Scholarly article on topic 'The genetics and pathology of mitochondrial disease'

The genetics and pathology of mitochondrial disease Academic research paper on "Biological sciences"

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Academic research paper on topic "The genetics and pathology of mitochondrial disease"

The genetics and pathology of mitochondrial disease

Charlotte L. Alston1, Mariana C. Rocha1, Nichola Z. Lax1, Doug M. Turnbull1 and

Robert W. Taylor11

1 Wellcome Trust Centre for Mitochondrial Research, Institute of Neuroscience, The Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK

<D O O

Correspondence to:

Professor Robert Taylor Wellcome Trust Centre for Mitochondrial Research Institute of Neuroscience The Medical School Newcastle University

Framlington Place Newcastle upon Tyne NE2 4HH UK

Phone: +44-191-2824375 Fax: +44-191-2824373 Email: robert.taylor@ncl.ac.uk

Abstract

itochondria are double membrane-bound organelles that are present in all nucleated eukaryotic cells and responsible for the production of cellular energy in the form of ATP. Mitochondrial function is under dual genetic control - the 16.6 kb mitochondrial genome encoding just 37 genes with the remaining ~1300 proteins of the mitoproteome encoded by nuclear genes. Mitochondrial dysfunction can arise due to defects in either mtDNA or nuclear mitochondrial genes, and can present in childhood or adulthood in association with vast clinical heterogeneity with symptoms affecting a single organ or tissue, or multisystem

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/path.4809_

involvement. There is no cure for mitochondrial disease for the vast majority of

itochondrial patients and a genetic diagnosis is therefore crucial for genetic counselling, recurrence risk calculation and it can modulate the clinical management of affected patients. ext-generation sequencing strategies are proving pivotal in the discovery of new disease enes and the diagnosis of clinically-affected patients; mutations in more than 250 genes have now been shown to cause mitochondrial disease and the biochemical, histochemical, mmunocytochemical and neuropathological characterisation of these patients has led to improved diagnostic testing strategies and novel diagnostic techniques. This review focuses on the current genetic landscape associated with mitochondrial disease before focussing on advances in studying associated mitochondrial pathology in two, clinically-relevant organs -skeletal muscle and brain.

Keywords: mitochondria, mitochondrial disease, mtDNA, respiratory chain deficiency, genetic diagnosis, muscle pathology, immunohistochemistry, neuropathology

ntroduction

itochondria are double membrane-bound organelles, present in all nucleated eukaryotic cells, responsible for numerous cellular processes including calcium homeostasis, iron-sulfur cluster biogenesis and apoptosis as well as the production of cellular energy (ATP) by oxidative phosphorylation (OXPHOS) [1, 2]. With bacterial origins, a historical symbiotic relationship evolved during which mitochondria became a normal constituent of eukaryotic iology [3]. Their ancestry remains apparent with their own multicopy genetic material (mtDNA), with copy number varying greatly between individuals and across different tissues from the same individual. The 16.6 kb circular mtDNA molecule encodes 13 subunits of the OXPHOS components, 22 mitochondrial tRNAs and 2 subunits of the mitoribosomes [4]. Additionally, the mitoproteome requires a further ~1300 nuclear-encoded proteins for comprising, assembling or supporting the five multimeric OXPHOS complexes (I-V) and ancillary mitochondrial processes [5]. It stands to reason that mitochondrial dysfunction can result from either mtDNA- or nuclear-encoded gene defects and can occur as a primary, congenital condition or a secondary, age-associated effect due to somatic mutation [6].

The umbrella term "mitochondrial disease" refers to a clinically heterogeneous group of primary mitochondrial disorders in which the tissues and organs that are most often affected are those with the highest energy demands. Clinical symptoms can arise in childhood or later n life, and can affect one organ in isolation or be multisystemic [7] and the minimum disease prevalence in adults is ~12.5 per 100,000 [8] and ~4.7 per 100,000 for children [9]. There is a general lack of genotype-phenotype correlations in many mitochondrial disorders which means that establishing a genetic diagnosis can be a complicated process and remains elusive for many patients. This review provides a concise update on three areas where there have been major advances in our understanding in recent years [10] - the molecular genetics, muscle pathology and neuropathology associated with mitochondrial disease - highlighting the range of new techniques that are improving the diagnosis of patients with suspected

itochondrial disease with the aim of providing options to families at risk of an otherwise incurable condition.

he genetics of mitochondrial disease itochondrial-encoded (mtDNA) mitochondrial disease

ike nuclear DNA, which is diploid and follows Mendelian laws of inheritance, mtDNA is xclusively maternally-inherited [11]. The multicopy nature of mtDNA gives rise to heteroplasmy, a unique aspect of mtDNA-associated genetics, which occurs when there is a mix of mutant and wildtype mtDNA molecules (heteroplasmy) in coexistence. By contrast,

omoplasmy occurs when all the mtDNA molecules have the same genotype. Heteroplasmic mutations often have a variable threshold, a level to which the cell can tolerate defective mtDNA molecules [12]. Where the mutation load exceeds this threshold, metabolic dysfunction and associated clinical symptoms occur. Point mutations and large-scale mtDNA deletions represent the two common causes of primary mtDNA disease, the former usually being maternally-inherited whilst mtDNA deletions typically arise de novo during embryonic development.

