Wellcome Centre for Mitochondrial Research

Human mtDNA shows striking regional variation, traditionally attributed to genetic drift. However, it is not easy to account for the fact that only two mtDNA lineages (M and N) left Africa to colonize Eurasia and that lineages A, C, D, and G show a 5-fold enrichment from central Asia to Siberia. As an alternative to drift, natural selection might have enriched for certain mtDNA lineages as people migrated north into colder climates. To test this hypothesis we analyzed 104 complete mtDNA sequences from all global regions and lineages. African mtDNA variation did not significantly deviate from the standard neutral model, but European, Asian, and Siberian plus Native American variations did. Analysis of amino acid substitution mutations (nonsynonymous, Ka) versus neutral mutations (synonymous, Ks) (kaks) for all 13 mtDNA protein-coding genes revealed that the ATP6 gene had the highest amino acid sequence variation of any human mtDNA gene, even though ATP6 is one of the more conserved mtDNA proteins. Comparison of the kaks ratios for each mtDNA gene from the tropical, temperate, and arctic zones revealed that ATP6 was highly variable in the mtDNAs from the arctic zone, cytochrome b was particularly variable in the temperate zone, and cytochrome oxidase I was notably more variable in the tropics. Moreover, multiple amino acid changes found in ATP6, cytochrome b, and cytochrome oxidase I appeared to be functionally significant. From these analyses we conclude that selection may have played a role in shaping human regional mtDNA variation and that one of the selective influences was climate.

As the second most common age related neurodegenerative disease after Alzheimer's disease, the health, social and economic impact resulting from Parkinson's disease will continue to increase alongside the longevity of the population. Ageing remains the biggest risk factor for developing idiopathic Parkinson's disease. Although research into the mechanisms leading to cell death in Parkinson's disease has shed light on many aspects of the pathogenesis of this disorder, we still cannot answer the fundamental question, what specific age related factors predispose some individuals to develop this common neurodegenerative disease. In this review we focus specifically on the neuronal population associated with the motor symptoms of Parkinson's disease, the dopaminergic neurons of the substantia nigra, and try to understand how ageing puts these neurons at risk to the extent that a slight change in protein metabolism or mitochondrial function can push the cells over the edge leading to catastrophic cell death and many of the symptoms seen in Parkinson's disease. We review the evidence that ageing is important for the development of Parkinson's disease and how age related decline leads to the loss of neurons within this disease, before describing exactly how advancing age may lead to substantia nigra neuronal loss and Parkinson's disease in some individuals.

OBJECTIVE: The prevalence of mitochondrial disease has proven difficult to establish, predominantly as a result of clinical and genetic heterogeneity. The phenotypic spectrum of mitochondrial disease has expanded significantly since the original reports that associated classic clinical syndromes with mitochondrial DNA (mtDNA) rearrangements and point mutations. The revolution in genetic technologies has allowed interrogation of the nuclear genome in a manner that has dramatically improved the diagnosis of mitochondrial disorders. We comprehensively assessed the prevalence of all forms of adult mitochondrial disease to include pathogenic mutations in both nuclear and mtDNA. METHODS: Adults with suspected mitochondrial disease in the North East of England were referred to a single neurology center from 1990 to 2014. For the midyear period of 2011, we evaluated the minimum prevalence of symptomatic nuclear DNA mutations and symptomatic and asymptomatic mtDNA mutations causing mitochondrial diseases. RESULTS: The minimum prevalence rate for mtDNA mutations was 1 in 5,000 (20 per 100,000), comparable with our previously published prevalence rates. In this population, nuclear mutations were responsible for clinically overt adult mitochondrial disease in 2.9 per 100,000 adults. INTERPRETATION: Combined, our data confirm that the total prevalence of adult mitochondrial disease, including pathogenic mutations of both the mitochondrial and nuclear genomes (≈1 in 4,300), is among the commonest adult forms of inherited neurological disorders. These figures hold important implications for the evaluation of interventions, provision of evidence-based health policies, and planning of future services.

