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Figure S1. STS-induced death is enhanced in UCP2hi MIA PaCa-2 and UCP2hi U-87 MG cells.

Cancer cells tilt their energy production away from oxidative phosphorylation (OXPHOS) toward glycolysis during malignant progression, even when aerobic metabolism is available. Reversing this phenomenon, known as the Warburg effect, may offer a generalized anticancer strategy. In this study, we show that overexpression of the mitochondrial membrane transport protein UCP2 in cancer cells is sufficient to restore a balance toward oxidative phosphorylation and to repress malignant phenotypes. Altered expression of glycolytic and oxidative enzymes mediated the effects of this metabolic shift. Notably, UCP2 overexpression increased signaling from the master energy-regulating kinase, adenosine monophosphate-activated protein kinase, while downregulating expression of hypoxia-induced factor. In support of recent new evidence about UCP2 function, we found that UCP2 did not function in this setting as a membrane potential uncoupling protein, but instead acted to control routing of mitochondria substrates. Taken together, our results define a strategy to reorient mitochondrial function in cancer cells toward OXPHOS that restricts their malignant phenotype. Cancer Res; 74(14); 3971–82. ©2014 AACR.

HIV-1 eradication is hindered by viral persistence in cell reservoirs, established not only in circulatory CD4+T-cells but also in tissue-resident macrophages. The nature of macrophage reservoirs and mechanisms of persistence despite combined anti-retroviral therapy (cART) remain unclear. Using genital mucosa from cART-suppressed HIV-1-infected individuals, we evaluated the implication of macrophage immunometabolic pathways in HIV-1 persistence. We demonstrate that ex vivo, macrophage tissue reservoirs contain transcriptionally active HIV-1 and viral particles accumulated in virus-containing compartments, and harbor an inflammatory IL-1R+S100A8+MMP7+M4-phenotype prone to glycolysis. Reactivation of infectious virus production and release from these reservoirs in vitro are induced by the alarmin S100A8, an endogenous factor produced by M4-macrophages and implicated in “sterile” inflammation. This process metabolically depends on glycolysis. Altogether, inflammatory M4-macrophages form a major tissue reservoir of replication-competent HIV-1, which reactivate viral production upon autocrine/paracrine S100A8-mediated glycolytic stimulation. This HIV-1 persistence pathway needs to be targeted in future HIV eradication strategies.

Assessing long-term mortality and identifying predictors of death in adults with mitochondrial diseases. We retrospectively included adult patients with genetically proven mitochondrial diseases referred to our centre between January 2000 and June 2016, and collected information relative to their genetic testing, clinical assessments, and vital status. We performed single and multiple variable analyses in search of predictors of total mortality, and calculated hazard ratios (HR) and 95% confidence intervals (CI). We included 267 patients (women 59%; median age 43.3 [31.3-54.2] years), including 111 with mitochondrial DNA (mtDNA) single large-scale deletions, 65 with m.3243A>G, 24 with m.8344A>G, 32 with other mtDNA point mutations, and 36 patients with nuclear genes mutations. Over a median follow-up of 8.9 years (0.3 to 18.7), 61 patients (22.8%) died, at a median age of 50.7 (37.9-51.9) years. Primary cause of death was cardiovascular disease in 16 patients (26.2%), respiratory in 11 (18.0%), and gastrointestinal in 5 (8.1%). By multiple variable analysis, diabetes (HR 2.75; 95% CI 1.46-5.18), intraventricular cardiac conduction defects (HR 3.38; 95% CI 1.71-6.76) and focal brain involvement (HR 2.39; 95% CI 1.25-4.57) were independent predictors of death. Adult patients with mitochondrial diseases present high morbidity that can be independently predicted by the presence of diabetes, intraventricular cardiac conduction defects, and focal brain involvement.

