 45]. Alpha synuclein has also been shown to affect mitochondrial dynamics and to decrease mitochondrial fusion. The fragmentation of the mitochondrial network by alpha synuclein was shown to be protected against by overexpression of pink1, parkin, and DJ-1 [46]. In human brains, a reduction in complex I of the respiratory chain has been reported in Parkinson’s disease (PD) substantia nigra [47–50]. Is this decline in complex I activity due to mutations of the mitochondrial genome? There are a few reports searching for mtDNA point mutations in the seven mitochondrial-encoded complex I genes in tissue samples from the frontal cortex of PD patients and age-matched controls. Low levels of several mtDNA point mutations were detected in the gene encoding MTND5 in PD compared with controls and the authors hypothesized that the cumulative effect of these low-level mtDNA mutations may cause PD [51, 52]. However, the actual effect of multiple low-level mtDNA mutations remains unknown. A study from our group and an independent group found high levels of mtDNA deletions (~50%) in neurons from aged individuals and patients with Parkinson’s disease [12, 25]. Individual cells were shown to contain different deletions, but within a single cell the mtDNA deletions were the same, indicating clonal expansion of a single mutagenic event. These mtDNA deletions were shown to be the cause of the respiratory chain deficiency observed in these cells. A follow-up study quantified the levels of mtDNA deletions in putamen, frontal cortex, and substantia nigra of patients with Alzheimer’s disease and age-matched controls (age range 63–89) [53]. It has also been shown that patients with mtDNA disease caused by mutations in POLG1 associated with PEO were also shown to co-segregate with Parkinsonian symptoms [54]. Neuropathological examination showed loss of dopaminergic neurons although without Lewy bodies, which are a characteristic hallmark of PD. Since then, there have been other reports of other autosomal dominant PEO patients with POLG1 mutations presenting with Parkinsonism, including the presence of Lewy bodies [55], which highlights a potential link between the accumulation of mtDNA mutations and cell loss in the substantia nigra [56–59]. It is intriguing that mtDNA deletions are detected in both PD patients and in those PEO patients with high levels of multiple mtDNA deletions caused by a mutation in POLG1 and the onset of Parkinsonism. The obvious common link between these two disease groups is the high levels of mtDNA deletions in substantia nigra neurons. It will be interesting to investigate a direct role of mtDNA deletions in PD in future experiments. To try to establish whether mitochondrial dysfunction can cause PD, a number of studies have investigated whether transferring the mitochondria from affected cells into normal ones transfers the defect. These cybrid studies have so far proved inconclusive. One study described that cybrids created using mtDNA from platelets of patients with PD formed inclusions that resembled Lewy bodies [60]; however other studies have failed to show any defects resulting from cytoplasmic transmission of a mitochondrial dysfunction [61, 62]. However, none of these studies identified the
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specific mtDNA mutation associated with the transmission of abnormalities. One major problem associated with these studies is the use of platelets as the source of mitochondria, as the major mitochondrial defects will be located in the brain regions affected, such as the substantia nigra.
5.5
mtDNA Mutations and Alzheimer’s Disease
Alzheimer’s disease (AD) is the most common form of dementia in older people. Mitochondrial dysfunction has also been well described in AD. There have been a number of reports of a reduction in complex IV, the terminal complex of the respiratory chain in affected brain regions in AD cases [63, 64] and an increase in respiratory chain deficient cells has been observed in the hippocampus and choroid plexus in AD cases compared to age-matched controls [65]. As in the PD studies, there have been a number of efforts to transfer the mitochondrial defect from affected cells into normal cells, but again this data has proved inconclusive [62, 66]. It is still uncertain as to the cause of the mitochondrial dysfunction observed in patients with AD. A single study reported an increase in mtDNA mutations in the control region in patients with AD compared to controls [30]. However, a larger study including more patients with AD did not observe any difference [67]. There have been some studies trying to identify whether any particular haplogroups confer susceptibility to developing AD (see Chap. 4). These studies have so far produced mixed results, with a suggestion of an excess of haplogroup J in AD patients from a French-Canadian population [68] whereas no such association has been observed in other studies [69]. These differences are most likely due to limitations in sample size. In AD as in PD there is an accumulation of protein in remaining neurons. The pathological changes in AD are amyloid plaques and neurofibrillary tangles, predominantly composed of tau. Amyloid, like alpha synuclein, has been shown to associate with mitochondria and is thought to relate to mitochondrial dysfunction [70]. There has been a single report describing a patient with mtDNA disease and multiple mtDNA deletions caused by a POLG1 mutation, but interestingly, this patient also developed Alzheimer’s symptoms before the patient died at age 60 [71]. The patient had high numbers of respiratory chain deficient cells and multiple deletions in different brain regions as expected from a patient with a POLG1 mutation. The patient had an APOE4/4 genotype, neurofibrillary tangles, and neuritic plaques in the hippocampus, entorhinal cortex, and amygdala plus evidence of phosphorylated tau in the neocortex. It is uncertain whether the development of AD symptoms in this patient is a coincidence or whether the high levels of somatic mutations may have caused premature aging and that could be reflected by the early onset of AD in this patient.
5.6
mtDNA Deletions and Amyotrophic Lateral Sclerosis
Amyotrophic Lateral Sclerosis (ALS) is caused by a progressive degeneration of the anterior horn cells of the spinal cord and cortical motor neurons. The primary cause of the neuronal cell death observed in patients with ALS remains uncertain. Some familial forms of ALS have point mutations in the superoxide dismutase 1 (SOD1)
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gene [72]. However, the cause of the majority of sporadic cases of ALS remains unknown. It is uncertain how mutated SOD1 leads to the selective cell loss of motor neurons, but there has been some suggestions that mitochondria may be involved as patients with sporadic ALS have clusters of dark abnormal mitochondria in anterior horn neurons [73]. An increase in respiratory chain deficient cells has also been detected in motor neurons [74]. It has been suggested that depletion of mtDNA may be responsible for the mitochondrial dysfunction observed in ALS [75], although more recently mtDNA deletions have also been detected in anterior spinal neurons [76].