Chronic inflammation is associated with normal and pathological ageing. Here we show that chronic, progressive low-grade inflammation induced by knockout of the nfkb1 subunit of the transcription factor NF-κB induces premature ageing in mice. We also show that these mice have reduced regeneration in liver and gut. nfkb1(-/-) fibroblasts exhibit aggravated cell senescence because of an enhanced autocrine and paracrine feedback through NF-κB, COX-2 and ROS, which stabilizes DNA damage. Preferential accumulation of telomere-dysfunctional senescent cells in nfkb1(-/-) tissues is blocked by anti-inflammatory or antioxidant treatment of mice, and this rescues tissue regenerative potential. Frequencies of senescent cells in liver and intestinal crypts quantitatively predict mean and maximum lifespan in both short- and long-lived mice cohorts. These data indicate that systemic chronic inflammation can accelerate ageing via ROS-mediated exacerbation of telomere dysfunction and cell senescence in the absence of any other genetic or environmental factor.

Cell senescence is an important tumour suppressor mechanism and driver of ageing. Both functions are dependent on the development of the senescent phenotype, which involves an overproduction of pro-inflammatory and pro-oxidant signals. However, the exact mechanisms regulating these phenotypes remain poorly understood. Here, we show the critical role of mitochondria in cellular senescence. In multiple models of senescence, absence of mitochondria reduced a spectrum of senescence effectors and phenotypes while preserving ATP production via enhanced glycolysis. Global transcriptomic analysis by RNA sequencing revealed that a vast number of senescent-associated changes are dependent on mitochondria, particularly the pro-inflammatory phenotype. Mechanistically, we show that the ATM, Akt and mTORC1 phosphorylation cascade integrates signals from the DNA damage response (DDR) towards PGC-1β-dependent mitochondrial biogenesis, contributing to aROS-mediated activation of the DDR and cell cycle arrest. Finally, we demonstrate that the reduction in mitochondrial content in vivo, by either mTORC1 inhibition or PGC-1β deletion, prevents senescence in the ageing mouse liver. Our results suggest that mitochondria are a candidate target for interventions to reduce the deleterious impact of senescence in ageing tissues.

Resetting of the epigenome in human primordial germ cells (hPGCs) is critical for development. We show that the transcriptional program of hPGCs is distinct from that in mice, with co-expression of somatic specifiers and naive pluripotency genes TFCP2L1 and KLF4. This unique gene regulatory network, established by SOX17 and BLIMP1, drives comprehensive germline DNA demethylation by repressing DNA methylation pathways and activating TET-mediated hydroxymethylation. Base-resolution methylome analysis reveals progressive DNA demethylation to basal levels in week 5-7 in vivo hPGCs. Concurrently, hPGCs undergo chromatin reorganization, X reactivation, and imprint erasure. Despite global hypomethylation, evolutionarily young and potentially hazardous retroelements, like SVA, remain methylated. Remarkably, some loci associated with metabolic and neurological disorders are also resistant to DNA demethylation, revealing potential for transgenerational epigenetic inheritance that may have phenotypic consequences. We provide comprehensive insight on early human germline transcriptional network and epigenetic reprogramming that subsequently impacts human development and disease.