Embryonic stem cells (ESC) have the unique ability to differentiate into all three germ cell layers. ESC transition through different states of pluripotency in response to growth factor signals and environmental cues before becoming terminally differentiated. Here, we demonstrated, by a multi-omic strategy, that the deubiquitinase USP9X regulates the developmental potential of ESC, and their transition from a naive to a more developmentally advance, or primed, state of pluripotency. We show that USP9X facilitates developmental gene expression and induces modifications of the mitochondrial bioenergetics, including decreased routing of pyruvate towards its oxidation and reduced respiration. In addition, USP9X binds to the pluripotency factor ESRRB, regulates its abundance and the transcriptional levels of a subset of its target genes. Finally, under permissive culture conditions, depletion of Usp9X accelerates cell differentiation in all cell lineages. We thus identified a new regulator of naive pluripotency and show that USP9X couples ESRRB pluripotency transcriptional network and cellular metabolism, both of which are important for ESC fate and pluripotency.

Objective To demonstrate the causal role in disease of the MT-TP m.15992A>T mutation observed in patients from 5 independent families. Methods Lactate measurement, muscle histology, and mitochondrial activities in patients; PCR-based analyses of the size, amount, and sequence of muscle mitochondrial DNA (mtDNA) and proportion of the mutation; respiration, mitochondrial activities, proteins, translation, transfer RNA (tRNA) levels, and base modification state in skin fibroblasts and cybrids; and reactive oxygen species production, proliferation in the absence of glucose, and plasma membrane potential in cybrids. Results All patients presented with severe exercise intolerance and hyperlactatemia. They were associated with prominent exercise-induced muscle swelling, conspicuous in masseter muscles (2 families), and/or with congenital cataract (2 families). MRI confirmed exercise-induced muscle edema. Muscle disclosed severe combined respiratory defect. Muscle mtDNA had normal size and amount. Its sequence was almost identical in all patients, defining the haplotype as J1c10, and sharing 31 variants, only 1 of which, MT-TP m.15992A>T, was likely pathogenic. The mutation was homoplasmic in all tissues and family members. Fibroblasts and cybrids with homoplasmic mutation had defective respiration, low complex III activity, and decreased tRNAPro amount. Their respiratory complexes amount and tRNAPro aminoacylation appeared normal. Low proliferation in the absence of glucose demonstrated the relevance of the defects on cybrid biology while abnormal loss of cell volume when faced to plasma membrane depolarization provided a link to the muscle edema observed in patients. Conclusions The homoplasmic MT-TP m.15992A>T mutation in the J1c10 haplotype causes exercise-induced muscle swelling and fatigue.

Synthetic lethality is an efficient mechanism-based approach to selectively target DNA repair defects. Excision repair cross-complementation group 1 (ERCC1) deficiency is frequently found in non–small-cell lung cancer (NSCLC), making this DNA repair protein an attractive target for exploiting synthetic lethal approaches in the disease. Using unbiased proteomic and metabolic high-throughput profiling on a unique in-house–generated isogenic model of ERCC1 deficiency, we found marked metabolic rewiring of ERCC1-deficient populations, including decreased levels of the metabolite NAD+ and reduced expression of the rate-limiting NAD+ biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT). We also found reduced NAMPT expression in NSCLC samples with low levels of ERCC1. These metabolic alterations were a primary effect of ERCC1 deficiency, and caused selective exquisite sensitivity to small-molecule NAMPT inhibitors, both in vitro — ERCC1-deficient cells being approximately 1,000 times more sensitive than ERCC1-WT cells — and in vivo. Using transmission electronic microscopy and functional metabolic studies, we found that ERCC1-deficient cells harbor mitochondrial defects. We propose a model where NAD+ acts as a regulator of ERCC1-deficient NSCLC cell fitness. These findings open therapeutic opportunities that exploit a yet-undescribed nuclear-mitochondrial synthetic lethal relationship in NSCLC models, and highlight the potential for targeting DNA repair/metabolic crosstalks for cancer therapy.

