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Mitochondrial dysfunction contributes to neurodegeneration in multiple sclerosis Maarten E. Witte1*, Don J. Mahad2*, Hans Lassmann3*, and Jack van Horssen1* 1
Department of Molecular Cell Biology and Immunology, VU University Medical Center, 1081BT, Amsterdam, The Netherlands Centre for Neuroregeneration, University of Edinburgh, Edinburgh, UK 3 Center for Brain Research, Medical University of Vienna, A-1090 Vienna, Austria 2
Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Current treatments are very effective in reducing the neuroinflammatory attack, but fail to significantly halt disease progression and associated loss of neuronal tissue. In recent years, it has become increasingly clear that dysfunctional mitochondria are important contributors to damage and loss of both axons and neurons. Observations in animal and histopathological studies suggest that infiltrating leukocytes and activated microglia play a central role in neuronal mitochondrial dysfunction. This review provides a comprehensive overview on the current knowledge regarding mitochondrial dysfunction in MS. Importantly, more insight into the cause and consequences of impaired mitochondrial function provide a basis for mitochondrial-targeted medicine to combat progressive MS. Multiple sclerosis MS is a chronic inflammatory disease of the central nervous system (CNS) and the most common cause of neurological disability in young adults, affecting over 2.5 million people worldwide [1]. First symptoms generally appear in early adulthood (20–40 years of age), including sensory disturbances, optic neuritis, and limb weakness, and approximately half of these patients need help walking within 15 years. As the disease progresses additional symptoms occur, such as fatigue, bladder dysfunction, and cognitive impairment. Depending on progression of clinical symptoms, three distinct MS subtypes can be distinguished. In relapsing-remitting (RR) MS (see Glossary), present in 80% of cases, patients suffer from clearly defined periods of neurological deficits followed by (partial) recovery [1]. In 65% of patients suffering from RRMS, the disease course gradually evolves into secondary progressive (SP) MS, characterised by progressive permanent neurological Corresponding author: Witte, M.E. ([email protected]). Keywords: multiple sclerosis; mitochondria; neurodegeneration; neuroinflammation; oxidative stress. * All authors contributed equally. 1471-4914/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molmed.2013.11.007
Glossary Astrocytes: the most prevalent type of neuroglia in the CNS. They have multiple crucial functions including providing trophic support to other CNS cells, regulating neuronal activity, and guarding CNS homeostasis. Demyelination: process of oligodendrocyte and myelin loss. Experimental autoimmune encephalomyelitis (EAE): a widely used animal model for MS, in which T cell mediated autoimmunity against myelin is experimentally induced. Grey matter: CNS tissue can be divided into white matter and grey matter; grey matter contains virtually all neuronal cells and less myelin than white matter. Inflammation: part of a complex biological response to harmful stimuli. Leber’s hereditary optic neuropathy (LHON): inherited disease, in which specific mutations in mitochondrial DNA cause degeneration of retinal ganglion cells and their axons. Microglia: the resident immune cells of the CNS and closely related to macrophages. Mitochondria: organelles responsible for oxidative energy metabolism in eukaryotic cells. They are also crucial in maintaining Ca2+ homeostasis and regulation of programmed cell death. N-Acetyl-aspartate (NAA): CNS specific metabolite that is only produced in neuronal mitochondria. Therefore, reduced NAA concentration as measured with magnetic resonance spectroscopy is considered to reflect loss of neuronal tissue. Neurodegeneration: in the broadest sense, neurodegeneration is the process of CNS tissue loss. Often the term neurodegeneration is used just for loss of neuronal tissue. Neurons: non-dividing cells that are electrically excitable and process and transmit information via electrochemical signals. Neurons can transmit signals both to other neurons and different effector cells (e.g., muscle cells). Oligodendrocytes: a population of glial cells that form the myelin sheaths that encapsulate axons, thereby facilitating fast saltatory conduction. Oxidative phosphorylation chain (OxPhos): five multi-subunit protein complexes located on the inner mitochondrial membrane, which together produce ATP through oxidation of nutrients. Oxidative stress: state reflecting an imbalance between the production and detoxification of ROS leading to cellular damage. Peroxisome proliferator-activated receptor gamma coactivator-1a (PGC-1a): transcriptional cofactor that can activate multiple transcription factors involved in energy metabolism. Therefore, PGC-1a is often considered the main regulator of energy metabolism. Primary progressive (PP) MS: MS subtype present in 20% of MS patients and characterised by a progressive disease course from onset. Reactive nitrogen species: highly reactive nitric oxide-derived molecules, which have important signalling functions but can also cause severe damage to various macromolecules and cells. Reactive oxygen species (ROS): chemically reactive molecules containing oxygen, which have important signalling functions but can also cause severe damage to various macromolecules and cells. Relapsing-remitting (RR) MS: MS subtype present in 80% of cases characterised by clearly defined periods of neurological deficits followed by (partial) recovery. Secondary progressive (SP) MS: MS subtype that follows after RRMS in 65% of cases and is characterised by progressive neurological symptoms. White matter: is one of the two components of the CNS and is predominantly composed of glial cells and myelinated axons.
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Review deficits. Furthermore, 20% of all MS patients have a progressive disease course from onset, known as primary progressive (PP) MS. It is known that both genetic and environmental factors contribute substantially to disease susceptibility; however, despite extensive research, the primary cause(s) and initial triggers of MS are still largely enigmatic. Thus far, only a few genes have been consistently shown to influence disease susceptibility, and those with the strongest effect are located on chromosome 6 and are part of the major histocompatibility complex class II (MHC II), namely HLA-DR15 and HLA-DQ6 [2]. It is now well documented that environmental factors, such as smoking, vitamin D status, and viral infections, have been linked to the occurrence of MS [3,4]. However, definitive proof of a pathogenic role for these factors is lacking. Histopathological analysis of MS brain autopsy material reveals focal demyelinated lesions scattered throughout the white and grey matter of the brain and spinal cord. The inflammatory demyelination observed in MS patients is generally believed to be initiated by autoreactive T cells activated in the periphery [5]. However, current immunomodulatory drugs are effective in reducing the number of inflammation-driven relapses during RRMS but fail to halt disease progression in progressive MS patients [6], which is primarily the result of neurodegenerative processes. Still, strong evidence exists that in progressive MS neurodegeneration is caused by inflammatory processes [7,8]. These processes, however, occur, for a large part, behind a closed blood–brain barrier, thereby explaining the failure of current therapeutics to slow down disease in the progressive phase [6]. This ineffectiveness of current MS therapeutics popularised a new concept of MS pathogenesis, the so-called inside-out hypothesis (Box 1) [9–11]. Either way, novel Box 1. Inside-out versus outside-in theories Histopathological studies evidently point to a pivotal role of both the innate and adaptive immune system in the pathogenesis of MS. In addition, genome-wide association studies showed a preponderance of immune-related genes associated with increased disease susceptibility, and current immunomodulatory drugs are highly effective in a majority of RRMS patients. Therefore, MS is generally considered a primary immune-driven disease in which autoreactive T cells and macrophages invade the CNS where they, together with activated microglia, attack myelin and/or oligodendrocytes (known as the ‘outside-in model’). In this model, neurodegeneration occurs both as a bystander effect of the inflammatory attack and as a consequence of the loss of myelin trophic support. This concept is now being challenged by those who argue that inflammatory processes in MS brains are secondary to a primary cytodegenerative process in resident CNS cells (e.g., oligodendrocytes and neurons/ axons) or alterations in the myelin–axon interaction (‘inside-out model’). Intriguingly, axonal alterations can be widespread in NAWM and the cortex without overt signs of infiltrating leukocytes. Another argument for the inside-out model is the ineffectiveness of the current anti-inflammatory therapies in progressive MS patients, where neurodegeneration drives disease progression. Nevertheless, throughout the course of MS active tissue injury remains strongly associated with the degree of inflammation. In NAWM and cortical lesions, where infiltrating immune cells are scarcely found, neurodegeneration is still ongoing and is associated with microglial activation. Because this process occurs behind a closed or repaired blood–brain barrier, microglial activation is out-of-reach for current immunomodulatory drugs. Although at this stage it is unclear which of the two opposing theories is correct, current data still appear to favour the outside-in model. 2
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therapeutics that effectively target the progressive phase of MS are needed, and therefore more insight into the mechanisms leading to neurodegeneration is essential. Evidence is now emerging that mitochondrial damage is an important feature of progressive MS and these studies strongly suggest that mitochondrial alterations contribute to neurodegenerative processes in MS [12]. In this review, we will systematically dissect the current literature regarding mitochondrial dysfunction in MS and address the potential of mitochondria as a therapeutic target in progressive MS. Lastly, we will point to important caveats in our knowledge and discuss future directions. Mitochondria and MS susceptibility Mitochondria are present in all eukaryotic cells and their prime function is to provide cells with energy, in the form of adenosine triphosphate (ATP), by oxidation of metabolic fuels. In addition, they are involved in many other important cellular processes, including fatty acid oxidation, apoptosis, and calcium homeostasis. Mitochondria are the sole carriers of non-nuclear and circular DNA in eukaryotic cells, which is a footprint of their bacterial origin. Mitochondrial DNA (mtDNA) encode 22 transfer RNAs, two ribosomal RNAs, and 13 essential proteins of the oxidative phosphorylation chain (OxPhos), the complex intramitochondrial machinery responsible for oxidative metabolism [13]. Mitochondria also constantly produce reactive oxygen species (ROS), which have important signalling functions. However, when the rate of ROS production exceeds the cellular antioxidant capacity it can cause oxidative stress and extensive damage to essential cell components [14]. The high energy requirements of the CNS make it particularly vulnerable to defects in mitochondrial function, as illustrated by the long list of neurological disorders caused by inherited genetic alterations in mtDNA and in nuclear genes encoding key mitochondrial proteins [15]. Importantly, extensive mitochondrial dysfunction and concomitant oxidative stress are key features of common neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease [16]. Interest in mitochondria in the context of MS was sparked by the finding that patients suffering from Leber’s hereditary optic neuropathy (LHON), which can be caused by mutations in mtDNA [17], have an increased risk of developing an MSlike disease, a condition known as Harding’s disease [18]. As a result, many studies have investigated the influence of mtDNA mutation on MS susceptibility (extensively reviewed in [19]). Most of these studies have focussed specifically on LHON-associated mutations; however, the results are rather contradictory. Various studies confirmed a weak association between secondary LHON mtDNA mutations and MS susceptibility, whereas other studies failed to find such an association. These differences might be partly explained by the very small sample size in most studies and differences in the studied population. Interestingly, several studies indicate that the occurrence of severe optic neuritis in a subset of MS patients is indeed linked to mtDNA mutations. This could be due to the association of both MS and LHON to a certain mtDNA haplotype (a distinct set of mtDNA polymorphisms) [20].