Leber hereditary optic neuropathy (LHON) and autosomal-dominant optic atrophy (DOA) are the two most common inherited optic neuropathies in the general population. Both disorders share striking pathological similarities, marked by the selective loss of retinal ganglion cells (RGCs) and the early involvement of the papillomacular bundle. Three mitochondrial DNA (mtDNA) point mutations; m.3460G>A, m.11778G>A, and m.14484T>C account for over 90% of LHON cases, and in DOA, the majority of affected families harbour mutations in the OPA1 gene, which codes for a mitochondrial inner membrane protein. Optic nerve degeneration in LHON and DOA is therefore due to disturbed mitochondrial function and a predominantly complex I respiratory chain defect has been identified using both in vitro and in vivo biochemical assays. However, the trigger for RGC loss is much more complex than a simple bioenergetic crisis and other important disease mechanisms have emerged relating to mitochondrial network dynamics, mtDNA maintenance, axonal transport, and the involvement of the cytoskeleton in maintaining a differential mitochondrial gradient at sites such as the lamina cribosa. The downstream consequences of these mitochondrial disturbances are likely to be influenced by the local cellular milieu. The vulnerability of RGCs in LHON and DOA could derive not only from tissue-specific, genetically-determined biological factors, but also from an increased susceptibility to exogenous influences such as light exposure, smoking, and pharmacological agents with putative mitochondrial toxic effects. Our concept of inherited mitochondrial optic neuropathies has evolved over the past decade, with the observation that patients with LHON and DOA can manifest a much broader phenotypic spectrum than pure optic nerve involvement. Interestingly, these phenotypes are sometimes clinically indistinguishable from other neurodegenerative disorders such as Charcot-Marie-Tooth disease, hereditary spastic paraplegia, and multiple sclerosis, where mitochondrial dysfunction is also thought to be an important pathophysiological player. A number of vertebrate and invertebrate disease models has recently been established to circumvent the lack of human tissues, and these have already provided considerable insight by allowing direct RGC experimentation. The ultimate goal is to translate these research advances into clinical practice and new treatment strategies are currently being investigated to improve the visual prognosis for patients with mitochondrial optic neuropathies.

Mitochondrial DNA (mtDNA) mutations are a major cause of genetic disease, but their prevalence in the general population is not known. We determined the frequency of ten mitochondrial point mutations in 3168 neonatal-cord-blood samples from sequential live births, analyzing matched maternal-blood samples to estimate the de novo mutation rate. mtDNA mutations were detected in 15 offspring (0.54%, 95% CI = 0.30–0.89%). Of these live births, 0.00107% (95% CI = 0.00087–0.0127) harbored a mutation not detected in the mother's blood, providing an estimate of the de novo mutation rate. The most common mutation was m.3243A→G. m.14484T→C was only found on sub-branches of mtDNA haplogroup J. In conclusion, at least one in 200 healthy humans harbors a pathogenic mtDNA mutation that potentially causes disease in the offspring of female carriers. The exclusive detection of m.14484T→C on haplogroup J implicates the background mtDNA haplotype in mutagenesis. These findings emphasize the importance of developing new approaches to prevent transmission. Mitochondrial DNA (mtDNA) mutations are a major cause of genetic disease, but their prevalence in the general population is not known. We determined the frequency of ten mitochondrial point mutations in 3168 neonatal-cord-blood samples from sequential live births, analyzing matched maternal-blood samples to estimate the de novo mutation rate. mtDNA mutations were detected in 15 offspring (0.54%, 95% CI = 0.30–0.89%). Of these live births, 0.00107% (95% CI = 0.00087–0.0127) harbored a mutation not detected in the mother's blood, providing an estimate of the de novo mutation rate. The most common mutation was m.3243A→G. m.14484T→C was only found on sub-branches of mtDNA haplogroup J. In conclusion, at least one in 200 healthy humans harbors a pathogenic mtDNA mutation that potentially causes disease in the offspring of female carriers. The exclusive detection of m.14484T→C on haplogroup J implicates the background mtDNA haplotype in mutagenesis. These findings emphasize the importance of developing new approaches to prevent transmission.

Across a variety of Mendelian disorders, ∼50-75% of patients do not receive a genetic diagnosis by exome sequencing indicating disease-causing variants in non-coding regions. Although genome sequencing in principle reveals all genetic variants, their sizeable number and poorer annotation make prioritization challenging. Here, we demonstrate the power of transcriptome sequencing to molecularly diagnose 10% (5 of 48) of mitochondriopathy patients and identify candidate genes for the remainder. We find a median of one aberrantly expressed gene, five aberrant splicing events and six mono-allelically expressed rare variants in patient-derived fibroblasts and establish disease-causing roles for each kind. Private exons often arise from cryptic splice sites providing an important clue for variant prioritization. One such event is found in the complex I assembly factor TIMMDC1 establishing a novel disease-associated gene. In conclusion, our study expands the diagnostic tools for detecting non-exonic variants and provides examples of intronic loss-of-function variants with pathological relevance.