Objective To explore potential spreading to peripheral nerves of the mitochondrial dysfunction in chronic progressive external ophthalmoplegia (CPEO) by assessing axonal excitability. Methods CPEO patients (n = 13) with large size deletion of mitochondrial DNA and matching healthy controls (n = 22) were included in a case-control study. Muscle strength was quantified using MRC sum-score and used to define two groups of patients: CPEO-weak and CPEO-normal (normal strength). Nerve excitability properties of median motor axons were assessed with the TROND protocol and changes interpreted with the aid of a model. Results Alterations of nerve excitability strongly correlated with scores of muscle strength. CPEO-weak displayed abnormal nerve excitability compared to CPEO-normal and healthy controls, with increased superexcitability and responses to hyperpolarizing current. Modeling indicated that the CPEO-weak recordings were best explained by an increase in the ‘Barrett-Barrett’ conductance across the myelin sheath. Conclusion CPEO patients with skeletal weakness presented sub-clinical nerve excitability changes, which were not consistent with axonal membrane depolarization, but suggested Schwann cell involvement. Significance This study provides new insights into the spreading of large size deletion of mitochondrial DNA to Schwann cells in CPEO patients.

Theileria annulata infects bovine leukocytes, transforming them into invasive, cancer-like cells that cause the widespread disease called tropical theileriosis. We report that in Theileria-transformed leukocytes hexokinase-2 (HK2) binds to B cell lymphoma-2-associated death promoter (BAD) only when serine (S) 155 in BAD is phosphorylated. We show that HK2 recruitment to BAD is abolished by a cell-penetrating peptide that acts as a non-phosphorylatable BAD substrate that inhibits endogenous S155 phosphorylation, leading to complex dissociation and ubiquitination and degradation of HK2 by the proteasome. As HK2 is a critical enzyme involved in Warburg glycolysis, its loss forces Theileria-transformed macrophages to switch back to HK1-dependent oxidative glycolysis that down-regulates macrophage proliferation only when they are growing on glucose. When growing on galactose, degradation of HK2 has no effect on Theileria-infected leukocyte proliferation, because metabolism of this sugar is independent of hexokinases. Thus, targeted disruption of the phosphorylation-dependent HK2/BAD complex may represent a novel approach to control Theileria-transformed leukocyte proliferation.

Polymerase γ catalytic subunit (POLG) gene encodes the enzyme responsible for mitochondrial DNA (mtDNA) synthesis. Mutations affecting POLG are the most prevalent cause of mitochondrial disease because of defective mtDNA replication and lead to a wide spectrum of clinical phenotypes characterized by mtDNA deletions or depletion. Enhancing mitochondrial deoxyribonucleoside triphosphate (dNTP) synthesis effectively rescues mtDNA depletion in different models of defective mtDNA maintenance due to dNTP insufficiency. In this study, we studied mtDNA copy number recovery rates following ethidium bromide-forced depletion in quiescent fibroblasts from patients harboring mutations in different domains of POLG. Whereas control cells spontaneously recovered initial mtDNA levels, POLG-deficient cells experienced a more severe depletion and could not repopulate mtDNA. However, activation of deoxyribonucleoside (dN) salvage by supplementation with dNs plus erythro-9-(2-hydroxy-3-nonyl) adenine (inhibitor of deoxyadenosine degradation) led to increased mitochondrial dNTP pools and promoted mtDNA repopulation in all tested POLG-mutant cells independently of their specific genetic defect. The treatment did not compromise POLG fidelity because no increase in multiple deletions or point mutations was detected. Our study suggests that physiologic dNTP concentration limits the mtDNA replication rate. We thus propose that increasing mitochondrial dNTP availability could be of therapeutic interest for POLG deficiency and other conditions in which mtDNA maintenance is challenged.-Blazquez-Bermejo, C., Carreno-Gago, L., Molina-Granada, D., Aguirre, J., Ramon, J., Torres-Torronteras, J., Cabrera-Perez, R., Martin, M. A., Dominguez-Gonzalez, C., de la Cruz, X., Lombes, A., Garcia-Arumi, E., Marti, R., Camara, Y. Increased dNTP pools rescue mtDNA depletion in human POLG-deficient fibroblasts.