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Review To our knowledge there is currently only one polymorphism in nuclear DNA encoding a mitochondrial protein that has been linked to MS susceptibility. This polymorphism, associated with MS in two independent cohorts of German patients [21], was found in the promoter region of uncoupling protein 2 (UCP2), a mitochondrial protein that is able to separate the mitochondrial membrane potential from ATP synthesis and thereby decrease ATP and ROS production. Because whole-genome sequencing is rapidly becoming less expensive, it is conceivable that other polymorphisms in nuclear DNA coding for mitochondrial proteins will be identified in the future. However, just like the polymorphism in UCP2, they are likely to only marginally contribute to MS susceptibility. Mitochondrial involvement in white matter damage White matter demyelination is considered the pathological hallmark of MS and white matter lesions are generally classified according to demyelination and inflammation status [22]. Lesions with ongoing demyelination by infiltrated macrophages and activated microglia are called active demyelinating lesions, which are accompanied by extensive oxidative damage [23–25]. Active demyelinating lesions are believed to gradually evolve into chronic active, slowly expanding lesions. Chronic active lesions are characterised by a completely demyelinated centre with ongoing inflammatory demyelination at the lesion edge. Eventually, inflammation resolves and the demyelinated area becomes known as a chronic inactive lesion. A substantial proportion of demyelinated lesions are remyelinated over the course of the disease [26], whereas nonremyelinated areas are filled with a gliotic scar. White matter lesions are surrounded by normal-appearing white matter (NAWM), which is defined as non-lesional regions devoid of marked changes in myelin protein expression. However, NAWM can be a deceptive term because activated microglia and axonal degeneration are frequently observed in these ‘normally’ myelinated areas. Besides damage to myelin and oligodendrocytes, the myelin-forming cells of the CNS, there is also extensive damage to axons in MS white matter [27] and the extent of axonal degeneration has been linked to permanent neurological disability in progressive MS patients [28,29]. The evidence presented here indicates an important role for mitochondrial dysfunction in driving axonal degeneration in MS white matter. White matter tissue injury in MS is most prominent in the foci of inflammatory demyelination where the extent of axon degeneration correlates with the severity of inflammation. In a subtype of acute MS lesions, the so-called pattern III or hypoxia-like lesions [30], diffuse mitochondrial respiratory chain defects are seen in axons, oligodendrocytes, and astrocytes [31]. Moreover, a recent study of inflammatory lesions in experimental autoimmune encephalomyelitis (EAE) (Box 2), a widely used MS animal model, identified acute mitochondrial injury as a critical step towards focal axonal degeneration [32]. Similar changes were also observed in postmortem MS tissue, suggesting that similar mitochondrial processes also play a role in active MS lesions. ROS and reactive nitrogen species (RNS), generated by activated microglia and
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Box 2. Experimental autoimmune encephalomyelitis: a valuable animal model of MS? Over a hundred years ago Pasteur developed a vaccine to treat rabies, a zoonotic, often fatal, disease. The vaccines were prepared by collecting spinal cord homogenates of rabbits infected with rabies virus. Although the vaccine was effective in many cases, some patients developed signs of paralysis. In the most severe cases patients died, and histopathological evaluation of the brain tissue revealed perivascular leukocyte infiltrates and demyelinated lesions. It is now well known that immunisation of susceptible animals, usually mice and rats, with either CNS homogenates or myelin proteins/peptides induces an autoimmune demyelinating disease called experimental autoimmune encephalomyelitis (EAE). Furthermore, EAE can be induced by adopted transfer of CD4+ T cells specific for myelin antigens. To date, EAE is still the most commonly used experimental animal model of MS. Based on the strain and antigens used, animals develop a variety of neurological symptoms ranging from a monophasic clinical episode of paralysis (acute EAE) to chronic-relapsing neurological periods and progressive disability (chronic EAE). In general, certain pathological key features of RRMS, such as glial cell activation, leukocyte infiltration, and axonal damage occur in most EAE models. However, inflammatory demyelination is mainly observed in chronic EAE models, although not extensively. The EAE model has been instrumental in providing important contributions to our understanding of key processes underlying neuroinflammation, including the process of leukocyte migration across the blood–brain barrier into the CNS. This has led to the development of effective anti-inflammatory and immunomodulatory therapies, of which some are currently successfully used in the clinic. Unfortunately, the main drawbacks of the current EAE models, even the chronic model, are that they do not accurately reproduce key aspects of progressive MS, such as cortical demyelination and neuronal loss.
macrophages through oxidative burst, are the most likely culprits of intra-axonal mitochondrial injury, because they are known to directly inhibit the respiratory chain (Figure 1A) [33,34]. Indeed, detoxification of ROS reversed mitochondrial pathology and rescued axons from degeneration in EAE [32]. Remarkably, even before influx of leukocytes, nitration of mitochondrial proteins has been observed in intact axons of EAE animals [35]. Conceivably, activation of microglia, which occurs rapidly after EAE induction when leukocyte infiltration is only present in meninges but not in the CNS parenchyme, induces these early mitochondrial alterations. This may explain why diffuse axonal loss in the spinal cord white matter associates with meningeal T cell infiltration [36]. Still, most axons survive the inflammatory demyelinating attack and become chronically demyelinated. Over time a proportion of these axons degenerate. Whereas degenerating chronically demyelinated axons in progressive MS cases contained injured mitochondria, over half of the intact and functional chronically demyelinated axons showed the opposite [37]. These demyelinated and otherwise uninjured axons contained mitochondria with increased size and respiratory chain enzyme activity [37,38]. Furthermore, uninjured and demyelinated axons in MS increased expression of an axon specific mitochondrial docking protein, syntaphilin. In vitro demyelination caused the motile mitochondria within axons to move in a retrograde and anterograde manner with greater speed [39]. This axonal mitochondrial response to demyelination, namely (i) increased mitochondrial presence, (ii) respiratory chain activity, and (iii) speed of motile mitochondria 3
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Figure 1. Mitochondrial dysfunction mediates axonal degeneration in multiple sclerosis (MS). (A) In inflammatory MS lesions, macrophages and activated microglia produce vast amounts of reactive oxygen species (ROS) and nitric oxide (NO), which subsequently induce mitochondrial dysfunction in both myelinated and demyelinated axons. In early stages, axonal mitochondrial dysfunction is reflected by swelling of intra-axonal mitochondria in intact axons. A substantial proportion of axons with swollen mitochondria will eventually degenerate. (B) Axons in chronic MS lesions have increased and redistributed their axolemmal Na+ channels, leading to an increased intra-axonal Na+ concentration. As a result, more ATP is required for the removal of excess Na+ by Na+/K+ ATPase (I). To meet the higher energy demand, mitochondrial content is increased in chronically demyelinated axons. However, due to cortical pathology or previous inflammatory damage, a proportion of mitochondria in chronically demyelinated axons are damaged (II). Over time, mitochondrial dysfunction accumulates to such an extent that ATP production becomes too low for Na+/K+ ATPase to remove excess intra-axonal Na+ (III). The rising Na+ concentrations inside axons lead to reversal of the axolemmal Na+/Ca2+ exchanger, which now pumps Na+ out and Ca2+ into the axon (IV). In turn, this leads to rising axonal Ca2+ concentrations, which will eventually trigger a set of several deleterious events, including further destabilisation of intra-axonal mitochondria and increased ROS production, which then contribute to axonal degeneration (V).