mtDNA point mutations

mtDNA point mutations (including small indel mutations) are a significant cause of human disease with estimated population prevalence of 1 in 200 [13]. Mutations have been reported in every mtDNA-encoded gene and have been associated with clinical symptoms ranging from non-syndromic sensorineural deafness to MELAS, a devastating syndromic

neurological condition whose predominant features Mitochondrial Encephalopathy, Lactic cidosis and Stroke like episodes - give rise to the acronym. Clinical symptoms can present in child or adulthood, and mutations can be inherited (~75% cases) or occur de novo (~25% cases) [14]. Maternally-transmitted mtDNA defects may involve a clinically-unaffected

other who harbours the familial mtDNA mutation below the threshold required for cellular dysfunction although her oocytes harbour varying mutation loads due to the selection ressures of the mitochondrial bottleneck [15]. It is therefore near-impossible to predict the recurrence risk for subsequent pregnancies although prenatal testing of embryonic tissues

sing chorionic villus biopsy or amniocentesis can provide an accurate measure of mtDNA heteroplasmy in the fetus which can inform reproductive choices [16]. The recurrence risk of 'e novo mtDNA point mutations is very low, but for the risk of germline mosaicism in maternal oocytes [14]

(~6 [17 m

Single, large-scale mtDNA deletions

Single large-scale mtDNA deletions have a population frequency of 1.5/100,000 [8] with hree main associated phenotypes: Chronic progressive external ophthalmoplegia (CPEO) 65% cases), Kearns Sayre Syndrome (~30% cases) and Pearson syndrome (<5% cases) ]. Pearson syndrome is the most severe presentation associated with single large scale tDNA deletions; patients present early in life with sideroblastic anaemia and pancreatic dysfunction and is often fatal in infancy [18]. Kearns Sayre syndrome patients present before age 20 years with ptosis and/or PEO and pigmentary retinopathy and may have multisystem i nvolvement including myopathy, ataxia or cardiac conduction defects [17]. PEO is the more enign presentation attributed to single mtDNA deletions, associated with ophthalmoplegia, ptosis and myopathy [19]. Unlike nuclear gene rearrangements, single large-scale mtDNA deletions often arise sporadically during embryonic development and have a low recurrence risk [20]. Clinically-affected women who harbour a large-scale mtDNA deletion have a low (<10%) risk of transmission [20] and prenatal testing is informative for at-risk pregnancies [16].

Secondary mtDNA mutations

Whilst large-scale mtDNA deletions and point mutations represent primary mtDNA defects, secondary defects are another common cause of mitochondrial disease. Defective mtDNA maintenance, transcription, protein translation or a defective ancillary process such as mitochondrial import can cause either quantitative (depletion of mtDNA copy number) or

qualitative (affecting mtDNA genome integrity, resulting in multiple large mtDNA deletions) effects. These result from mutations affecting nuclear genes and inheritance occurs in a Mendelian (or de novo) fashion.

uclear mitochondrial disease

The majority of the genes in the mitoproteome are encoded by the nuclear genome [5] and ollow Mendelian inheritance patterns. De novo, X-linked, dominant and recessive inheritance cases have been reported in the literature [21-24]. The first nuclear mitochondrial gene mutation was identified in SDHA, encoding a structural subunit of complex II in 1995 [25], and there has been monumental progress in the discovery of mitochondrial disease candidate genes since. New proteomic and transcriptomic approaches are being applied to models of human disease to uncover new candidates [26, 27], whilst patient analyses are

alidating their involvement in human pathology [28]. The traditional approach of linkage analysis using multiple affected family members has given way to massively parallel sequencing strategies including whole exome sequencing (WES), either of affected singletons orproband-parent trios, and new disease genes are still emerging over 20 years on. Of the 1300 proteins in the mitoproteome, mutations have been reported in over 250 genes [29], and not only are new genes being reported but also new mechanisms involving genes already mplicated in human disease through alternative pathways [30]. It is apparent that more severe clinical phenotypes are often associated with recessive defects, presumably due to varying heteroplasmy levels in clinically-affected tissues and the dichotomous effect of ecessive mutations; as such, mtDNA mutations are more common in adults whilst nuclear gene defects are overrepresented in paediatric cases [31].

In this review, we delineate the nuclear-encoded mitochondrial disease genes into those that cause isolated and those that cause multiple respiratory chain complex deficiencies for simplicity and brevity.