Background and purpose Our aim was to determine the prognostic value of urine and blood heteroplasmy in patients with the m.3243A>G mutation. Methods Adults with the m.3243A>G mutation referred to our institution between January 2000 and May 2014 were retrospectively included. The relationship between their baseline clinical characteristics, their mutation load in urine and blood, and major adverse events (MAEs) during follow-up, defined as medical complications requiring a hospitalization or complicated by death, was studied. Results Of the 43 patients (age 45.6 ± 13.3 years) included in the study, 36 patients were symptomatic, including nine with evidence of focal brain involvement, and seven were asymptomatic. Over a 5.5 ± 4.0 year mean follow-up duration, 14 patients (33%) developed MAEs. Patients with MAEs had a higher mutation load than others in urine (60.1% ± 13.8% vs. 40.6% ± 26.2%, P = 0.01) and in blood (26.9% ± 18.4% vs. 16.0% ± 12.1%, P = 0.03). Optimal cutoff values for the prediction of MAEs were 45% for urine and 35% for blood. In multivariate analysis, mutation load in urine ≥45% [odds ratio 25.3; 95% confidence interval (CI) 1.1–567.8; P = 0.04], left ventricular hypertrophy (odds ratio 16.7; 95% CI 1.3– 222.5; P = 0.03) and seizures (odds ratio 48.3; 95% CI 2.5–933; P = 0.01) were associated with MAEs. Conclusions Patients with the m.3243A>G mutation are at high risk of MAEs, which can be independently predicted by mutation load in urine ≥45%, a personal history of seizures, and left ventricular hypertrophy.

Mitotane (also termed o,p'‑DDD) is the most effective therapy for advanced adrenocortical carcinoma (ACC). Mitotane‑induced dyslipidemia is treated with statins. Mitotane and statins are known to exert anti‑proliferative effects in vitro; however, the effects of statins have never been directly evaluated in patients with ACC and ACC cells, at least to the best of our knowledge. Thus, in this study, we aimed to examine the effects of the rosuvastatin on ACC cells. It has been shown that the combined use of mitotane and statins significantly increases the tumor control rate in patients with ACC; however, it would be of interest to elucidate the molecular mechanisms involved in this potentiation. In this study, we examined the effects of mitotane, rosuvastatin and their combination in NCI‑H295R human ACC cells using proliferation assays, gene expression analyses and free intracellular cholesterol measurements. The results revealed that mitotane dose‑dependently reduced cell viability, induced apoptosis and increased intracellular free cholesterol levels, considered as one of the key features of mitotane action, while rosuvastatin alone reduced cell viability and increased apoptosis at high concentrations. We also demonstrated that rosuvastatin potentiated the effects of mitotane by reducing cell viability, inducing apoptosis, increasing intracellular free cholesterol levels, and by decreasing the expression of 3‑hydroxy‑3‑methylglutaryl‑CoA reductase (HMGCR) and ATP binding cassette subfamily a member 1 (ABCA1), genes involved in cholesterol metabolism, and inhibiting steroidogenesis. Collectively, potentiating the effects of mitotane with the use of rosuvastatin may provide novel therapeutic strategies for ACC, given that the combination of these drugs, pending clinical validation, may lead to the better management of ACC.