within demyelinated axons, has been consistently in disease models of demyelination and dysmyelination, and it therefore appears to be an adaptive or compensatory phenomenon to the disturbance of myelin. In MS, the absence of myelin in otherwise intact axons alters the distribution of ion channels, in particular sodium channels, which supposedly causes an increased intra-axonal energy demand also known as ‘virtual hypoxia’ [40], which in turn leads to the observed changes in mitochondria. However, although markedly less than in inflammatory MS lesions, axons still degenerate in chronic MS lesions, and this is thought to be caused by the eventual failure of intra-axonal mitochondria to fulfil the need of axons for ATP (Figure 1B) [34,41]. The grey matter mitochondrial abnormalities located within neuronal cell bodies (soma) in MS, and 4
discussed below, raise the interesting possibility that axon degeneration in progressive MS may also be due to the inability of the axon to mount and maintain the mitochondrial response to demyelination as a result of the inability of the neuronal soma to supply the axon with healthy mitochondria. Alternatively, mitochondrial damage sustained previously during active white matter inflammation and/or ongoing mild inflammation is also likely to contribute to the eventual failure of intra-axonal mitochondria to support chronically demyelinated axons. Interestingly, imaging of newly forming lesions in the spinal cord of MS patients revealed similar mitochondrial alterations as found in postmortem and animal studies. In newly forming inflammatory MS lesions, the concentration of N-acetyl-aspartate (NAA) decreased significantly,
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Review reflecting decreased neuronal mitochondrial function as seen in active MS lesions. As the inflammation cleared over time, NAA concentration again increased reflecting increased mitochondrial presence and activity in chronically demyelinated MS lesions [41,42]. However, because NAA contributes to myelin formation by oligodendrocytes, an increase in NAA might also reflect remyelination. Currently, studies looking into mitochondrial changes in NAWM and preactive lesions are lacking. Those studies are warranted to elucidate if mitochondria already react to the subtle changes occurring in NAWM and/or contribute to lesion initiation in preactive lesions. Intriguingly, nitration of mitochondrial proteins has been observed as early as 3 days after sensitisation in EAE, suggesting that mitochondrial dysfunction precedes leukocyte infiltration within the CNS parenchyme [35]. Unfortunately, activation of microglia, which also occurs before influx of leukocytes in EAE [43], was not assessed and therefore the likely relation between mitochondrial protein nitration and microglial activation remains unresolved. Mitochondrial involvement in grey matter damage Although damage to grey matter structures in MS has long been overlooked, it occurs early in disease and is extensive in many MS patients [44–46]. Cortical demyelination can reach up to 75% of the total cortex and even exceeds the extent of white matter demyelination in a subset of patients [47,48]. Postmortem studies revealed that cortical lesions differ markedly from white matter lesions. Grey matter lesions generally lack blood–brain barrier alterations and abundant infiltration of blood-borne leukocytes [49–51]. However, a recent study analysing biopsy material from patients with early fulminant MS reported that a large proportion of grey matter lesions in these patients contained prominent leukocyte infiltration [52]. It remains to be determined if this is specific for early fulminant MS, or also occurs in the more common forms of early MS. Besides demyelination, neurodegeneration is also widespread in the MS cortex. Imaging studies revealed that cortical atrophy is detectable early in disease, but increases strikingly in the progressive stage of the disease [45,53,54]. Several studies have now established cortical atrophy as a correlate of clinical disability, emphasising the clinical relevance of cortical pathology in MS [55,56]. It was shown that cortical thinning is the combined result of glial cell loss, neuronal loss, and reduced synaptic density, which were most prevalent in cortical lesions [57]. However, neuronal injury and loss can also be extensive in normal-appearing grey matter (NAGM) [8,58]. Thus, neurodegeneration occurs, at least partly, independent of local demyelination. Other processes proposed to contribute to neurodegeneration are (distant) axonal damage, local meningeal inflammation [8,59], the presence of persistent microglial activation [23,51,60], and mitochondrial dysfunction [34]. Microarray analysis of the postmortem non-demyelinated motor cortex from MS patients revealed decreased gene expression of 26 nuclear-encoded subunits of the OxPhos chain [61], which coincided with a significant reduction in activity of OxPhos complexes I and III. In situ hybridisation showed that this decrease occurs
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specifically in cortical neurons. Later, it was observed that the MS cerebral cortex contained neurons devoid of complex IV activity [62]. These so-called respiratory deficient neurons were present in both NAGM and cortical lesions, and harboured high levels of clonally expanded mtDNA deletions [62]. Damage to mtDNA and reduced activity of the OxPhos complexes may be initiated by oxidative and nitrosative stress. Furthermore, damage to neuronal mtDNA in MS also contributes to reduced activities of OxPhos complexes I, III, and IV, because mtDNA codes for essential subunits of these OxPhos complexes. However, it does not explain the reduction of various nuclear-encoded OxPhos subunits in the MS cortex [61]. Expression of key OxPhos genes is tightly coordinated by a number of transcription factors, including nuclear respiratory factor 1, -2, oestrogen-related receptor a and peroxisome proliferator-activated receptors (PPARs) (for an extensive review, see [63]). A decrease in a transcription factor complex containing nuclear respiratory factor 2 (NRF-2) in MS grey matter samples has been observed, which correlated with reduced expression of OxPhos genes and increased oxidative damage [64]. Recently, a significant reduction of peroxisome proliferator-activated receptor gamma coactivator-1a (PGC-1a) in NAGM samples was identified in progressive MS cases [65]. PGC-1a is a transcriptional cofactor that binds and activates nuclear transcription factors involved in mitochondrial function. PGC1a expression was reduced in cortical neurons of MS patients and correlated with local neuronal density, indicating a possible role in neurodegeneration [65]. Knockdown of neuronal PGC-1a induced a decrease in nuclearencoded OxPhos subunits, as observed in the MS cortex, and increased neuronal ROS production, thereby further enhancing the already existing oxidative stress. In addition, reduced PGC-1a resulted in decreased expression of mitochondrial antioxidants [65]. Because ROS and nitric oxide production by microglia can be extensive in the cortex of MS patients [66], we propose a mechanism where reduced neuronal PGC-1a directly induces mitochondrial dysfunction by decreased transcription of key mitochondrial proteins and augments mitochondrial susceptibility to oxidative/nitrosative damage (Figure 2). Importantly, the presence of mtDNA damage and nitrotyrosine residues in mitochondrial proteins strongly indicate increased oxidative and nitrosative damage to mitochondria in the MS cortex [61,67]. Over time, damage to mitochondria will further impair neuronal mitochondrial function in the MS cortex, ultimately leading to neuronal death (Figure 2). A recent study linked neuronal mitochondrial dysfunction in the MS cortex to local failure of myelination by oligodendrocytes via reduced NAA production, further emphasising the importance of mitochondrial dysfunction in MS pathology [68]. Currently, it is unclear what causes the decrease in neuronal PGC-1a expression in the MS cortex. Intriguingly, PGC-1a levels are also markedly reduced in ‘classic’ neurodegenerative disorders including Huntington’s disease, Parkinson’s disease, and Alzheimer’s disease [69– 73]. Extensive microglial activation is an early and persistent phenomenon in these neurodegenerative diseases [74], and it is therefore conceivable that inflammatory 5
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Figure 2. Mitochondrial dysfunction mediates neurodegeneration in the multiple sclerosis (MS) cortex. In the MS cortex, two distinct but interrelated pathways contribute to neuronal mitochondrial dysfunction. One mechanism is by production of reactive oxygen species (ROS) and nitric oxide (NO) by continuously activated microglia in the MS cortex (I). This will lead to inhibition of several oxidative phosphorylation (OxPhos) complexes (a, inset) and damage to mitochondrial DNA (mtDNA) in neuronal mitochondria (b, inset). Damage to mtDNA will further impair OxPhos function leading to reduced ATP production (c, inset). Secondly, decreased expression of neuronal PGC-1a, a transcriptional coactivator, leads to reduced transcription of OxPhos subunits and mitochondrial antioxidants (II). We propose that chronic microglial activation present in the MS cortex eventually leads to reduced neuronal PGC-1a expression. As a consequence, neuronal mitochondria have a reduced capacity to produce ATP (d, inset) and detoxify intramitochondrial ROS (e, inset; O2, superoxide), which can eventually fully compromise mitochondrial function and lead to neuronal cell death. Importantly, dysfunctional mitochondria in the neuronal soma could also have deleterious effects on (demyelinated) axons, because the neuronal cell body cannot supply its axon with sufficient healthy mitochondria (f).
factors produced by microglia mediate reduced neuronal PGC-1a expression. It should be noted that inflammatory factors present in the MS cortex, such as tumour necrosis factor a (TNFa), are also likely to alter neuronal mitochondrial function via other mechanisms. Although there is currently no experimental evidence for such a mechanism in MS, it has been shown in other settings that TNFa can alter neuronal mitochondrial distribution and induce mitochondria-mediated apoptosis via TNF receptor-associated death domains (TRADDs) [75,76]. Concluding remarks and future perspectives Over the past few years interest in the role of mitochondria in MS pathogenesis has been steadily increasing, and the importance of mitochondrial dysfunction is now widely 6
acknowledged [40,77,78]. We propose that mitochondrial dysfunction contributes to neurodegeneration in MS via three distinct but interrelated mechanisms: (i) in acute, inflammatory MS lesions, intra-axonal mitochondria are damaged by inflammation-derived ROS and nitric oxide, leading to severe mitochondrial dysfunction and subsequent axonal injury (Figure 1A); (ii) in chronically demyelinated axons, energy demand is increased several-fold compared with normally myelinated axons, and at a certain point, intra-axonal mitochondria cannot meet the energy demand due to accumulating mitochondrial dysfunction, setting off a cascade of deleterious events leading to axonal degeneration and loss (Figure 1B); and (iii) high levels of mtDNA mutation, reduced neuronal expression of PGC-1a, and increased nitric oxide and ROS production by
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Review Box 3. Outstanding questions In EAE, mitochondrial alterations precede the influx of leukocytes into the brain parenchyma. In MS, do mitochondrial alterations already occur in NAWM? Is mitochondrial dysfunction in neurons directly related to local inflammatory events, such as cortical microglial activation or meningeal inflammation? Which molecular pathways are responsible for reduced neuronal PGC-1a expression and subsequent mitochondrial dysfunction in the MS cortex? Are treatments aimed at improving or protecting neuronal/axonal mitochondrial function able to reduce neurodegeneration and thereby slowing down disease progression in MS patients?