Isolated respiratory chain complex deficiencies

Histochemical and biochemical evidence of an isolated respiratory chain complex deficiency can be suggestive of a mutation affecting either a structural subunit or an assembly/ancillary factor of one of the five OXPHOS complexes. Our current knowledge of the structural subunits and ancillary factors for each complex is summarized in Figure 1.

Isolated complex I deficiency

Complex I (NADH dehydrogenase) is composed of 44 structural subunits (7 of which are mtDNA encoded) with at least fourteen ancillary/assembly factors [32, 33]. Isolated complex I deficiency represents the biochemical phenotype for ~30% of paediatric patients [34], of

hich 70-80% have a nuclear gene defect [35]. Clinical symptoms associated with complex I deficiency are heterogeneous although the prognosis is typically poor with rapid progression. Lactic acidosis is a common feature, although is often present with another symptoms such as in cardiomyopathy or leukodystrophy. Mutations have been identified in 19 of the 37 structural subunits, and in 10 of 14 identified assembly factors; Although there are a few exceptions, such as the p.Trp22Arg NDUFB3 [36], the p.Gly212Val TMEM126B European founder mutation [28, 37] and the p.Cys115Tyr NDUFS6 Caucasus Jewish founder mutation [38], studies report the majority of complex I deficiency mutations as private and non-ecurrent [39]. NDUFS2 and ACAD9 mutations account for a significant proportion of diagnoses although it is likely that clearer genetic diagnostic trends will emerge from large diagnostic NGS datasets [40].

solated complex II deficiency

uccinate dehydrogenase (SDH), unlike any of the other complexes of the mitochondrial XPHOS system, is entirely nuclear encoded and is involved in both the tricarboxylic cycle (where it metabolises succinate to fumarate) and the respiratory chain (transferring electrons from FADH2 to reduce ubiquinone to ubiquinol). Complex II deficiency is rare (2-8% itochondrial disease cases [41, 42]) with <50 patients reported. Biallelic mutations have een associated with congenital metabolic presentations predominantly affecting either the CNS or heart (hypertrophic cardiomyopathy, leukodystrophy, Leigh syndrome and encephalopathy) [43] whereas heterozygous mutations are associated with cancer susceptibility, particularly pheochromocytoma and paraganglioma [44]. Although initially believed to have distinct gene-phenotype relationships (SDHA and SDHAF1 linked to mitochondrial disease, and SDHB/SDHC/SDHD/SDHAF2 with cancer susceptibility) it is emerging that there is phenotypic overlap, prompting tumour surveillance of unaffected relatives heterozygous for SDHx mutations [45, 46].

Isolated complex III deficiency

Ubiquinol-cytochrome c oxidoreductase, Complex III of the respiratory chain, functions as a homodimer to transfer electrons from ubiquinol to cytochrome b, then to cytochrome c.

pre; b

Complex III is composed of 11 structural subunits plus two haem groups and the Rieske iron sulphur protein. Exercise intolerance is the clinical phenotype reported for >50% patients with mutations in the mtDNA-encoded MTCYB gene but cardiomyopathy and encephalomyopathy are also noted [47]. Pathogenic mutations have been reported in four of t he nuclear-encoded structural subunits plus five assembly/ancillary factors [48] with presentations including developmental delay, encephalopathy, lactic acidosis liver dysfunction, renal tubulopathy and muscle weakness [48, 49].

solated complex IV deficiency

Cytochrome c oxidase (COX), complex IV of the respiratory chain, is embedded in the inner mitochondrial membrane and functions as a dimer, with two copper binding sites, two heme groups, one magnesium ion and one zinc ion [50]. Complex IV pumps protons across the inner mitochondrial membrane, contributing to the proton motive force for ATP synthase exploitation, and donates electrons to oxygen at the respiratory chain termini to form water. Complex IV has 13 structural subunits, and at least 26 additional proteins involved in assembly and biogenesis [51]. NDUFA4 was originally described as a complex I subunit but as since been reassigned to complex IV following functional studies [52] supported by the presence of NDUFA4 defects in a patient with severe COX deficiency [53]. Mutations have een reported in structural COX subunits but most defects affect biogenesis/assembly roteins. Some proteins are linked tightly with specific aspects of COX biogenesis (e.g. COA6, involved in copper-dependent COX2 biogenesis [54]) and others have more diverse oles [55]. Clinically, presentations are often early onset and devastating, predominantly affecting the heart and CNS (e.g. SURF1, in which >80 different mutations have been reported to cause Leigh syndrome [56]) although a milder Charcot Marie Tooth phenotype has been associated with biallelic COX6A1 variants [57].

Isolated complex V deficiency

ATP synthase, complex V, is the multimeric molecular motor that drives ATP production through phosphorylation of ADP. Utilising the proton motive force generated by electron transport and proton pumping by the respiratory chain, the 600kDa complex consists 13 different subunits (some of which have different isoforms, e.g. ATP5G1/ATP5G2/ATP5G3 encode subunit c isoforms) and involves at least 3 ancillary factors. Defects have been reported in just four nuclear complex V genes to date with varied clinical phenotypes. The most common defects involve TMEM70, including a Roma TMEM70 founder mutation

causing lactic acidosis and cardiomyopathy [58], although encephalopathy and cataracts have been reported in other populations [59].