OBJECTIVE: Diverse and variable clinical features, a loose genotype-phenotype relationship, and presentation to different medical specialties have all hindered attempts to gauge the epidemiological impact of mitochondrial DNA (mtDNA) disease. Nevertheless, a clear understanding of its prevalence remains an important goal, particularly about planning appropriate clinical services. Consequently, the aim of this study was to accurately define the prevalence of mtDNA disease (primary mutation occurs in mtDNA) in the working-age population of the North East of England. METHODS: Adults with suspected mitochondrial disease in the North East of England were referred to a single neurology center for investigation from 1990 to 2004. Those with pathogenic mtDNA mutations were identified and pedigree analysis performed. For the midyear period of 2001, we calculated the minimum point prevalence of mtDNA disease for adults of working age (>16 and <60/65 years for female/male patients, respectively). RESULTS: In this population, we found that 9.2 in 100,000 people have clinically manifest mtDNA disease, making this one of the commonest inherited neuromuscular disorders. In addition, a further 16.5 in 100,000 children and adults younger than retirement age are at risk for development of mtDNA disease. INTERPRETATION: Through detailed pedigree analysis and active family tracing, we have been able to provide revised minimum prevalence figures for mtDNA disease. These estimates confirm that mtDNA disease is a common cause of chronic morbidity and is more prevalent than has been previously appreciated.

Abstract Senescent cells drive age-related tissue dysfunction partially through the induction of a chronic senescence-associated secretory phenotype (SASP) 1 . Mitochondria are major regulators of the SASP; however, the underlying mechanisms have not been elucidated 2 . Mitochondria are often essential for apoptosis, a cell fate distinct from cellular senescence. During apoptosis, widespread mitochondrial outer membrane permeabilization (MOMP) commits a cell to die 3 . Here we find that MOMP occurring in a subset of mitochondria is a feature of cellular senescence. This process, called minority MOMP (miMOMP), requires BAX and BAK macropores enabling the release of mitochondrial DNA (mtDNA) into the cytosol. Cytosolic mtDNA in turn activates the cGAS–STING pathway, a major regulator of the SASP. We find that inhibition of MOMP in vivo decreases inflammatory markers and improves healthspan in aged mice. Our results reveal that apoptosis and senescence are regulated by similar mitochondria-dependent mechanisms and that sublethal mitochondrial apoptotic stress is a major driver of the SASP. We provide proof-of-concept that inhibition of miMOMP-induced inflammation may be a therapeutic route to improve healthspan.

Major advances in understanding the pathogenesis of inherited metabolic disease caused by mitochondrial DNA mutations have yet to translate into treatments of proven efficacy. Leber's hereditary optic neuropathy is the most common mitochondrial DNA disorder causing irreversible blindness in young adult life. Anecdotal reports support the use of idebenone in Leber's hereditary optic neuropathy, but this has not been evaluated in a randomized controlled trial. We conducted a 24-week multi-centre double-blind, randomized, placebo-controlled trial in 85 patients with Leber's hereditary optic neuropathy due to m.3460G>A, m.11778G>A, and m.14484T>C or mitochondrial DNA mutations. The active drug was idebenone 900 mg/day. The primary end-point was the best recovery in visual acuity. The main secondary end-point was the change in best visual acuity. Other secondary end-points were changes in visual acuity of the best eye at baseline and changes in visual acuity for both eyes in each patient. Colour-contrast sensitivity and retinal nerve fibre layer thickness were measured in subgroups. Idebenone was safe and well tolerated. The primary end-point did not reach statistical significance in the intention to treat population. However, post hoc interaction analysis showed a different response to idebenone in patients with discordant visual acuities at baseline; in these patients, all secondary end-points were significantly different between the idebenone and placebo groups. This first randomized controlled trial in the mitochondrial disorder, Leber's hereditary optic neuropathy, provides evidence that patients with discordant visual acuities are the most likely to benefit from idebenone treatment, which is safe and well tolerated.