activated cortical microglia synergistically induce neuronal mitochondrial dysfunction, driving the neurodegenerative process in the MS cortex (Figure 2). Although there is some evidence for an important role of mitochondria in different stages of MS, much remains to be uncovered (Box 3). First, a thorough study of mitochondrial function in NAWM is warranted to elucidate if, in analogy to EAE [35], mitochondrial alterations precede the influx of leukocytes into the lesions themselves. Moreover, levels of microglial inflammation in NAGM can differ greatly between and in patients. A correlation between the extent of microglial activation and mitochondrial alterations, for example, the extent of mtDNA damage, and PGC-1a expression in NAGM might shed light on the causative role of activated microglia in mediating neuronal mitochondrial injury. Also, a better understanding of the molecular pathways leading to mitochondrial dysfunction could open up new therapeutic targets to combat neurodegeneration in MS. However, the lack of a suitable animal model for progressive MS presents a major hurdle [6]. Most MS animal models accurately mimic parts of the inflammatory reaction seen in patients, but fail to reproduce important aspects of progressive MS, such as cortical neurodegeneration. Development of an animal model that more accurately reflects progressive MS is needed to fully comprehend the role of mitochondria in MS disease progression and test the potential of novel therapeutic strategies aimed at improving the mitochondrial metabolism. Current approved treatments for MS are all immunomodulatory or anti-inflammatory, and have little or no effect on progressive MS, where neuroinflammation is partly compartmentalised behind a closed blood–brain barrier, and therefore inaccessible for many therapeutics, and neurodegeneration drives clinical deterioration [6]. Thus, there is a great need for therapeutics that combat neurodegeneration, and several studies have now shown that mitochondria provide us with a potential therapeutic target. For instance, gene delivery of superoxide dismutase 2, which scavenges superoxide in mitochondria, preserved axonal integrity in EAE optic nerves [79]. Moreover, knockout of cyclophilin D and p66ShcA, both important regulators of the mitochondrial permeability transition pore and consequently mitochondria-mediated cell death, significantly reduced axonal damage in animals suffering from EAE [80,81]. This is believed to be the result of increased resistance to oxidative and nitrosative stress of axonal mitochondria and an increased
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ability of axonal mitochondria to sequester Ca2+. Very recently, two studies reported protective properties of MitoQ, a synthetic antioxidant that specifically accumulates in mitochondria, in two different EAE models [82,83]. Because levels of inflammation were not altered by MitoQ, it is conceivable that increased mitochondrial protection against ROS is sufficient to reduce axonal damage. Therefore, more insight into the inflammatory factors that drive ROS and nitric oxide production by macrophages/microglia will also provide interesting targets for intervention. Another potentially interesting therapeutic candidate is PGC-1a, because boosting its expression and/or activity will have a broad range of beneficial effects on mitochondrial function and protection. Thus far, several compounds have been shown to enhance PGC-1a activity and thereby increase neuronal survival in animal models for various neurodegenerative disorders, including EAE [84–86]. However, these compounds, including resveratrol and thialozidines, have many other targets and therefore the specific effects of increased PGC-1a activity is difficult to establish. Taken together, the current literature strongly suggests that therapeutic strategies aimed at improving mitochondrial function in MS are worthwhile to pursue but warrant additional research. References 1 Compston, A. and Coles, A. (2008) Multiple sclerosis. Lancet 372, 1502– 1517 2 Hauser, S.L. and Oksenberg, J.R. (2006) The neurobiology of multiple sclerosis: genes, inflammation, and neurodegeneration. Neuron 52, 61–76 3 DeLorenze, G.N. et al. (2006) Epstein–Barr virus and multiple sclerosis: evidence of association from a prospective study with longterm follow-up. Arch. Neurol. 63, 839–844 4 Marrie, R.A. (2004) Environmental risk factors in multiple sclerosis aetiology. Lancet Neurol. 3, 709–718 5 Steinman, L. et al. (2002) Multiple sclerosis: deeper understanding of its pathogenesis reveals new targets for therapy. Annu. Rev. Neurosci. 25, 491–505 6 Lassmann, H. et al. (2012) Progressive multiple sclerosis: pathology and pathogenesis. Nat. Rev. Neurol. 8, 647–656 7 Frischer, J.M. et al. (2009) The relation between inflammation and neurodegeneration in multiple sclerosis brains. Brain 132, 1175–1189 8 Magliozzi, R. et al. (2010) A gradient of neuronal loss and meningeal inflammation in multiple sclerosis. Ann. Neurol. 68, 477–493 9 Geurts, J.J. et al. (2010) Multiple sclerosis as an ‘‘inside-out’’ disease. Ann. Neurol. 68, 767–768 10 Trapp, B.D. and Nave, K.A. (2008) Multiple sclerosis: an immune or neurodegenerative disorder? Annu. Rev. Neurosci. 31, 247–269 11 Stys, P.K. et al. (2012) Will the real multiple sclerosis please stand up? Nat. Rev. Neurosci. 13, 507–514 12 Su, K.G. et al. (2009) Axonal degeneration in multiple sclerosis: the mitochondrial hypothesis. Curr. Neurol. Neurosci. Rep. 9, 411–417 13 Wallace, D.C. (1992) Diseases of the mitochondrial DNA. Annu. Rev. Biochem. 61, 1175–1212 14 Lin, M.T. and Beal, M.F. (2006) Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443, 787–795 15 DiMauro, S. et al. (2013) The clinical maze of mitochondrial neurology. Nat. Rev. Neurol. 9, 429–444 16 Beal, M.F. (1995) Aging, energy, and oxidative stress in neurodegenerative diseases. Ann. Neurol. 38, 357–366 17 Wallace, D.C. et al. (1988) Mitochondrial DNA mutation associated with Leber’s hereditary optic neuropathy. Science 242, 1427–1430 18 Olsen, N.K. et al. (1995) Leber’s hereditary optic neuropathy associated with a disorder indistinguishable from multiple sclerosis in a male harbouring the mitochondrial DNA 11778 mutation. Acta Neurol. Scand. 91, 326–329 7
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49 Van Horssen, J. et al. (2007) The blood–brain barrier in cortical multiple sclerosis lesions. J. Neuropathol. Exp. Neurol. 66, 321–328 50 Bo, L. et al. (2003) Intracortical multiple sclerosis lesions are not associated with increased lymphocyte infiltration. Mult. Scler. 9, 323–331 51 Peterson, J.W. et al. (2001) Transected neurites, apoptotic neurons, and reduced inflammation in cortical multiple sclerosis lesions. Ann. Neurol. 50, 389–400 52 Lucchinetti, C.F. et al. (2011) Inflammatory cortical demyelination in early multiple sclerosis. N. Engl. J. Med. 365, 2188–2197 53 Fisher, E. et al. (2008) Gray matter atrophy in multiple sclerosis: a longitudinal study. Ann. Neurol. 64, 255–265 54 Fisniku, L.K. et al. (2008) Gray matter atrophy is related to long-term disability in multiple sclerosis. Ann. Neurol. 64, 247–254 55 Bonati, U. et al. (2011) Cervical cord and brain grey matter atrophy independently associate with long-term MS disability. J. Neurol. Neurosurg. Psychiatry 82, 471–472 56 Rudick, R.A. et al. (2009) Gray matter atrophy correlates with MS disability progression measured with MSFC but not EDSS. J. Neurol. Sci. 282, 106–111 57 Wegner, C. et al. (2006) Neocortical neuronal, synaptic, and glial loss in multiple sclerosis. Neurology 67, 960–967 58 Clements, R.J. et al. (2008) Distribution of parvalbumin and calretinin immunoreactive interneurons in motor cortex from multiple sclerosis post-mortem tissue. Exp. Brain Res. 187, 459–465 59 Howell, O.W. et al. (2011) Meningeal inflammation is widespread and linked to cortical pathology in multiple sclerosis. Brain 134, 2755–2771 60 Lassmann, H. and van Horssen, J. (2011) The molecular basis of neurodegeneration in multiple sclerosis. FEBS Lett. 585, 3715–3723 61 Dutta, R. et al. (2006) Mitochondrial dysfunction as a cause of axonal degeneration in multiple sclerosis patients. Ann. Neurol. 59, 478–489 62 Campbell, G.R. et al. (2011) Mitochondrial DNA deletions and neurodegeneration in multiple sclerosis. Ann. Neurol. 69, 481–492 63 Scarpulla, R.C. (2008) Transcriptional paradigms in mammalian mitochondrial biogenesis and function. Physiol. Rev. 88, 611–638 64 Pandit, A. et al. (2009) Impaired regulation of electron transport chain subunit genes by nuclear respiratory factor 2 in multiple sclerosis. J. Neurol. Sci. 279, 14–20 65 Witte, M.E. et al. (2013) Reduced expression of PGC-1a partly underlies mitochondrial changes and correlates with neuronal loss in multiple sclerosis cortex. Acta Neuropathol. 