Multiple respiratory chain defects

itochondrial function is regulated and maintained by around 1300 nuclear encoded genes; hese nuclear-encoded genes are translated by cytosolic translational machinery and the 5'

itochondrial targeting sequence directs transport of the translated proteins into the mitochondrion where they are required for diverse functions. These include the transcription of mitochondrial mRNA (e.g. POLRMT [60]), mitochondrial DNA maintenance (e.g. POLG [61]), regulation of mitochondrial dNTP pools (e.g. RRM2B [62]), cellular signalling (e.g. IRT1 [63]) and the translation of mitochondrial proteins. Numerous subgroups of proteins are involved in mitochondrial gene translation; mt-aminoacyl tRNA synthetases that are esponsible for charging each mitochondrial tRNA molecule with the appropriate amino acid, (e.g. AARS2 [64]), proteins involved in RNA processing (e.g. MTPAP [65]), mitoribosomal proteins (e.g. MRPL44 [66]) and those involved in mt-tRNA modification (e.g. TRMU [67]). efects in approximately 250 nuclear-encoded mitochondrial genes have now been reported n association with multiple respiratory chain defects and clinical mitochondrial disease [29].

genetic diagnostic pathway for these disorders is complex and WES is often the most uccessful strategy [68].

Non-OXPHOS mitochondrial disease ot all mitochondrial disease patients have evidence of respiratory chain enzyme dysfunction ut have other evidence of mitochondrial disease, such as elevated lactates, suggestive MRI brain changes and multisystem involvement. Genetic causes include defective enzymes of the rebs cycle (e.g. ACO2 [69]) or cofactor transport (e.g. SLC19A3 [70]).

Molecular genetic analysis of mitochondrial disease

In the absence of effective treatments, provision of a firm genetic diagnosis facilitates genetic counselling and access to reproductive options for patients and their families. Given the small size of the mtDNA genome, this is often sequenced in suspected mitochondrial disease patients to exclude a primary mtDNA defect before nuclear genes are scrutinised. NGS-based testing is becoming more prevalent [71] and also provides an accurate measure of mtDNA heteroplasmy. NGS-technologies are revolutionising the genetic testing pipeline in the

diagnostic genetic laboratory with Sanger sequencing of candidate nuclear genes on a sequential basis being replaced with powerful, high throughput analysis. A variety of options are currently being implemented - targeted panels of candidate genes [36], unbiased WES [72] and whole genome sequencing (WGS) [73] (Figure 2). Custom, panel based NGS trategies can be very successful for providing a rapid genetic diagnosis in the clinical setting, ut this success depends upon the degree of characterisation to ensure the appropriate andidate genes are targeted. Stratification according to respiratory chain defect can be appropriate for many patients in whom muscle biopsy is available, but even then it may be misleading - a number of patients with an isolated complex I deficiency in fact have a defect of mitochondrial translation [40]; moreover, this strategy can be ineffective for genes that exhibit inconsistent biochemical profiles [74]. Stratification according to clinical phenotype is similarly complicated by genetic heterogeneity [75].

Despite a proven track record in a research setting and the increasing availability of affordable NGS options to diagnostic laboratories, the case has yet to be made regarding the clinical validity of unrestricted WES within a diagnostic setting. One solution to the ¿ratification dilemma, and one that has been successfully implemented for the analysis of other heterogeneous Mendelian disorders - is a combination of unbiased WES with targeted nalysis of "virtual" gene panels [76, 77]; this allows informative reporting of negative results and removes the possibility of incidental findings. Further analysis of the WES data for patients lacking a diagnosis following virtual panel analysis could be subsequently ndertaken in a research setting. Indeed, most of the candidate genes included on diagnostic irtual panels have their origins in research which has been incredibly fruitful in elucidating genes involved in human pathology, including heterogeneous mitochondrial clinical phenotypes such as cardiomyopathy with mutations identified in AARS2 [78], MRPL3 [79], MTO1 [80] and ACAD9 [72]. New candidate genes continue to be discovered in a research setting and are then included in diagnostic screening, one success is exemplified by the report of patients harbouring TMEM126B mutations, a candidate gene identified by research-based complexome profiling [27, 28, 37]. Similarly, characterisation of predicted mitochondrial proteins of unknown function is another critical strategy for identifying potential disease candidate genes [26].