There is an intense debate concerning whether selection or demographics has been most important in shaping the sequence variation observed in modern human mitochondrial DNA (mtDNA). Purifying selection is thought to be important in shaping mtDNA sequence evolution, but the strength of this selection has been debated, mainly due to the threshold effect of pathogenic mtDNA mutations and an observed excess of new mtDNA mutations in human population data. We experimentally addressed this issue by studying the maternal transmission of random mtDNA mutations in mtDNA mutator mice expressing a proofreading-deficient mitochondrial DNA polymerase. We report a rapid and strong elimination of nonsynonymous changes in protein-coding genes; the hallmark of purifying selection. There are striking similarities between the mutational patterns in our experimental mouse system and human mtDNA polymorphisms. These data show strong purifying selection against mutations within mtDNA protein-coding genes. To our knowledge, our study presents the first direct experimental observations of the fate of random mtDNA mutations in the mammalian germ line and demonstrates the importance of purifying selection in shaping mitochondrial sequence diversity.

We have performed a detailed population study of patients with genetic muscle disease in the northern region of England. Our current clinic population comprises over 1100 patients in whom we have molecularly characterized 31 separate muscle disease entities. Diagnostic clarity achieved through careful delineation of clinical features supported by histological, immunological and genetic analysis has allowed us to reach a definitive diagnosis in 75.7% of our patients. We have compared our case profile with that from Walton and Nattrass' seminal study from 1954, also of the northern region, together with data from other more recent studies from around the world. Point prevalence figures for each of the five major disease categories are comparable with those from other recent studies. Myotonic dystrophies are the most common, comprising 28.6% of our clinic population with a point prevalence of 10.6/100,000. Next most frequent are the dystrophinopathies and facioscapulohumeral muscular dystrophy making up 22.9% (8.46/100,000) and 10.7% (3.95/100,000) of the clinic population, respectively. Spinal muscular atrophy patients account for 5.1% or 1.87/100,000 patients. Limb girdle muscular dystrophy, which was described for the first time in the paper by Walton and Nattrass (1954) and comprised 17% of their clinic population, comprises 6.2% of our clinic population at a combined prevalence of 2.27/100,000. The clinic population included patients with 12 other muscle disorders. These disorders ranged from a point prevalence of 0.89/100 000 for the group of congenital muscular dystrophies to conditions with only two affected individuals in a population of three million. For the first time our study provides epidemiological information for X-linked Emery-Dreifuss muscular dystrophy and the collagen VI disorders. Each of the X-linked form of Emery-Dreifuss muscular dystrophy and Ullrich muscular dystrophy has a prevalence of 0.13/100,000, making both very rare. Bethlem myopathy was relatively more common with a prevalence of 0.77/100,000. Overall our study provides comprehensive epidemiological information on individually rare inherited neuromuscular conditions in Northern England. Despite the deliberate exclusion of relatively common groups such as hereditary motor and sensory neuropathy (40/100,000) and mitochondrial disorders (9.2/100,000), the combined prevalence is 37.0/100,000, demonstrating that these disorders, taken as a group, encompass a significant proportion of patients with chronic disease. The study also illustrates the immense diagnostic progress since the first regional survey over 50 years ago by Walton and Nattrass.