125, 231–243 66 Fischer, M.T. et al. (2013) Disease-specific molecular events in cortical multiple sclerosis lesions. Brain 136, 1799–1815 67 Broadwater, L. et al. (2011) Analysis of the mitochondrial proteome in multiple sclerosis cortex. Biochim. Biophys. Acta 1812, 630–641 68 Li, S. et al. (2013) Decreased NAA in gray matter is correlated with decreased availability of acetate in white matter in postmortem multiple sclerosis cortex. Neurochem. Res. 38, 2385–2396 69 Cameron, B. and Landreth, G.E. (2010) Inflammation, microglia, and Alzheimer’s disease. Neurobiol. Dis. 37, 503–509 70 Moller, T. (2010) Neuroinflammation in Huntington’s disease. J. Neural Transm. 117, 1001–1008 71 Qin, W. et al. (2009) PGC-1a expression decreases in the Alzheimer disease brain as a function of dementia. Arch. Neurol. 66, 352–361 72 Rona-Voros, K. and Weydt, P. (2010) The role of PGC-1a in the pathogenesis of neurodegenerative disorders. Curr. Drug Targets 11, 1262–1269 73 Tansey, M.G. and Goldberg, M.S. (2010) Neuroinflammation in Parkinson’s disease: its role in neuronal death and implications for therapeutic intervention. Neurobiol. Dis. 37, 510–518 74 Dal-Bianco, A. et al. (2008) Multiple sclerosis and Alzheimer’s disease. Ann. Neurol. 63, 174–183 75 Stommel, E.W. et al. (2007) Tumor necrosis factor-a induces changes in mitochondrial cellular distribution in motor neurons. Neuroscience 146, 1013–1019 76 Swarup, V. et al. (2007) Tumor necrosis factor receptor-1-induced neuronal death by TRADD contributes to the pathogenesis of Japanese encephalitis. J. Neurochem. 103, 771–783 77 Campbell, G.R. and Mahad, D.J. (2011) Mitochondria as crucial players in demyelinated axons: lessons from neuropathology and experimental demyelination. Autoimmune Dis. 2011, 262847 78 Stadelmann, C. (2011) Multiple sclerosis as a neurodegenerative disease: pathology, mechanisms and therapeutic implications. Curr. Opin. Neurol. 24, 224–229
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Review 79 Qi, X. et al. (2007) Suppression of mitochondrial oxidative stress provides long-term neuroprotection in experimental optic neuritis. Invest. Ophthalmol. Vis. Sci. 48, 681–691 80 Forte, M. et al. (2007) Cyclophilin D inactivation protects axons in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Proc. Natl. Acad. Sci. U.S.A. 104, 7558–7563 81 Su, K.G. et al. (2012) Genetic inactivation of the p66 isoform of ShcA is neuroprotective in a murine model of multiple sclerosis. Eur. J. Neurosci. 35, 562–571 82 Davies, A.L. et al. (2013) Neurological deficits caused by tissue hypoxia in neuroinflammatory disease. Ann. Neurol. http://dx.doi.org/10.1002/ ana.24006
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83 Mao, P. et al. (2013) MitoQ, a mitochondria-targeted antioxidant, delays disease progression and alleviates pathogenesis in an experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. Biochim. Biophys. Acta 1832, 2322–2331 84 Hondares, E. et al. (2006) Thiazolidinediones and rexinoids induce peroxisome proliferator-activated receptor-coactivator (PGC)-1a gene transcription: an autoregulatory loop controls PGC-1a expression in adipocytes via peroxisome proliferator-activated receptor-gamma coactivation. Endocrinology 147, 2829–2838 85 Jin, F. et al. (2008) Neuroprotective effect of resveratrol on 6-OHDAinduced Parkinson’s disease in rats. Eur. J. Pharmacol. 600, 78–82 86 Shindler, K.S. et al. (2010) Oral resveratrol reduces neuronal damage in a model of multiple sclerosis. J. Neuroophthalmol. 30, 328–339
9
Review
Mitochondrial dysfunction contributes to neurodegeneration in multiple sclerosis Maarten E. Witte1*, Don J. Mahad2*, Hans Lassmann3*, and Jack van Horssen1* 1
Department of Molecular Cell Biology and Immunology, VU University Medical Center, 1081BT, Amsterdam, The Netherlands Centre for Neuroregeneration, University of Edinburgh, Edinburgh, UK 3 Center for Brain Research, Medical University of Vienna, A-1090 Vienna, Austria 2
Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Current treatments are very effective in reducing the neuroinflammatory attack, but fail to significantly halt disease progression and associated loss of neuronal tissue. In recent years, it has become increasingly clear that dysfunctional mitochondria are important contributors to damage and loss of both axons and neurons. Observations in animal and histopathological studies suggest that infiltrating leukocytes and activated microglia play a central role in neuronal mitochondrial dysfunction. This review provides a comprehensive overview on the current knowledge regarding mitochondrial dysfunction in MS. Importantly, more insight into the cause and consequences of impaired mitochondrial function provide a basis for mitochondrial-targeted medicine to combat progressive MS. Multiple sclerosis MS is a chronic inflammatory disease of the central nervous system (CNS) and the most common cause of neurological disability in young adults, affecting over 2.5 million people worldwide [1]. First symptoms generally appear in early adulthood (20–40 years of age), including sensory disturbances, optic neuritis, and limb weakness, and approximately half of these patients need help walking within 15 years. As the disease progresses additional symptoms occur, such as fatigue, bladder dysfunction, and cognitive impairment. Depending on progression of clinical symptoms, three distinct MS subtypes can be distinguished. In relapsing-remitting (RR) MS (see Glossary), present in 80% of cases, patients suffer from clearly defined periods of neurological deficits followed by (partial) recovery [1]. In 65% of patients suffering from RRMS, the disease course gradually evolves into secondary progressive (SP) MS, characterised by progressive permanent neurological Corresponding author: Witte, M.E. ([email protected]). Keywords: multiple sclerosis; mitochondria; neurodegeneration; neuroinflammation; oxidative stress. * All authors contributed equally. 1471-4914/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molmed.2013.11.007
Glossary Astrocytes: the most prevalent type of neuroglia in the CNS. They have multiple crucial functions including providing trophic support to other CNS cells, regulating neuronal activity, and guarding CNS homeostasis. Demyelination: process of oligodendrocyte and myelin loss. Experimental autoimmune encephalomyelitis (EAE): a widely used animal model for MS, in which T cell mediated autoimmunity against myelin is experimentally induced. Grey matter: CNS tissue can be divided into white matter and grey matter; grey matter contains virtually all neuronal cells and less myelin than white matter. Inflammation: part of a complex biological response to harmful stimuli. Leber’s hereditary optic neuropathy (LHON): inherited disease, in which specific mutations in mitochondrial DNA cause degeneration of retinal ganglion cells and their axons. Microglia: the resident immune cells of the CNS and closely related to macrophages. Mitochondria: organelles responsible for oxidative energy metabolism in eukaryotic cells. They are also crucial in maintaining Ca2+ homeostasis and regulation of programmed cell death. N-Acetyl-aspartate (NAA): CNS specific metabolite that is only produced in neuronal mitochondria. Therefore, reduced NAA concentration as measured with magnetic resonance spectroscopy is considered to reflect loss of neuronal tissue. Neurodegeneration: in the broadest sense, neurodegeneration is the process of CNS tissue loss. Often the term neurodegeneration is used just for loss of neuronal tissue. Neurons: non-dividing cells that are electrically excitable and process and transmit information via electrochemical signals. Neurons can transmit signals both to other neurons and different effector cells (e.g., muscle cells). Oligodendrocytes: a population of glial cells that form the myelin sheaths that encapsulate axons, thereby facilitating fast saltatory conduction. Oxidative phosphorylation chain (OxPhos): five multi-subunit protein complexes located on the inner mitochondrial membrane, which together produce ATP through oxidation of nutrients. Oxidative stress: state reflecting an imbalance between the production and detoxification of ROS leading to cellular damage. Peroxisome proliferator-activated receptor gamma coactivator-1a (PGC-1a): transcriptional cofactor that can activate multiple transcription factors involved in energy metabolism. Therefore, PGC-1a is often considered the main regulator of energy metabolism. Primary progressive (PP) MS: MS subtype present in 20% of MS patients and characterised by a progressive disease course from onset. Reactive nitrogen species: highly reactive nitric oxide-derived molecules, which have important signalling functions but can also cause severe damage to various macromolecules and cells. Reactive oxygen species (ROS): chemically reactive molecules containing oxygen, which have important signalling functions but can also cause severe damage to various macromolecules and cells. Relapsing-remitting (RR) MS: MS subtype present in 80% of cases characterised by clearly defined periods of neurological deficits followed by (partial) recovery. Secondary progressive (SP) MS: MS subtype that follows after RRMS in 65% of cases and is characterised by progressive neurological symptoms. White matter: is one of the two components of the CNS and is predominantly composed of glial cells and myelinated axons.