Although relatively common in children, severe acute lactic acidosis is rare in adults with mitochondrial myopathies. We report here three cases, aged 27, 32 and 32 years, who developed life-threatening metabolic crisis with severe lactic acidosis, requiring hospitalisation in intensive care unit. Plasma lactates were elevated 10 to 15 fold normal values, necessitating extra-renal dialysis. By contrast CK levels were moderately increased (3 to 5N). No triggering factor was identified, but retrospectively all patients reported long-lasting mild muscle fatigability and weakness before their acute metabolic crisis. All of them recovered after prolonged intensive care but resting lactate levels remained elevated. Muscle biopsy showed ragged-red and COX-negative fibers in two patients and mild lipidosis in the third one. Heteroplasmic pathogenic point mutations were detected in MT-TL1 (m.3280G>A;m.3258C>T) and MT-TK (m.8363A>G). Life-threatening lactic acidosis may thus be a major inaugural clinical manifestation in adults with mitochondrial myopathies. Prolonged intensive care may lead to a dramatic and sustained improvement and is mandatory in such cases.

Mammalian respiratory complex I (CI) biogenesis requires both nuclear and mitochondria-encoded proteins and is mostly organized in respiratory supercomplexes. Among the CI proteins encoded by the mitochondrial DNA, NADH-ubiquinone oxidoreductase chain 1 (ND1) is a core subunit, evolutionary conserved from bacteria to mammals. Recently, ND1 has been recognized as a pivotal subunit in maintaining the structural and functional interaction among the hydrophilic and hydrophobic CI arms. A critical role of human ND1 both in CI biogenesis and in the dynamic organization of supercomplexes has been depicted, although the proof of concept is still missing and the critical amount of ND1 protein necessary for a proper assembly of both CI and supercomplexes is not defined. By exploiting a unique model in which human ND1 is allotopically re-expressed in cells lacking the endogenous protein, we demonstrated that the lack of this protein induces a stall in the multi-step process of CI biogenesis, as well as the alteration of supramolecular organization of respiratory complexes. We also defined a mutation threshold for the m.3571insC truncative mutation in mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1 (MT-ND1), below which CI and its supramolecular organization is recovered, strengthening the notion that a certain amount of human ND1 is required for CI and supercomplexes biogenesis.

We describe two patients with mitochondrial DNA mutations in the gene encoding cytochrome b (m.15579A>G, p.Tyr278Cys and m.15045G>A p.Arg100Gln), which presented as a pure myopathic form (exercise intolerance), with an onset in childhood. Diagnosis was delayed, because acylcarnitine profile showed an increase in medium and long-chain acylcarnitines, suggestive of multiple acyl-CoA dehydrogenase deficiency, riboflavin transporter deficiency or FAD metabolism disorder. Implication of cytochrome b in fatty acid oxidation, and physiopathology of the mutations are discussed.

Mitochondrial diseases, defined as the diseases due to oxidative phosphorylation defects, are the most frequent inborn errors of metabolism. Their clinical presentation is highly diverse. Their diagnosis is difficult. It relies on metabolic parameters, histological anomalies and enzymatic assays showing defective activity, all of which are both inconstant and relatively unspecific. Most mitochondrial diseases have a genetic origin. Candidate genes are very numerous, located either in the mitochondrial genome or the nuclear DNA. Pathophysiological mechanisms of mitochondrial diseases are still the matter of much debate. Those underlying the tissue-specificity of diseases due to the alterations of a ubiquitously expressed gene are discussed including (i) quantitative aspect of the expression of the causal gene or its partners when appropriate, (ii) quantitative aspects of the bioenergetic function in each tissue, and (iii) tissue distribution of heteroplasmic mitochondrial DNA alterations.

OBJECTIVES: The common data elements (CDE) project was developed by the National Institute of Neurological Disorders and Stroke (NINDS) to provide clinical researchers with tools to improve data quality and allow for harmonization of data collected in different research studies. CDEs have been created for several neurological diseases; the aim of this project was to develop CDEs specifically curated for mitochondrial disease (Mito) to enhance clinical research. METHODS: Nine working groups (WGs), composed of international mitochondrial disease experts, provided recommendations for Mito clinical research. They initially reviewed existing NINDS CDEs and instruments, and developed new data elements or instruments when needed. Recommendations were organized, internally reviewed by the Mito WGs, and posted online for external public comment for a period of eight weeks. The final version was again reviewed by all WGs and the NINDS CDE team prior to posting for public use. RESULTS: The NINDS Mito CDEs and supporting documents are publicly available on the NINDS CDE website (https://commondataelements.ninds.nih.gov/), organized into domain categories such as Participant/Subject Characteristics, Assessments, and Examinations. CONCLUSION: We developed a comprehensive set of CDE recommendations, data definitions, case report forms (CRFs), and guidelines for use in Mito clinical research. The widespread use of CDEs is intended to enhance Mito clinical research endeavors, including natural history studies, clinical trial design, and data sharing. Ongoing international collaboration will facilitate regular review, updates and online publication of Mito CDEs, and support improved consistency of data collection and reporting.