Abstract Ageing is the biggest risk factor for cardiovascular disease. Cellular senescence, a process driven in part by telomere shortening, has been implicated in age‐related tissue dysfunction. Here, we address the question of how senescence is induced in rarely dividing/post‐mitotic cardiomyocytes and investigate whether clearance of senescent cells attenuates age‐related cardiac dysfunction. During ageing, human and murine cardiomyocytes acquire a senescent‐like phenotype characterised by persistent DNA damage at telomere regions that can be driven by mitochondrial dysfunction and crucially can occur independently of cell division and telomere length. Length‐independent telomere damage in cardiomyocytes activates the classical senescence‐inducing pathways, p21 CIP and p16 INK4a , and results in a non‐canonical senescence‐associated secretory phenotype, which is pro‐fibrotic and pro‐hypertrophic. Pharmacological or genetic clearance of senescent cells in mice alleviates detrimental features of cardiac ageing, including myocardial hypertrophy and fibrosis. Our data describe a mechanism by which senescence can occur and contribute to age‐related myocardial dysfunction and in the wider setting to ageing in post‐mitotic tissues.

Recent sequencing studies have extensively explored the somatic alterations present in the nuclear genomes of cancers. Although mitochondria control energy metabolism and apoptosis, the origins and impact of cancer-associated mutations in mtDNA are unclear. In this study, we analyzed somatic alterations in mtDNA from 1675 tumors. We identified 1907 somatic substitutions, which exhibited dramatic replicative strand bias, predominantly C > T and A > G on the mitochondrial heavy strand. This strand-asymmetric signature differs from those found in nuclear cancer genomes but matches the inferred germline process shaping primate mtDNA sequence content. A number of mtDNA mutations showed considerable heterogeneity across tumor types. Missense mutations were selectively neutral and often gradually drifted towards homoplasmy over time. In contrast, mutations resulting in protein truncation undergo negative selection and were almost exclusively heteroplasmic. Our findings indicate that the endogenous mutational mechanism has far greater impact than any other external mutagens in mitochondria and is fundamentally linked to mtDNA replication.

Mitochondrial DNA instability disorders are responsible for a large clinical spectrum, among which amyotrophic lateral sclerosis-like symptoms and frontotemporal dementia are extremely rare. We report a large family with a late-onset phenotype including motor neuron disease, cognitive decline resembling frontotemporal dementia, cerebellar ataxia and myopathy. In all patients, muscle biopsy showed ragged-red and cytochrome c oxidase-negative fibres with combined respiratory chain deficiency and abnormal assembly of complex V. The multiple mitochondrial DNA deletions found in skeletal muscle revealed a mitochondrial DNA instability disorder. Patient fibroblasts present with respiratory chain deficiency, mitochondrial ultrastructural alterations and fragmentation of the mitochondrial network. Interestingly, expression of matrix-targeted photoactivatable GFP showed that mitochondrial fusion was not inhibited in patient fibroblasts. Using whole-exome sequencing we identified a missense mutation (c.176C>T; p.Ser59Leu) in the CHCHD10 gene that encodes a coiled-coil helix coiled-coil helix protein, whose function is unknown. We show that CHCHD10 is a mitochondrial protein located in the intermembrane space and enriched at cristae junctions. Overexpression of a CHCHD10 mutant allele in HeLa cells led to fragmentation of the mitochondrial network and ultrastructural major abnormalities including loss, disorganization and dilatation of cristae. The observation of a frontotemporal dementia-amyotrophic lateral sclerosis phenotype in a mitochondrial disease led us to analyse CHCHD10 in a cohort of 21 families with pathologically proven frontotemporal dementia-amyotrophic lateral sclerosis. We identified the same missense p.Ser59Leu mutation in one of these families. This work opens a novel field to explore the pathogenesis of the frontotemporal dementia-amyotrophic lateral sclerosis clinical spectrum by showing that mitochondrial disease may be at the origin of some of these phenotypes.