5.7
Consequences of mtDNA Mutations
Mitochondrial function is intimately linked to many essential neuronal processes such as protein metabolism, synaptic function, and axonal transport. Therefore, neurons with high levels of mtDNA mutations which cause mitochondrial dysfunction may encounter problems in these functions (Fig. 5.2). Protein aggregation is an important feature in many neurodegenerative diseases and can occur through the inability to adequately clear the abnormal aggregates through the ubiquitin proteasome system (UPS) or autophagy (see Chaps. 6 and 9). Interestingly, abnormalities of both autophagy and the UPS are found in the presence of mitochondrial dysfunction. For example, mitochondrial dysfunction can lead to a loss of ATP, which results in a decrease in the ratio of ATP to AMP. Subsequently, AMP-activated protein kinase (AMPK) acts as a master sensor of intracellular energy status and reduces the activity of the mammalian target of rapamycin complex 1 (mTORC1), which is a major positive modulator of mitochondrial metabolism and biogenesis [77, 78]. Autophagy is also the main method for the removal of defective mitochondria in a process referred to as mitophagy. It has been suggested that Parkin, a gene involved in some forms of PD, may signal the selective removal of defective mitochondria within a cell [79]. In this study, it was shown that long-term overexpression of Parkin can eliminate mitochondria with deleterious COXI mutations in heteroplasmic cybrid cells, enrich wild-type, and restore COX activity. Mitochondria also have crucial roles in both apoptosis and calcium metabolism. Apoptosis is associated with high levels of mtDNA mutations, mitochondrial proliferation, and “ragged red fibres” in patients’ skeletal muscle with mtDNA disease [80]. Therefore, this data provides a direct link between high levels of mtDNA mutations and cell death. Synaptic function and maintenance is also likely to be impaired with mitochondrial dysfunction since the ATP generated by mitochondrial oxidative metabolism is crucial for axonal and dendritic transport (see Chap. 10), release and reuptake of neurotransmitters and for reserve pool mobilization during intense stimulation. A study showed that embryonic stem cell cybrids created with an mtDNA mutation in complex I were unaffected by calcium transient in response to single glutamatergic stimuli, but did alter in response to repeated stimuli with each successive calcium transient decaying ever more slowly in the mutant cells [81]. Thus, it was proposed
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mtDNA Mutations in Brain Aging and Neurodegeneration
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mtDNA mutations
Clonal expansion
Mitochondrial dysfunction
Protein metabolism
Changes in ATP levels
Axonal transport
Calcium handling
Synaptic function
Neuronal dysfunction
Neurodegeneration
Fig. 5.2 Schematic diagram to display some essential cell functions that may be compromised by high levels of mtDNA mutations
that calcium handling deficits may contribute to the pathological phenotype in patients with mitochondrial disease. It is therefore highly possible that synaptic problems are present in those respiratory chain deficient cells observed in aging and neurodegenerative disease. Another study utilizing embryonic stem cell cybrids containing a mtDNA mutation in complex I had significantly increased mitochondrial membrane potential which collapsed in response to oligomycin [82]. However, cells with a less severe complex IV mutation exhibited similar results to controls in cells. These results suggested that the mitochondrial membrane potential was maintained by the FoF1 ATPase operating in “reverse” mode. ROS production was increased in both of the mutant cell
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lines, where as glutathione was depleted in the complex I mutant only. Subsequently, neuronal death was increased in the complex I mutant only, which was abrogated by the addition of ROS scavengers. Therefore, neurons with a severe complex I mutation maintain a high membrane potential by FoF1 ATPase activity combined with an impaired respiratory chain causing oxidative stress which promotes cell death [82].
5.8
Conclusions
There is an increasing body of evidence demonstrating the accumulation of mtDNA mutations in aging and neurodegenerative disease. But do they play any role in the neurodegeneration observed in these situations? We have described how high levels of mtDNA mutations are responsible for mtDNA disease, where prominent neurodegeneration is observed. There is also evidence to show that synaptic function is compromised with pathogenic mtDNA mutations. Mechanisms such as autophagy exist to clear dysfunctional mitochondria; however, when this fails or the system is overloaded, high levels of mtDNA mutations in cells have been shown to lead to cell death. Future work is needed to further investigate the role of mtDNA mutations in neurodegeneration. Also a better understanding into the mechanisms of mtDNA mutation generation and clonal expansion is crucial if we are to try and prevent them accumulating.
References 1. Bogenhagen DF, Rousseau D, Burke S. The layered structure of human mitochondrial DNA nucleoids. J Biol Chem. 2008;283:3665–75. 2. Taylor RW, Turnbull DM. Mitochondrial DNA mutations in human disease. Nat Rev Genet. 2005;6(5):389–402. 3. Diaz F, Bayona-Bafaluy MP, Rana M, et al. Human mitochondrial DNA with large deletions repopulates organelles faster than full-length genomes under relaxed copy number control. Nucleic Acids Res. 2002;30(21):4626–33. 4. Fukui H, Moraes CT. Mechanisms of formation and accumulation of mitochondrial DNA deletions in aging neurons. Hum Mol Genet. 2009;18(6):1028–36. 5. Elson JL, Samuels DC, Turnbull DM, et al. Random intracellular drift explains the clonal expansion of mitochondrial DNA mutations with age. Am J Hum Genet. 2001;68:802–6. 6. Schaefer AM, McFarland R, Blakely EL, He L, Whittaker RG, Taylor RW, et al. P