Hydrogen sulfide (H2S) is the third gasotransmitter discovered. Sulfide shares with the two others (NO and CO) the same inhibiting properties towards mitochondrial respiration. However, in contrast with NO or CO, sulfide at concentrations lower than the toxic (μM) level is an hydrogen donor and a substrate for mitochondrial respiration. This is due to the activity of a sulfide quinone reductase found in a large majority of mitochondria. An ongoing study of the metabolic state of liver in obese patients allowed us to evaluate the sulfide oxidation capacity with twelve preparations of human liver mitochondria. The results indicate relatively high rates of sulfide oxidation with a large variability between individuals. These observations made with isolated mitochondria appear in agreement with the main characteristics of sulfide oxidation as established before with the help of cellular models.

Aims: Storkhead box 1 (STOX1) is a winged-helix transcription factor that is implicated in the genetic forms of a high-prevalence human gestational disease, pre-eclampsia. STOX1 overexpression confers pre-eclampsia-like transcriptomic features to trophoblastic cell lines and pre-eclampsia symptoms to pregnant mice. The aim of this work was to evaluate the impact of STOX1 on free radical equilibrium and mitochondrial function, both in vitro and in vivo. Results: Transcriptome analysis of STOX1-transgenic versus nontransgenic placentas at 16.5 days of gestation revealed alterations of mitochondria-related pathways. Placentas overexpressing STOX1 displayed altered mitochondrial mass and were severely biased toward protein nitration, indicating nitroso-redox imbalance in vivo. Trophoblast cells overexpressing STOX1 displayed an increased mitochondrial activity at 20% O2 and in hypoxia, despite reduction of the mitochondrial mass in the former. STOX1 overexpression is, therefore, associated with hyperactive mitochondria, resulting in increased free radical production. Moreover, nitric oxide (NO) production pathways were activated, resulting in peroxynitrite formation. At low oxygen pressure, STOX1 overexpression switched the free radical balance from reactive oxygen species (ROS) to reactive nitrogen species (RNS) in the placenta as well as in a trophoblast cell line. Innovation: In pre-eclamptic placentas, NO interacts with ROS and generates peroxynitrite and nitrated proteins as end products. This process will deprive the maternal organism of NO, a crucial vasodilator molecule. Conclusion: Our data posit STOX1 as a genetic switch in the ROS/RNS balance and suggest an explanation for elevated blood pressure in pre-eclampsia. Antioxid. Redox Signal. 21, 819–834.

Introduction The prevalence of arterial hypertension in mitochondrial diseases remains unknown. Mitochondrial component is suspected in the genetics of hypertension in the general population. Materials and methods Between January 2000 and May 2014, we retrospectively included patients with genetically proven mitochondrial diseases. We recorded clinical, genetic and cardiac exploration data, including the measure of arterial pressure. Results We included 260 patients in the study (mean age = 44, women = 158). The prevalence of hypertension was 41.5%. The prevalence of hypertension by sex and age was higher than that observed in the general population for all groups. The prevalence of hypertension was significantly higher inpatients with MELAS mutations (66%) and MERRFs mutations (61%). Inpatients with MELAS mutation, the presence of hypertension was significantly associated with age and mutation rate in the blood (OR = 1.12; P = 0.02) in multivariate analysis. Conclusion The prevalence of hypertension was more important inpatients having a mitochondrial disease. The increased risk was more important in patient with MELAS or MERRF and depended on the rate of heteroplasmy.