Multiple sclerosis is a chronic inflammatory disease of the central nervous system, associated with demyelination and neurodegeneration. The mechanisms of tissue injury are poorly understood, but recent data suggest that mitochondrial injury may play an important role in this process. Mitochondrial injury can be triggered by reactive oxygen and nitric oxide species, and we recently provided evidence for oxidative damage of oligodendrocytes and dystrophic axons in early stages of active multiple sclerosis lesions. In this study, we identified potential sources of reactive oxygen and nitrogen species through gene expression in carefully staged and dissected lesion areas and by immunohistochemical analysis of protein expression. Genome-wide microarrays confirmed mitochondrial injury in active multiple sclerosis lesions, which may serve as an important source of reactive oxygen species. In addition, we found differences in the gene expression levels of various nicotinamide adenine dinucleotide phosphate oxidase subunits between initial multiple sclerosis lesions and control white matter. These results were confirmed at the protein level by means of immunohistochemistry, showing upregulation of the subunits gp91phox, p22phox, p47phox, nicotinamide adenine dinucleotide phosphate oxidase 1 and nicotinamide adenine dinucleotide phosphate oxidase organizer 1 in activated microglia in classical active as well as slowly expanding lesions. The subunits gp91phox and p22phox were constitutively expressed in microglia and were upregulated in the initial lesion. In contrast, p47phox, nicotinamide adenine dinucleotide phosphate oxidase 1 and nicotinamide adenine dinucleotide phosphate oxidase organizer 1 expression were more restricted to the zone of initial damage or to lesions from patients with acute or early relapsing/remitting multiple sclerosis. Double labelling showed co-expression of the nicotinamide adenine dinucleotide phosphate oxidase subunits in activated microglia and infiltrated macrophages, suggesting the assembly of functional complexes. Our data suggest that the inflammation-associated oxidative burst in activated microglia and macrophages plays an important role in demyelination and free radical-mediated tissue injury in the pathogenesis of multiple sclerosis.

BACKGROUND: Mitochondrial respiratory chain disorders are the most prevalent group of inherited neurometabolic diseases. They present with central and peripheral neurological features usually in association with other organ involvement including the eye, the heart, the liver, and kidneys, diabetes mellitus and sensorineural deafness. Current treatment is largely supportive and the disorders progress relentlessly causing significant morbidity and premature death. Vitamin supplements, pharmacological agents and exercise therapy have been used in isolated cases and small clinical trials, but the efficacy of these interventions is unclear. The first review was carried out in 2003, and identified six clinical trials. This major update was carried out to identify new studies and grade the original studies for potential bias in accordance with revised Cochrane Collaboration guidelines. OBJECTIVES: To determine whether there is objective evidence to support the use of current treatments for mitochondrial disease. SEARCH METHODS: We searched the Cochrane Neuromuscular Disease Group Specialized Register (4 July 2011), CENTRAL (2011, Issue 2, MEDLINE (1966 to July 2011), and EMBASE (January 1980 to July 2011), and contacted experts in the field. SELECTION CRITERIA: We included randomised controlled trials (including cross-over studies). Two of the authors independently selected abstracts for further detailed review. Further review was performed independently by all five authors to decide which trials fit the inclusion criteria and graded risk of bias. Participants included males and females of any age with a confirmed diagnosis of mitochondrial disease based upon muscle histochemistry, respiratory chain complex analysis of tissues or cell lines or DNA studies. Interventions included any pharmacological agent, dietary modification, nutritional supplement, exercise therapy or other treatment. The review authors excluded studies at high risk of bias in any category. The primary outcome measures included an change in muscle strength and/or endurance, or neurological clinical features. Secondary outcome measures included quality of life assessments, biochemical markers of disease and negative outcomes. DATA COLLECTION AND ANALYSIS: Two of the authors (GP and PFC) independently identified studies for further evaluation from all abstracts within the search period. For those studies identified for further review, all five authors then independently assessed which studies met the entry criteria. For the included studies, we extracted details of the number of randomised participants, treatment, study design, study category, allocation concealment and other risk of bias criteria, and participant characteristics. Analysis was based on intention-to-treat data. We planned to use meta-analysis, but this did not prove necessary. MAIN RESULTS: The authors reviewed 1335 abstracts, and from these identified 21 potentially eligible abstracts. Upon detailed review, 12 studies fulfilled the entry criteria. Of these, eight were new studies that had been published since the previous version of this review. Two studies which were included in the previous version of this review were excluded because of potential for bias. The comparability of the included studies is extremely low because of differences in the specific diseases studied, differences in the therapeutic agents used, dosage, study design, and outcomes. The methodological quality of included studies was generally high, although risk of bias was unclear in random sequence generation and allocation concealment for most studies. Otherwise, the risk of bias was low for most studies in the other categories. Serious adverse events were uncommon, except for peripheral nerve toxicity in a long-term trial of dichloroacetate (DCA) in adults.One trial studied high-dose coenzyme Q10 without clinically meaningful improvement (although there were multiple biochemical, physiologic, and neuroimaging outcomes, in 30 participants). Three trials used creatine monohydrate alone, with one reporting evidence of improved measures of muscle strength and post-exercise lactate, but the other two reported no benefit (total of 38 participants). One trial studied the effects of a combination of coenzyme Q10, creatine monohydrate, and lipoic acid and reported a statistically significant improvement in biochemical markers and peak ankle dorsiflexion strength, but overall no clinical improvement in 16 participants. Five trials studied the effects of DCA: three trials in children showed a statistically significant improvement in secondary outcome measures of mitochondrial metabolism (venous lactate in three trials, and magnetic resonance spectroscopy (MRS) in one trial; total of 63 participants). One trial of short-term DCA in adults demonstrated no clinically relevant improvement (improved venous lactate but no change in physiologic, imaging, or questionnaire findings, in eight participants). One longer-term DCA trial in adults was terminated prematurely due to peripheral nerve toxicity without clinical benefit (assessments included the GATE score, venous lactate and MRS, in 30 participants). One trial using dimethylglycine showed no significant effect (measurements of venous lactate and oxygen consumption (VO(2)) in five participants). One trial using a whey-based supplement showed statistically significant improvement in markers of free radical reducing capacity but no clinical benefit (assessments included the Short Form 36 Health Survey (SF-36) questionnaire and UK Medical Research Council (MRC) muscle strength, in 13 participants). AUTHORS' CONCLUSIONS: Despite identifying eight new trials there is currently no clear evidence supporting the use of any intervention in mitochondrial disorders. Further research is needed to establish the role of a wide range of therapeutic approaches. We suggest further research should identify novel agents to be tested in homogeneous study populations with clinically relevant primary endpoints.