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Review deficits. Furthermore, 20% of all MS patients have a progressive disease course from onset, known as primary progressive (PP) MS. It is known that both genetic and environmental factors contribute substantially to disease susceptibility; however, despite extensive research, the primary cause(s) and initial triggers of MS are still largely enigmatic. Thus far, only a few genes have been consistently shown to influence disease susceptibility, and those with the strongest effect are located on chromosome 6 and are part of the major histocompatibility complex class II (MHC II), namely HLA-DR15 and HLA-DQ6 [2]. It is now well documented that environmental factors, such as smoking, vitamin D status, and viral infections, have been linked to the occurrence of MS [3,4]. However, definitive proof of a pathogenic role for these factors is lacking. Histopathological analysis of MS brain autopsy material reveals focal demyelinated lesions scattered throughout the white and grey matter of the brain and spinal cord. The inflammatory demyelination observed in MS patients is generally believed to be initiated by autoreactive T cells activated in the periphery [5]. However, current immunomodulatory drugs are effective in reducing the number of inflammation-driven relapses during RRMS but fail to halt disease progression in progressive MS patients [6], which is primarily the result of neurodegenerative processes. Still, strong evidence exists that in progressive MS neurodegeneration is caused by inflammatory processes [7,8]. These processes, however, occur, for a large part, behind a closed blood–brain barrier, thereby explaining the failure of current therapeutics to slow down disease in the progressive phase [6]. This ineffectiveness of current MS therapeutics popularised a new concept of MS pathogenesis, the so-called inside-out hypothesis (Box 1) [9–11]. Either way, novel Box 1. Inside-out versus outside-in theories Histopathological studies evidently point to a pivotal role of both the innate and adaptive immune system in the pathogenesis of MS. In addition, genome-wide association studies showed a preponderance of immune-related genes associated with increased disease susceptibility, and current immunomodulatory drugs are highly effective in a majority of RRMS patients. Therefore, MS is generally considered a primary immune-driven disease in which autoreactive T cells and macrophages invade the CNS where they, together with activated microglia, attack myelin and/or oligodendrocytes (known as the ‘outside-in model’). In this model, neurodegeneration occurs both as a bystander effect of the inflammatory attack and as a consequence of the loss of myelin trophic support. This concept is now being challenged by those who argue that inflammatory processes in MS brains are secondary to a primary cytodegenerative process in resident CNS cells (e.g., oligodendrocytes and neurons/ axons) or alterations in the myelin–axon interaction (‘inside-out model’). Intriguingly, axonal alterations can be widespread in NAWM and the cortex without overt signs of infiltrating leukocytes. Another argument for the inside-out model is the ineffectiveness of the current anti-inflammatory therapies in progressive MS patients, where neurodegeneration drives disease progression. Nevertheless, throughout the course of MS active tissue injury remains strongly associated with the degree of inflammation. In NAWM and cortical lesions, where infiltrating immune cells are scarcely found, neurodegeneration is still ongoing and is associated with microglial activation. Because this process occurs behind a closed or repaired blood–brain barrier, microglial activation is out-of-reach for current immunomodulatory drugs. Although at this stage it is unclear which of the two opposing theories is correct, current data still appear to favour the outside-in model. 2
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therapeutics that effectively target the progressive phase of MS are needed, and therefore more insight into the mechanisms leading to neurodegeneration is essential. Evidence is now emerging that mitochondrial damage is an important feature of progressive MS and these studies strongly suggest that mitochondrial alterations contribute to neurodegenerative processes in MS [12]. In this review, we will systematically dissect the current literature regarding mitochondrial dysfunction in MS and address the potential of mitochondria as a therapeutic target in progressive MS. Lastly, we will point to important caveats in our knowledge and discuss future directions. Mitochondria and MS susceptibility Mitochondria are present in all eukaryotic cells and their prime function is to provide cells with energy, in the form of adenosine triphosphate (ATP), by oxidation of metabolic fuels. In addition, they are involved in many other important cellular processes, including fatty acid oxidation, apoptosis, and calcium homeostasis. Mitochondria are the sole carriers of non-nuclear and circular DNA in eukaryotic cells, which is a footprint of their bacterial origin. Mitochondrial DNA (mtDNA) encode 22 transfer RNAs, two ribosomal RNAs, and 13 essential proteins of the oxidative phosphorylation chain (OxPhos), the complex intramitochondrial machinery responsible for oxidative metabolism [13]. Mitochondria also constantly produce reactive oxygen species (ROS), which have important signalling functions. However, when the rate of ROS production exceeds the cellular antioxidant capacity it can cause oxidative stress and extensive damage to essential cell components [14]. The high energy requirements of the CNS make it particularly vulnerable to defects in mitochondrial function, as illustrated by the long list of neurological disorders caused by inherited genetic alterations in mtDNA and in nuclear genes encoding key mitochondrial proteins [15]. Importantly, extensive mitochondrial dysfunction and concomitant oxidative stress are key features of common neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease [16]. Interest in mitochondria in the context of MS was sparked by the finding that patients suffering from Leber’s hereditary optic neuropathy (LHON), which can be caused by mutations in mtDNA [17], have an increased risk of developing an MSlike disease, a condition known as Harding’s disease [18]. As a result, many studies have investigated the influence of mtDNA mutation on MS susceptibility (extensively reviewed in [19]). Most of these studies have focussed specifically on LHON-associated mutations; however, the results are rather contradictory. Various studies confirmed a weak association between secondary LHON mtDNA mutations and MS susceptibility, whereas other studies failed to find such an association. These differences might be partly explained by the very small sample size in most studies and differences in the studied population. Interestingly, several studies indicate that the occurrence of severe optic neuritis in a subset of MS patients is indeed linked to mtDNA mutations. This could be due to the association of both MS and LHON to a certain mtDNA haplotype (a distinct set of mtDNA polymorphisms) [20].
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Review To our knowledge there is currently only one polymorphism in nuclear DNA encoding a mitochondrial protein that has been linked to MS susceptibility. This polymorphism, associated with MS in two independent cohorts of German patients [21], was found in the promoter region of uncoupling protein 2 (UCP2), a mitochondrial protein that is able to separate the mitochondrial membrane potential from ATP synthesis and thereby decrease ATP and ROS production. Because whole-genome sequencing is rapidly becoming less expensive, it is conceivable that other polymorphisms in nuclear DNA coding for mitochondrial proteins will be identified in the future. However, just like the polymorphism in UCP2, they are likely to only marginally contribute to MS susceptibility. Mitochondrial involvement in white matter damage White matter demyelination is considered the pathological hallmark of MS and white matter lesions are generally classified according to demyelination and inflammation status [22]. Lesions with ongoing demyelination by infiltrated macrophages and activated microglia are called active demyelinating lesions, which are accompanied by extensive oxidative damage [23–25]. Active demyelinating lesions are believed to gradually evolve into chronic active, slowly expanding lesions. Chronic active lesions are characterised by a completely demyelinated centre with ongoing inflammatory demyelination at the lesion edge. Eventually, inflammation resolves and the demyelinated area becomes known as a chronic inactive lesion. A substantial proportion of demyelinated lesions are remyelinated over the course of the disease [26], whereas nonremyelinated areas are filled with a gliotic scar. White matter lesions are surrounded by normal-appearing white matter (NAWM), which is defined as non-lesional regions devoid of marked changes in myelin protein expression. However, NAWM can be a deceptive term because activated microglia and axonal degeneration are frequently observed in these ‘normally’ myelinated areas. Besides damage to myelin and oligodendrocytes, the myelin-forming cells of the CNS, there is also extensive damage to axons in MS white matter [27] and the extent of axonal degeneration has been linked to permanent neurological disability in progressive MS patients [28,29]. The evidence presented here indicates an important role for mitochondrial dysfunction in driving axonal degeneration in MS white matter. White matter tissue injury in MS is most prominent in the foci of inflammatory demyelination where the extent of axon degeneration correlates with the severity of inflammation. In a subtype of acute MS lesions, the so-called pattern III or hypoxia-like lesions [30], diffuse mitochondrial respiratory chain defects are seen in axons, oligodendrocytes, and astrocytes [31]. Moreover, a recent study of inflammatory lesions in experimental autoimmune encephalomyelitis (EAE) (Box 2), a widely used MS animal model, identified acute mitochondrial injury as a critical step towards focal axonal degeneration [32]. Similar changes were also observed in postmortem MS tissue, suggesting that similar mitochondrial processes also play a role in active MS lesions. ROS and reactive nitrogen species (RNS), generated by activated microglia and
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Box 2. Experimental autoimmune encephalomyelitis: a valuable animal model of MS? Over a hundred years ago Pasteur developed a vaccine to treat rabies, a zoonotic, often fatal, disease. The vaccines were prepared by collecting spinal cord homogenates of rabbits infected with rabies virus. Although the vaccine was effective in many cases, some patients developed signs of paralysis. In the most severe cases patients died, and histopathological evaluation of the brain tissue revealed perivascular leukocyte infiltrates and demyelinated lesions. It is now well known that immunisation of susceptible animals, usually mice and rats, with either CNS homogenates or myelin proteins/peptides induces an autoimmune demyelinating disease called experimental autoimmune encephalomyelitis (EAE). Furthermore, EAE can be induced by adopted transfer of CD4+ T cells specific for myelin antigens. To date, EAE is still the most commonly used experimental animal model of MS. Based on the strain and antigens used, animals develop a variety of neurological symptoms ranging from a monophasic clinical episode of paralysis (acute EAE) to chronic-relapsing neurological periods and progressive disability (chronic EAE). In general, certain pathological key features of RRMS, such as glial cell activation, leukocyte infiltration, and axonal damage occur in most EAE models. However, inflammatory demyelination is mainly observed in chronic EAE models, although not extensively. The EAE model has been instrumental in providing important contributions to our understanding of key processes underlying neuroinflammation, including the process of leukocyte migration across the blood–brain barrier into the CNS. This has led to the development of effective anti-inflammatory and immunomodulatory therapies, of which some are currently successfully used in the clinic. Unfortunately, the main drawbacks of the current EAE models, even the chronic model, are that they do not accurately reproduce key aspects of progressive MS, such as cortical demyelination and neuronal loss.
macrophages through oxidative burst, are the most likely culprits of intra-axonal mitochondrial injury, because they are known to directly inhibit the respiratory chain (Figure 1A) [33,34]. Indeed, detoxification of ROS reversed mitochondrial pathology and rescued axons from degeneration in EAE [32]. Remarkably, even before influx of leukocytes, nitration of mitochondrial proteins has been observed in intact axons of EAE animals [35]. Conceivably, activation of microglia, which occurs rapidly after EAE induction when leukocyte infiltration is only present in meninges but not in the CNS parenchyme, induces these early mitochondrial alterations. This may explain why diffuse axonal loss in the spinal cord white matter associates with meningeal T cell infiltration [36]. Still, most axons survive the inflammatory demyelinating attack and become chronically demyelinated. Over time a proportion of these axons degenerate. Whereas degenerating chronically demyelinated axons in progressive MS cases contained injured mitochondria, over half of the intact and functional chronically demyelinated axons showed the opposite [37]. These demyelinated and otherwise uninjured axons contained mitochondria with increased size and respiratory chain enzyme activity [37,38]. Furthermore, uninjured and demyelinated axons in MS increased expression of an axon specific mitochondrial docking protein, syntaphilin. In vitro demyelination caused the motile mitochondria within axons to move in a retrograde and anterograde manner with greater speed [39]. This axonal mitochondrial response to demyelination, namely (i) increased mitochondrial presence, (ii) respiratory chain activity, and (iii) speed of motile mitochondria 3
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Figure 1. Mitochondrial dysfunction mediates axonal degeneration in multiple sclerosis (MS). (A) In inflammatory MS lesions, macrophages and activated microglia produce vast amounts of reactive oxygen species (ROS) and nitric oxide (NO), which subsequently induce mitochondrial dysfunction in both myelinated and demyelinated axons. In early stages, axonal mitochondrial dysfunction is reflected by swelling of intra-axonal mitochondria in intact axons. A substantial proportion of axons with swollen mitochondria will eventually degenerate. (B) Axons in chronic MS lesions have increased and redistributed their axolemmal Na+ channels, leading to an increased intra-axonal Na+ concentration. As a result, more ATP is required for the removal of excess Na+ by Na+/K+ ATPase (I). To meet the higher energy demand, mitochondrial content is increased in chronically demyelinated axons. However, due to cortical pathology or previous inflammatory damage, a proportion of mitochondria in chronically demyelinated axons are damaged (II). Over time, mitochondrial dysfunction accumulates to such an extent that ATP production becomes too low for Na+/K+ ATPase to remove excess intra-axonal Na+ (III). The rising Na+ concentrations inside axons lead to reversal of the axolemmal Na+/Ca2+ exchanger, which now pumps Na+ out and Ca2+ into the axon (IV). In turn, this leads to rising axonal Ca2+ concentrations, which will eventually trigger a set of several deleterious events, including further destabilisation of intra-axonal mitochondria and increased ROS production, which then contribute to axonal degeneration (V).