Objective: To report that homoplasmic deleterious mutations in the mitochondrial DNA MT-ATP6/8 genes may be responsible for acute episodes of limb weakness mimicking periodic paralysis due to channelopathies and dramatically responding to acetazolamide. Methods: Mitochondrial DNA sequencing and restriction PCR, oxidative phosphorylation functional assays, reactive oxygen species metabolism, and patch-clamp technique in cultured skin fibroblasts. Results: Occurrence of a typical MELAS (mitochondrial encephalopathy with lactic acidosis and stroke-like episodes) syndrome in a single member of a large pedigree with episodic weakness associated with a later-onset distal motor neuropathy led to the disclosure of 2 deleterious mitochondrial DNA mutations. The MT-ATP6 m.9185T>C p.Leu220Pro mutation, previously associated with Leigh syndrome, was present in all family members, while the MT-TL1 m.3271T>C mutation, a known cause of MELAS syndrome, was observed in the sole patient with MELAS presentation. Significant defect of complexes V and I as well as oxidative stress were observed in both primary fibroblasts and cybrid cells with 100% m.9185T>C mutation. Permanent plasma membrane depolarization and altered permeability to K + in fibroblasts provided a link with the paralysis episodes. Screening of 9 patients, based on their clinical phenotype, identified 4 patients with similar deleterious MT-ATP6 mutations (twice m.9185T>C and once m.9176T>C or m.8893T>C). A fifth patient presented with an original potentially deleterious MT-ATP8 mutation (m.8403T>C). All mutations were associated with almost-normal complex V activity but significant oxidative stress and permanent plasma membrane depolarization. Conclusion: Homoplasmic mutations in the MT-ATP6/8 genes may cause episodic weakness responding to acetazolamide treatment.

Many individuals with abnormalities of mitochondrial respiratory chain complex III remain genetically undefined. Here, we report mutations (c.288G>T [p.Trp96Cys] and c.643C>T [p.Leu215Phe]) in CYC1, encoding the cytochrome c1 subunit of complex III, in two unrelated children presenting with recurrent episodes of ketoacidosis and insulin-responsive hyperglycemia. Cytochrome c1, the heme-containing component of complex III, mediates the transfer of electrons from the Rieske iron-sulfur protein to cytochrome c. Cytochrome c1 is present at reduced levels in the skeletal muscle and skin fibroblasts of affected individuals. Moreover, studies on yeast mutants and affected individuals’ fibroblasts have shown that exogenous expression of wild-type CYC1 rescues complex III activity, demonstrating the deleterious effect of each mutation on cytochrome c1 stability and complex III activity.

Objectives: To determine the long-term incidence of cardiac life-threatening complications and death in patients with the m.3243A>G mutation, and to identify cardiac prognostic factors. Methods: We retrospectively included patients carrying the m.3243A>G mutation who were admitted to the Neuromuscular Disease Clinic of Pitie Salpetriere Hospital between January 1992 and December 2010. We collected information relative to their yearly neurologic and cardiac investigations, their mutation load in blood, urine, and muscle at initial admission, and the occurrence of cardiac life-threatening adverse events and death during follow-up. Results: Forty-one patients (median age = 47 years [36–55 years], men = 13) were included, of whom 38 had clinical manifestations of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) and 3 were asymptomatic. One patient had a personal history of cardiac transplantation. Cardiac investigations displayed left ventricular hypertrophy, left ventricular dysfunction, or both abnormalities in 18 patients, along with Wolff-Parkinson-White syndrome in 7, conduction system disease in 4, and atrial fibrillation in 1. Over a median 5-year (3–9 years) follow-up period, 11 patients died, including 3 due to heart failure; 7 had life-threatening adverse events, including 6 hospitalizations for severe heart failure and 1 resuscitated cardiac arrest. By multivariate analysis, left ventricular hypertrophy was the only parameter independently associated with occurrence of cardiac adverse events. Conclusion: Patients with the m.3243A>G mutation have a high incidence of cardiac death and life-threatening adverse events. Left ventricular hypertrophy was the only parameter independently associated with occurrence of these events.