Additional neurological features have recently been described in seven families transmitting pathogenic mutations in OPA1, the most common cause of autosomal dominant optic atrophy. However, the frequency of these syndromal 'dominant optic atrophy plus' variants and the extent of neurological involvement have not been established. In this large multi-centre study of 104 patients from 45 independent families, including 60 new cases, we show that extra-ocular neurological complications are common in OPA1 disease, and affect up to 20% of all mutational carriers. Bilateral sensorineural deafness beginning in late childhood and early adulthood was a prominent manifestation, followed by a combination of ataxia, myopathy, peripheral neuropathy and progressive external ophthalmoplegia from the third decade of life onwards. We also identified novel clinical presentations with spastic paraparesis mimicking hereditary spastic paraplegia, and a multiple sclerosis-like illness. In contrast to initial reports, multi-system neurological disease was associated with all mutational subtypes, although there was an increased risk with missense mutations [odds ratio = 3.06, 95% confidence interval = 1.44-6.49; P = 0.0027], and mutations located within the guanosine triphosphate-ase region (odds ratio = 2.29, 95% confidence interval = 1.08-4.82; P = 0.0271). Histochemical and molecular characterization of skeletal muscle biopsies revealed the presence of cytochrome c oxidase-deficient fibres and multiple mitochondrial DNA deletions in the majority of patients harbouring OPA1 mutations, even in those with isolated optic nerve involvement. However, the cytochrome c oxidase-deficient load was over four times higher in the dominant optic atrophy + group compared to the pure optic neuropathy group, implicating a causal role for these secondary mitochondrial DNA defects in disease pathophysiology. Individuals with dominant optic atrophy plus phenotypes also had significantly worse visual outcomes, and careful surveillance is therefore mandatory to optimize the detection and management of neurological disability in a group of patients who already have significant visual impairment.