Investigating muscle pathology associated with mitochondrial disease

As discussed above, the laboratory investigation of suspected mitochondrial disease is complex and algorithms employ a multidisciplinary approach using clinical and functional studies to guide genetic analysis [81]. Although mitochondrial disorders are characterised by a wide spectrum of clinical presentations, owing to its high metabolic requirements, muscle is requently affected - either exclusively (e.g. myopathy, chronic progressive external ophthalmoplegia) or as a predominant feature in multi-systemic phenotypes [81, 82]. In both scenarios, muscle involvement can arise from nuclear-encoded or mtDNA-encoded mutations andthe association of distinctive histopathological hallmarks defines muscle as excellent post-mitotic surrogate for the study of many multisystem mitochondrial disorders. Diagnostic centres specialising in mitochondrial disorders employ numerous techniques to assess mitochondrial function including the assessment of individual mitochondrial OXPHOS activities in vitro [83] Although useful for identifying wide-spread mitochondrial defects, this echnique has some limitations; it requires large quantities of muscle (typically 50-100mg tissue) and may fail to detect subtle OXPHOS deficiencies, especially when only a few muscle fibres are affected (e.g. mild mosaic deficiencies). Furthermore, only complexes I-IV canbe reliably assessed in frozen muscle.

histological and histochemical examination of serially-sectioned muscle can provide rucial evidence of mitochondrial pathology. Haematoxylin & Eosin (H&E) and modified Gomori trichrome assess basic muscle morphology, providing information on fibre size and the presence of any abnormal inclusions or central nuclei which are indicative of muscle

enervation (Figure 3). The modified Gomori trichrome stain [84, 85] specifically highlights connective tissue (light blue), muscle fibres (blue) and mitochondria (red) and allows detection of ragged-red fibres (RRF) [86]. RRF are characterised by a "fibre cracking" appearance and abnormal subsarcolemmal proliferation of mitochondria resulting from a compensatory response to a respiratory chain biochemical defect [87]. RRF can either show normal activities of oxidative enzyme activities (often reported in association with the m.3243A>G mutation or some sporadic MTCYB mutations) or cytochrome c oxidase (COX)-deficient associated with a wide-range of mtDNA-related genetic disorders [88]. They represent a characteristic histopathological feature of mitochondrial disorders, however, they are not entirely diagnostic as these are also seen with normal ageing [6] and other muscle conditions [89, 90].

to a tro a

Sequential COX/SDH histochemistry is the standard method to assess mitochondrial espiratory chain function in muscle cryosections [91, 92], assessing the activities of the partially mtDNA-encoded complex IV (COX), and the fully nuclear-encoded complex II (SDH). By combining both reactions in a single slide (see Figure 3A), fibres or cells with

itochondrial dysfunction are easily identifiable and are seen as a mosaic reduction or loss of COX activity with preserved SDH activity (blue fibres), indicative of an underlying mtDNA-elated abnormality [93, 94]. The absence of routine histochemical assays to evaluate other OXPHOS complexes, such as complex I which is the largest and most commonly affected OXPHOS complex in mitochondrial disorders [95], has prompted the recent development of a novel high-throughput immunofluorescence assay to fill within the diagnostic repertoire [96]. This technique enables accurate quantification of the two most commonly affected OXPHOS components, namely complexes I and IV [97], together with a mitochondrial mass arker (porin) in individual muscle fibres on a single 10p,m tissue section (Figure 3B). The semi-automatic quantification of a large number of muscle fibres is facilitated by labelling aminin to define fibre boundaries. Image analysis is exclusively based on intensity measurements, increasing its accuracy and reliability, and is automated (http://iah-devext.ncl.ac.uk/immuno/). We are currently optimising the immunodetection of antibodies o assess complex III and complex V to better quantify the full extent of mitochondrial espiratory deficiency in patient muscle sections but the opportunity to assess this at a single fibre level shows great potential for both diagnosis and research applications (Figure 4).

Neuropathology associated with mitochondrial disease

Neurological symptoms are particularly common and may be devastating in patients with mitochondrial disease, including sensorineural deafness, cerebellar ataxia, peripheral neuropathy, dementia and epilepsy [81]. In recent years, a number of neuropathological studies have documented the characteristic features of neurodegeneration in mitochondrial disease and these have spurred the development of novel tools to understand the mechanisms underlying neural dysfunction and cell death.

New insights into mechanisms of neurodegeneration

Upon neuropathological investigation, the brains from patients with mitochondrial disease often show atrophy, cortical lesions, evidence of neuronal cell loss and mitochondrial OXPHOS abnormalities in remaining cells. Patients with the heteroplasmic m.3243A>G