within demyelinated axons, has been consistently in disease models of demyelination and dysmyelination, and it therefore appears to be an adaptive or compensatory phenomenon to the disturbance of myelin. In MS, the absence of myelin in otherwise intact axons alters the distribution of ion channels, in particular sodium channels, which supposedly causes an increased intra-axonal energy demand also known as ‘virtual hypoxia’ [40], which in turn leads to the observed changes in mitochondria. However, although markedly less than in inflammatory MS lesions, axons still degenerate in chronic MS lesions, and this is thought to be caused by the eventual failure of intra-axonal mitochondria to fulfil the need of axons for ATP (Figure 1B) [34,41]. The grey matter mitochondrial abnormalities located within neuronal cell bodies (soma) in MS, and 4
discussed below, raise the interesting possibility that axon degeneration in progressive MS may also be due to the inability of the axon to mount and maintain the mitochondrial response to demyelination as a result of the inability of the neuronal soma to supply the axon with healthy mitochondria. Alternatively, mitochondrial damage sustained previously during active white matter inflammation and/or ongoing mild inflammation is also likely to contribute to the eventual failure of intra-axonal mitochondria to support chronically demyelinated axons. Interestingly, imaging of newly forming lesions in the spinal cord of MS patients revealed similar mitochondrial alterations as found in postmortem and animal studies. In newly forming inflammatory MS lesions, the concentration of N-acetyl-aspartate (NAA) decreased significantly,
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Review reflecting decreased neuronal mitochondrial function as seen in active MS lesions. As the inflammation cleared over time, NAA concentration again increased reflecting increased mitochondrial presence and activity in chronically demyelinated MS lesions [41,42]. However, because NAA contributes to myelin formation by oligodendrocytes, an increase in NAA might also reflect remyelination. Currently, studies looking into mitochondrial changes in NAWM and preactive lesions are lacking. Those studies are warranted to elucidate if mitochondria already react to the subtle changes occurring in NAWM and/or contribute to lesion initiation in preactive lesions. Intriguingly, nitration of mitochondrial proteins has been observed as early as 3 days after sensitisation in EAE, suggesting that mitochondrial dysfunction precedes leukocyte infiltration within the CNS parenchyme [35]. Unfortunately, activation of microglia, which also occurs before influx of leukocytes in EAE [43], was not assessed and therefore the likely relation between mitochondrial protein nitration and microglial activation remains unresolved. Mitochondrial involvement in grey matter damage Although damage to grey matter structures in MS has long been overlooked, it occurs early in disease and is extensive in many MS patients [44–46]. Cortical demyelination can reach up to 75% of the total cortex and even exceeds the extent of white matter demyelination in a subset of patients [47,48]. Postmortem studies revealed that cortical lesions differ markedly from white matter lesions. Grey matter lesions generally lack blood–brain barrier alterations and abundant infiltration of blood-borne leukocytes [49–51]. However, a recent study analysing biopsy material from patients with early fulminant MS reported that a large proportion of grey matter lesions in these patients contained prominent leukocyte infiltration [52]. It remains to be determined if this is specific for early fulminant MS, or also occurs in the more common forms of early MS. Besides demyelination, neurodegeneration is also widespread in the MS cortex. Imaging studies revealed that cortical atrophy is detectable early in disease, but increases strikingly in the progressive stage of the disease [45,53,54]. Several studies have now established cortical atrophy as a correlate of clinical disability, emphasising the clinical relevance of cortical pathology in MS [55,56]. It was shown that cortical thinning is the combined result of glial cell loss, neuronal loss, and reduced synaptic density, which were most prevalent in cortical lesions [57]. However, neuronal injury and loss can also be extensive in normal-appearing grey matter (NAGM) [8,58]. Thus, neurodegeneration occurs, at least partly, independent of local demyelination. Other processes proposed to contribute to neurodegeneration are (distant) axonal damage, local meningeal inflammation [8,59], the presence of persistent microglial activation [23,51,60], and mitochondrial dysfunction [34]. Microarray analysis of the postmortem non-demyelinated motor cortex from MS patients revealed decreased gene expression of 26 nuclear-encoded subunits of the OxPhos chain [61], which coincided with a significant reduction in activity of OxPhos complexes I and III. In situ hybridisation showed that this decrease occurs
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specifically in cortical neurons. Later, it was observed that the MS cerebral cortex contained neurons devoid of complex IV activity [62]. These so-called respiratory deficient neurons were present in both NAGM and cortical lesions, and harboured high levels of clonally expanded mtDNA deletions [62]. Damage to mtDNA and reduced activity of the OxPhos complexes may be initiated by oxidative and nitrosative stress. Furthermore, damage to neuronal mtDNA in MS also contributes to reduced activities of OxPhos complexes I, III, and IV, because mtDNA codes for essential subunits of these OxPhos complexes. However, it does not explain the reduction of various nuclear-encoded OxPhos subunits in the MS cortex [61]. Expression of key OxPhos genes is tightly coordinated by a number of transcription factors, including nuclear respiratory factor 1, -2, oestrogen-related receptor a and peroxisome proliferator-activated receptors (PPARs) (for an extensive review, see [63]). A decrease in a transcription factor complex containing nuclear respiratory factor 2 (NRF-2) in MS grey matter samples has been observed, which correlated with reduced expression of OxPhos genes and increased oxidative damage [64]. Recently, a significant reduction of peroxisome proliferator-activated receptor gamma coactivator-1a (PGC-1a) in NAGM samples was identified in progressive MS cases [65]. PGC-1a is a transcriptional cofactor that binds and activates nuclear transcription factors involved in mitochondrial function. PGC1a expression was reduced in cortical neurons of MS patients and correlated with local neuronal density, indicating a possible role in neurodegeneration [65]. Knockdown of neuronal PGC-1a induced a decrease in nuclearencoded OxPhos subunits, as observed in the MS cortex, and increased neuronal ROS production, thereby further enhancing the already existing oxidative stress. In addition, reduced PGC-1a resulted in decreased expression of mitochondrial antioxidants [65]. Because ROS and nitric oxide production by microglia can be extensive in the cortex of MS patients [66], we propose a mechanism where reduced neuronal PGC-1a directly induces mitochondrial dysfunction by decreased transcription of key mitochondrial proteins and augments mitochondrial susceptibility to oxidative/nitrosative damage (Figure 2). Importantly, the presence of mtDNA damage and nitrotyrosine residues in mitochondrial proteins strongly indicate increased oxidative and nitrosative damage to mitochondria in the MS cortex [61,67]. Over time, damage to mitochondria will further impair neuronal mitochondrial function in the MS cortex, ultimately leading to neuronal death (Figure 2). A recent study linked neuronal mitochondrial dysfunction in the MS cortex to local failure of myelination by oligodendrocytes via reduced NAA production, further emphasising the importance of mitochondrial dysfunction in MS pathology [68]. Currently, it is unclear what causes the decrease in neuronal PGC-1a expression in the MS cortex. Intriguingly, PGC-1a levels are also markedly reduced in ‘classic’ neurodegenerative disorders including Huntington’s disease, Parkinson’s disease, and Alzheimer’s disease [69– 73]. Extensive microglial activation is an early and persistent phenomenon in these neurodegenerative diseases [74], and it is therefore conceivable that inflammatory 5
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Figure 2. Mitochondrial dysfunction mediates neurodegeneration in the multiple sclerosis (MS) cortex. In the MS cortex, two distinct but interrelated pathways contribute to neuronal mitochondrial dysfunction. One mechanism is by production of reactive oxygen species (ROS) and nitric oxide (NO) by continuously activated microglia in the MS cortex (I). This will lead to inhibition of several oxidative phosphorylation (OxPhos) complexes (a, inset) and damage to mitochondrial DNA (mtDNA) in neuronal mitochondria (b, inset). Damage to mtDNA will further impair OxPhos function leading to reduced ATP production (c, inset). Secondly, decreased expression of neuronal PGC-1a, a transcriptional coactivator, leads to reduced transcription of OxPhos subunits and mitochondrial antioxidants (II). We propose that chronic microglial activation present in the MS cortex eventually leads to reduced neuronal PGC-1a expression. As a consequence, neuronal mitochondria have a reduced capacity to produce ATP (d, inset) and detoxify intramitochondrial ROS (e, inset; O2, superoxide), which can eventually fully compromise mitochondrial function and lead to neuronal cell death. Importantly, dysfunctional mitochondria in the neuronal soma could also have deleterious effects on (demyelinated) axons, because the neuronal cell body cannot supply its axon with sufficient healthy mitochondria (f).