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mutation and a MELAS phenotype often develop foci of cortical necrosis on the surface of the brain (Figure 5A). These are often referred to as ischaemic-like lesions since they resemble stroke penumbra but do not conform to a particular vascular territory. It is proposed hat these lesions evolve during stroke-like episodes and may be initiated by mitochondrial espiratory abnormalities in neurons which act to alter the balance of excitation and inhibition in neural networks promoting neuronal hyperexcitability [98]. This is important since seizures are frequently detected on EEG in patients who have had a stroke-like episode [99]. Although focal necrotic changes associated with m.3243A>G have been commonly documented, it is important to note that patients harbouring other genetic defects (e.g. the m.8344A>G mutation [100] and autosomal recessive POLG mutations [101, 102]) also develop cortical lesions suggesting shared mechanisms underpinning their formation. These lesions typically affect posterior brain regions, including the occipital, parietal and temporal obes and feature microvacuolation and neuronal cell dropout (Figure 5B, C), neuronal eosinophilia, astrogliosis and secondary myelin loss. Recent studies propose a vulnerability of GABAergic interneurons could underpin neuronal hyperexcitability since dramatic downregulation of OXPHOS subunits comprising complexes I and IV has been observed within interneurons (Figure 5D) [103]. While other theories suggest that aggregation of abnormally enlarged mitochondria and presence of mitochondrial respiratory chain bnormalities in the cerebral microvasculature may contribute to impaired cerebral perfusion [104, 105]. Though the precise mechanisms are not known, the emergence of lesions in the brain reflect an acute process leading to rapid neuronal loss which can occur on the ackground of more chronic and protracted cell loss throughout the brain.

The cerebellum is frequently involved in mitochondrial disease with many patients developing cerebellar ataxia. Neuropathologically, the cerebellum reveals signs of lesions (Figure 6A), similar to those observed in the cortex, global Purkinje cell dropout (Figure 6B), and loss of dentate nucleus neurons [106]. Recent work has shown downregulation of protein subunits comprising complex I in remaining Purkinje cells, their GABAergic synapses and dentate nucleus neurons (Figure 6C). In conjunction, there is evidence of neuronal network remodelling with thickened dendritic arborisations, axonal torpedoes and altered synaptic density [107-109]. There is a distinct lack of correlation between the severity of cell loss and heteroplasmy level of mutated mtDNA in surviving neurons suggesting other factors must be important in determining cell loss [110].

Patients harbouring a single large-scale mtDNA deletion may develop Kearns-Sayre syndrome (KSS) which is associated with severe demyelination and spongiosis of the white matter tracts of the brain, including the cerebrum, cerebellum, spinal cord and brainstem [111]. The loss of myelin is proposed to be due to a specific vulnerability of mature digodendrocytes, the myelin-producing glia, where a loss of respiratory chain activity due to he mtDNA deletion causes a distal oligodendrogliopathy and subsequent loss of myelin roducts [112]. It is not known why the mtDNA deletion preferentially affects oligodendrocytes.

In summary, neuropathological studies have shown that neuronal cell loss can occur via two different processes, an acute event such as in stroke-like lesions or a global, protracted loss of cells. There is no evidence of protein accumulation within neurons, surviving neurons requently show respiratory chain deficiency including downregulation of complex I subunits and there is a lack of correlation of cell loss and mtDNA heteroplasmy in remaining neurons.

Tools to aid study of mitochondrial neuropathology

ecently a number of novel methods have been developed to provide further insights into potential mechanisms of neurodegeneration, particularly for understanding the early events eading to irreversible neuronal cell loss. CLARITY (Clear Lipid-exchanged Acrylamide-hybridised Rigid Imaging/Immunostaining//« szYw-hybridisation-compatible Tissue hYdrogel) has paved the way for large volumes of archived, post-mortem material to be interrogated ith three-dimensional analysis of the neuronal networks [113]. This will enable a greater nderstanding of neuronal vulnerability in mitochondrial disease [114]. Recent development of induced pluripotent stem (iPS) cell technology allows the cellular transfection of human patient fibroblasts with four key transcription factors to confer pluripotency. These pluripotent cells can subsequently be differentiated into neurons and glial cells and the effects of both the nuclear and mitochondrial genome can be investigated to understand disease mechanisms, efficacy of drug treatment, and cell replacement therapies [115, 116]. Additionally, a number of transgenic mouse models utilising Cre/Lox technology to selectively knock out nuclear mitochondrial genes within specific populations of neurons and glial cells will be promising for the understanding of specific disease mechanisms [117-119].

Challenges for the future

Developing an effective treatment for mitochondrial disease is an enormous challenge that is dependent upon the integration of clinical understanding of disease progression, molecular genetic mechanisms, and neuropathological features in mitochondrial disease. Patient-based clinical, molecular genetic and histopathology studies can then inform development of appropriate disease model systems to understand mechanisms and treatment to ultimately improve the lives of patients with mitochondrial disease.

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Acknowledgements

Work in our laboratories is supported by a Wellcome Trust Strategic Award (096919/Z/11/Z), the MRC Centre for Neuromuscular Diseases (G0601943), Newcastle University Centre for Ageing and Vitality (supported by the Biotechnology and Biological Sciences Research Council and Medical Research Council (G016354/1)), the UK NIHR

iomedical Research Centre in Age and Age Related Diseases award to the Newcastle upon Tyne Hospitals NHS Foundation, the MRC/ESPRC Newcastle Molecular Pathology Node, the UK National Health Service Highly Specialised "Rare Mitochondrial Disorders of Adults and Children" service and the Lily Foundation. CLA is in receipt of a National Institute for Health Research (NIHR) doctoral fellowship (NIHR-HCS-D12-03-04). The views expressed are those of the authors and not necessarily of the NHS, NIHR, or the Department of Health. The authors would like to thank Alexia Chrysostomou, Hannah Rosa and Amy Vincent for contributing images shown within the Figures.

Author contributions

authors contributed to the drafting of the manuscript and its critical revision for important intellectual content

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Figure Legends

igure 1. Schematic of the OXPHOS complexes, their component subunits and associated ancillary factors. Multimeric protein complexes I-IV shuttle electrons along the respiratory chain, facilitated by the reduction of co-factors Coenzyme Q1o (Q) and ytochrome c (cyt c). Electron transfer is coupled to the transfer of protons (H+) across the inner mitochondrial membrane to generate a proton motive force which is used by Complex V (ATP synthase) to synthesise ATP. Characterisation of OXPHOS complexes has identified he constitutive subunits that are either mtDNA or nuclear-encoded, and many of the nuclear-encoded proteins involved in complex assembly, biogenesis or ancillary function; genes in which mutations have been identified are shown in bold and the first report of disease-causing mutations is referenced accordingly in blue.

igure 2. Next generation sequencing strategies employed in the genetic diagnosis of mitochondrial disease. A, Whole genome sequencing (WGS) analyses all coding and non-coding regions of the genome, whilst whole exome sequencing (WES; B) only targets the coding exons plus immediate intron-exon boundaries. C, Target capture facilitates equencing of a predetermined genomic region or list of candidate disease genes. Non-coding/intronic regions are shaded grey, exons of candidate genes are shaded blue and exons f non-candidate genes are shaded pink.

Figure 3. Histological, histochemical and immunohistochemical hallmarks of

itochondrial pathology in primary mtDNA-related disease. A, serial skeletal muscle (vastus lateralis) sections from a patient with a single, large-scale mtDNA deletion were stained for Haematoxylin & Eosin (H&E) and modified Gomori trichrome to assess basic muscle morphology and the presence of ragged-red fibres, respectively. The individual COX, SDH and sequential COX/SDH histochemical reactions show fibres manifesting mitochondrial accumulation and focal COX-deficiency. B, the lack of histochemical assays to assess other OXPHOS complex activities prompted the development of a quadruple immunofluorescence assay which can quantitate the expression of complex I (NDUFB8 subunit), complex IV (COXI subunit), laminin and a mitochondrial mass marker (porin), all within a single 10p,m section. A highlighted COX-deficient fibre (*) demonstrates focal accumulation of sarcolemmal mitochondria around the periphery of the fibre, and down-regulated expression of both complex I and IV proteins. (All images taken at 20X objective magnification).

igure 4. Current and future applications of a quantitative, quadruple OXPHOS immunofluorescence assay. Given its capacity to interrogate expression of both complex I and IV - and additional OXPHOS components - at a single muscle fibre level, we believe the uadruple immunofluorescence assay can be applied to several areas of diagnostic and research activity in the laboratory to help investigate the role of mitochondrial biochemical efects [96]. We are already implementing this methodology in a diagnostic setting, alidating the assay with biopsies from patients exhibiting a range of mtDNA-related and nuclear genetic diagnosis of mitochondrial disease. The assay also shows promise as a powerful tool to investigate the mitochondrial pathological changes observed in ageing and other myopathies (e.g. myofibrillar myopathies [90]), to investigate molecular disease mechanisms and mitochondrial disease progression, as well as an extremely sensitive utcome measure in clinical therapeutic intervention studies (e.g. pharmacological agents or exercise) aimed at improving muscle oxidative capacity in patients with mitochondrial disease.

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igure 5. Neuropathological changes associated with stroke-like episodes in patients with mitochondrial disease. A, Extensive cortical necrosis affecting the occipital, temporal ndparietal lobes in a brain from a patient harbouring the m.3243A>G mutation. Microscopic analysis reveals atrophy, microvacuolation and severe neuronal loss in the frontal cortex of a patient with m.3243A>G mutation (B; Cresyl fast violet staining) and in he temporal cortex of a patient with m.8344A>G mutation (C; Cresyl fast violet staining). espiratory chain abnormalities include downregulation of subunits comprising complex I (red; NDUFB8 subunit) and complex IV (green; COXI) relative to intact mitochondrial mass (magenta; porin) in inhibitory interneurons (blue; GAD 65-67) in a patient harbouring autosomal recessive POLG mutations (D; Scale bar = 10 |im).