factors produced by microglia mediate reduced neuronal PGC-1a expression. It should be noted that inflammatory factors present in the MS cortex, such as tumour necrosis factor a (TNFa), are also likely to alter neuronal mitochondrial function via other mechanisms. Although there is currently no experimental evidence for such a mechanism in MS, it has been shown in other settings that TNFa can alter neuronal mitochondrial distribution and induce mitochondria-mediated apoptosis via TNF receptor-associated death domains (TRADDs) [75,76]. Concluding remarks and future perspectives Over the past few years interest in the role of mitochondria in MS pathogenesis has been steadily increasing, and the importance of mitochondrial dysfunction is now widely 6
acknowledged [40,77,78]. We propose that mitochondrial dysfunction contributes to neurodegeneration in MS via three distinct but interrelated mechanisms: (i) in acute, inflammatory MS lesions, intra-axonal mitochondria are damaged by inflammation-derived ROS and nitric oxide, leading to severe mitochondrial dysfunction and subsequent axonal injury (Figure 1A); (ii) in chronically demyelinated axons, energy demand is increased several-fold compared with normally myelinated axons, and at a certain point, intra-axonal mitochondria cannot meet the energy demand due to accumulating mitochondrial dysfunction, setting off a cascade of deleterious events leading to axonal degeneration and loss (Figure 1B); and (iii) high levels of mtDNA mutation, reduced neuronal expression of PGC-1a, and increased nitric oxide and ROS production by
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Review Box 3. Outstanding questions In EAE, mitochondrial alterations precede the influx of leukocytes into the brain parenchyma. In MS, do mitochondrial alterations already occur in NAWM? Is mitochondrial dysfunction in neurons directly related to local inflammatory events, such as cortical microglial activation or meningeal inflammation? Which molecular pathways are responsible for reduced neuronal PGC-1a expression and subsequent mitochondrial dysfunction in the MS cortex? Are treatments aimed at improving or protecting neuronal/axonal mitochondrial function able to reduce neurodegeneration and thereby slowing down disease progression in MS patients?
activated cortical microglia synergistically induce neuronal mitochondrial dysfunction, driving the neurodegenerative process in the MS cortex (Figure 2). Although there is some evidence for an important role of mitochondria in different stages of MS, much remains to be uncovered (Box 3). First, a thorough study of mitochondrial function in NAWM is warranted to elucidate if, in analogy to EAE [35], mitochondrial alterations precede the influx of leukocytes into the lesions themselves. Moreover, levels of microglial inflammation in NAGM can differ greatly between and in patients. A correlation between the extent of microglial activation and mitochondrial alterations, for example, the extent of mtDNA damage, and PGC-1a expression in NAGM might shed light on the causative role of activated microglia in mediating neuronal mitochondrial injury. Also, a better understanding of the molecular pathways leading to mitochondrial dysfunction could open up new therapeutic targets to combat neurodegeneration in MS. However, the lack of a suitable animal model for progressive MS presents a major hurdle [6]. Most MS animal models accurately mimic parts of the inflammatory reaction seen in patients, but fail to reproduce important aspects of progressive MS, such as cortical neurodegeneration. Development of an animal model that more accurately reflects progressive MS is needed to fully comprehend the role of mitochondria in MS disease progression and test the potential of novel therapeutic strategies aimed at improving the mitochondrial metabolism. Current approved treatments for MS are all immunomodulatory or anti-inflammatory, and have little or no effect on progressive MS, where neuroinflammation is partly compartmentalised behind a closed blood–brain barrier, and therefore inaccessible for many therapeutics, and neurodegeneration drives clinical deterioration [6]. Thus, there is a great need for therapeutics that combat neurodegeneration, and several studies have now shown that mitochondria provide us with a potential therapeutic target. For instance, gene delivery of superoxide dismutase 2, which scavenges superoxide in mitochondria, preserved axonal integrity in EAE optic nerves [79]. Moreover, knockout of cyclophilin D and p66ShcA, both important regulators of the mitochondrial permeability transition pore and consequently mitochondria-mediated cell death, significantly reduced axonal damage in animals suffering from EAE [80,81]. This is believed to be the result of increased resistance to oxidative and nitrosative stress of axonal mitochondria and an increased
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ability of axonal mitochondria to sequester Ca2+. Very recently, two studies reported protective properties of MitoQ, a synthetic antioxidant that specifically accumulates in mitochondria, in two different EAE models [82,83]. Because levels of inflammation were not altered by MitoQ, it is conceivable that increased mitochondrial protection against ROS is sufficient to reduce axonal damage. Therefore, more insight into the inflammatory factors that drive ROS and nitric oxide production by macrophages/microglia will also provide interesting targets for intervention. Another potentially interesting therapeutic candidate is PGC-1a, because boosting its expression and/or activity will have a broad range of beneficial effects on mitochondrial function and protection. Thus far, several compounds have been shown to enhance PGC-1a activity and thereby increase neuronal survival in animal models for various neurodegenerative disorders, including EAE [84–86]. However, these compounds, including resveratrol and thialozidines, have many other targets and therefore the specific effects of increased PGC-1a activity is difficult to establish. Taken together, the current literature strongly suggests that therapeutic strategies aimed at improving mitochondrial function in MS are worthwhile to pursue but warrant additional research. References 1 Compston, A. and Coles, A. (2008) Multiple sclerosis. Lancet 372, 1502– 1517 2 Hauser, S.L. and Oksenberg, J.R. (2006) The neurobiology of multiple sclerosis: genes, inflammation, and neurodegeneration. Neuron 52, 61–76 3 DeLorenze, G.N. et al. (2006) Epstein–Barr virus and multiple sclerosis: evidence of association from a prospective study with longterm follow-up. Arch. Neurol. 63, 839–844 4 Marrie, R.A. (2004) Environmental risk factors in multiple sclerosis aetiology. Lancet Neurol. 3, 709–718 5 Steinman, L. et al. (2002) Multiple sclerosis: deeper understanding of its pathogenesis reveals new targets for therapy. Annu. Rev. Neurosci. 25, 491–505 6 Lassmann, H. et al. (2012) Progressive multiple sclerosis: pathology and pathogenesis. Nat. Rev. Neurol. 8, 647–656 7 Frischer, J.M. et al. (2009) The relation between inflammation and neurodegeneration in multiple sclerosis brains. Brain 132, 1175–1189 8 Magliozzi, R. et al. (2010) A gradient of neuronal loss and meningeal inflammation in multiple sclerosis. Ann. Neurol. 68, 477–493 9 Geurts, J.J. et al. (2010) Multiple sclerosis as an ‘‘inside-out’’ disease. Ann. Neurol. 68, 767–768 10 Trapp, B.D. and Nave, K.A. (2008) Multiple sclerosis: an immune or neurodegenerative disorder? Annu. Rev. Neurosci. 31, 247–269 11 Stys, P.K. et al. (2012) Will the real multiple sclerosis please stand up? Nat. Rev. Neurosci. 13, 507–514 12 Su, K.G. et al. (2009) Axonal degeneration in multiple sclerosis: the mitochondrial hypothesis. Curr. Neurol. Neurosci. Rep. 9, 411–417 13 Wallace, D.C. (1992) Diseases of the mitochondrial DNA. Annu. Rev. Biochem. 61, 1175–1212 14 Lin, M.T. and Beal, M.F. (2006) Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443, 787–795 15 DiMauro, S. et al. (2013) The clinical maze of mitochondrial neurology. Nat. Rev. Neurol. 9, 429–444 16 Beal, M.F. (1995) Aging, energy, and oxidative stress in neurodegenerative diseases. Ann. Neurol. 38, 357–366 17 Wallace, D.C. et al. (1988) Mitochondrial DNA mutation associated with Leber’s hereditary optic neuropathy. Science 242, 1427–1430 18 Olsen, N.K. et al. (1995) Leber’s hereditary optic neuropathy associated with a disorder indistinguishable from multiple sclerosis in a male harbouring the mitochondrial DNA 11778 mutation. Acta Neurol. Scand. 91, 326–329 7
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Review 79 Qi, X. et al. (2007) Suppression of mitochondrial oxidative stress provides long-term neuroprotection in experimental optic neuritis. Invest. Ophthalmol. Vis. Sci. 48, 681–691 80 Forte, M. et al. (2007) Cyclophilin D inactivation protects axons in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Proc. Natl. Acad. Sci. U.S.A. 104, 7558–7563 81 Su, K.G. et al. (2012) Genetic inactivation of the p66 isoform of ShcA is neuroprotective in a murine model of multiple sclerosis. Eur. J. Neurosci. 35, 562–571 82 Davies, A.L. et al. (2013) Neurological deficits caused by tissue hypoxia in neuroinflammatory disease. Ann. Neurol. http://dx.doi.org/10.1002/ ana.24006
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83 Mao, P. et al. (2013) MitoQ, a mitochondria-targeted antioxidant, delays disease progression and alleviates pathogenesis in an experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. Biochim. Biophys. Acta 1832, 2322–2331 84 Hondares, E. et al. (2006) Thiazolidinediones and rexinoids induce peroxisome proliferator-activated receptor-coactivator (PGC)-1a gene transcription: an autoregulatory loop controls PGC-1a expression in adipocytes via peroxisome proliferator-activated receptor-gamma coactivation. Endocrinology 147, 2829–2838 85 Jin, F. et al. (2008) Neuroprotective effect of resveratrol on 6-OHDAinduced Parkinson’s disease in rats. Eur. J. Pharmacol. 600, 78–82 86 Shindler, K.S. et al. (2010) Oral resveratrol reduces neuronal damage in a model of multiple sclerosis. J. Neuroophthalmol. 30, 328–339
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