Figure 6. Cerebellar pathology in patients with the m.3243A>G mutation. Numerous areas of necrosis are evident throughout the cerebellar cortex of a patient in comparison to control cerebellum (A; H&E staining). Extreme neuronal loss is detected microscopically affecting Purkinje cells and granule cells in patient cortex (B; Cresyl fast violet staining; Scale bar = 100 |im). C, in dentate nucleus neurons and in GABAergic (blue; GAD 65-67) synapses (magenta; synaptophysin) from Purkinje cells, there is downregulation of complex I (green; NDUFA13) relative to mitochondrial mass (red; COX4I2; Scale bar = 10 |im).

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164. Saada A, Vogel RO, Hoefs SJ, van den Brand MA, Wessels HJ, Willems PH, et al. Mutations in NDUFAF3 (C3ORF60), encoding an NDUFAF4 (C6ORF66)-interacting complex I assembly protein, cause fatal neonatal mitochondrial disease. Amer J Hum Genet 2009; 84: 718-727.

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:igure 1

intermembrane space

inner mitochondrial membrane

mitochondrial matrix

2H++1/2 02 T

I OXPHOS Component Complex I Complex II \ Complex III Complex IV Complex V

mtDNA structural MTND1 [120| MTND211211 MTND3|I22] M7CrS[127] M7COf|l28] Л»7Л7«[131]

subunit genes MTND41I23] MTND4L[]24] М7С02Ц2Э] МТАТРЦ\32]

MTND5[125] М7ЛЮ6И26] МТСОЗ |l 30]

Nuclear structural M>UFSf[133] «OUFS2[134] S0ÍM[25] UQCflS[153] COXJ|157] АТР5АЦ7Щ

subunit genes W>!/FSJ[135] NDUFS4[436] NDUFS5 SDH8|151] UQCRC1 COX5A АТР5В

NDUFSt [137] AIDUFS7[138] SDHC CVCi[156] COX5B АТР5С1

ЛШ1ДЗД[139] NDUFAIt 140] SDHD[152| !/OCRC2|154] СО*«Л]57] ATP5D

NDUFA2p41] NDUFA3 NDUFA5 UQCRFS1 COY6fl[158] Л7Р5Е[161]

NDUFA6 NDUFA7 NDUFA8 UQCRH COX6C ATP5F1

NDUFAS |142] NDUFA10[U3] UQCKQÍ155] COX7A ATP5G1 АТРОвг

NOUFA11 (21] NDUFA12{ 144] uocnio COX78 [1591 ATP5G3

NDUFA1H145] NDUFAB1 UQCR11 COX7C ATPÖH

NDUFV1UAB] NDUFV2[Ï47] NDUFV3 C0*«|160] ATP5I

NDUFB1 NDUFB2 (VDl/FBJ[148] АТР50

NDUFB4 NDUFBÖ NDUFB6 NDUFB7 ATPÖJ ATP5J2

NDUFB8 NDUFB9{ÏV}\ NDUFB10 ATPÖL ATP5L2

MDUFBIIII50] NDUFC1 NDUFC2

Assembly factor and NDUFAF1{№2\ NDUFAF2[163] SD/MFÍI41] Bcsium СОЛ1 COAJ]173] COA4 СОД511Г4] ATPAF1

ancillary protein NDUFAF3\ 164] MDUFA«[165] SDHAF2 1таМ7]169] COAS[175] COA7 СОХ10Ц76] СОХП Л7РЛЯ2Ц89]

genes NDUFAFSl\œ] NDUFAF6\m] SDHAF3 UQCC1 COX14[]77] CO>Я5Ц78] COX16 COX17 ТМ£М70[53]

NDUFAFJ гохяеощеа] SDHAF4 UQCC21170] COXIS СОХ19 COX20|179] SCOÍ|180]

JCJDSpO] ECSIT UQCC3\ 7'] SC02[1S1] SUHFf|182] PFT117

NUBPLvss] тмЕмтвт. 37] 7TCfS[172] /_ЯРРЯС|181] PE71ÍM[184] CEP»9|185]

TIMMDC1 C17orf89 PTCD2 T¿COf[l86] ОХАЯ АРОРТЦ18Т]

NDUFA4Í53] MS™*[188J

Figure 2

Whole genome sequencing') (WGS)

Whole exome sequencing (WES)

Targeted capture

Figure 3 A

* HwSrt * WM &

NDUFB8 (Complex I) COX1 (Complex IV)

Overlay

Investigating mitochondrial pathology in ageing and other muscle diseases

Sarcopenla Myofibrillar myopathy

Improving the diagnosis of mitochondrial disease

Outcome measure in

clinical intervention

studies

E 5 -i0' s w

Understanding disease mechanisms

single, large-scale m.3243A>G mutation mtDNA deletion

Monitoring disease progression in mitochondrial disease

Figure 5

c 'W. : .

>... -

' Jl , Çrj '

■ .:-'-'.-• ' V v . v • r . ....., v . . IjjftfflBffli HHHHMH

Figure 6

A Control subject Patient with m.3243A>G B Control subject Patient with m.3243A>G

Overlay Synapses GABA Mitochondrial mass NDUFA13 subunit