Deleterious consequences of heterozygous OPA1 mutations responsible for autosomal dominant optic atrophy remain a matter of debate. Primary skin fibroblasts derived from patients have shown diverse mitochondrial alterations that were however difficult to resolve in a unifying scheme. To address the potential use of these cells as disease model, we undertook parallel and quantitative analyses of the diverse reported alterations in four fibroblast lines harboring different OPA1 mutations, nonsense or missense, in the guanosine triphosphatase or the C-terminal coiled-coil domains. We tackled several factors potentially underlying discordant reports and showed that fibroblasts with heterozygous OPA1 mutations present with several mitochondrial alterations. These included defective mitochondrial fusion during pharmacological challenge with the protonophore carbonyl cyanide m‐chlorophenyl hydrazone, significant mitochondrial elongation with decreased OPA1 and DRP1 proteins, and abnormal mitochondrial fragmentation during glycolysis shortage or exogenous oxidative stress. Respiratory complex IV activity and subunits steady-state were decreased without alteration of the mitochondrial deoxyribonucleic acid size, amount or transcription. Physical link between OPA1 protein and oxidative phosphorylation was shown by reciprocal immunoprecipitation. Altered cristae structure coexisted with normal response to pro-apoptotic stimuli and expression of Bax or Bcl2 proteins. Skin fibroblasts with heterozygous OPA1 mutations thus share significant mitochondrial remodeling, and may therefore be useful for analyzing disease pathophysiology. Identifying whether the observed alterations are also present in ganglion retinal cells, and which of them underlies their degeneration process remains however an essential goal for therapeutic strategy.

We investigated a family in which the index subject presented with severe congenital lactic acidosis and dysmorphic features associated with a cytochrome c oxidase (COX)-assembly defect and a specific decrease in the synthesis of COX I, the subunit that nucleates COX assembly. Using a combination of microcell-mediated chromosome transfer, homozygosity mapping, and transcript profiling, we mapped the gene defect to chromosome 12 and identified a homozygous missense mutation (c.88G>A) in C12orf62. C12orf62 was not detectable by immunoblot analysis in subject fibroblasts, and retroviral expression of the wild-type C12orf62 cDNA rescued the biochemical phenotype. Furthermore, siRNA-mediated knockdown of C12orf 62 recapitulated the biochemical defect in control cells and exacerbated it in subject cells. C12orf62 is apparently restricted to the vertebrate lineage. It codes for a very small (6 kDa), uncharacterized, single-transmembrane protein that localizes to mitochondria and elutes in a complex of ∼110 kDa by gel filtration. COX I, II, and IV coimmunoprecipated with an epitope-tagged version of C12orf62, and 2D blue-native-polyacrylamide-gel-electrophoresis analysis of newly synthesized mitochondrial COX subunits in subject fibroblasts showed that COX assembly was impaired and that the nascent enzyme complex was unstable. We conclude that C12orf62 is required for coordination of the early steps of COX assembly with the synthesis of COX I.

Neonatal cardiomyopathies due to mitochondrial oxidative phosphorylation (OXPHOS) defects are extremely severe conditions which can be either isolated or included in a multi-organ disease, with or without metabolic crises, of which profound lactic acidosis is the prominent feature. Cardiomyopathy is more often hypertrophic than dilated. Antenatal manifestations such as fetal cardiomyopathy, arrhythmia and/or hydrops have been reported. Pathophysiological mechanisms are complex, going beyond ATP deficiency of the high-energy-consuming neonatal myocardium. Birth is a key metabolic period when the myocardium switches ATP production from anaerobic glycolysis to mitochondrial fatty acid oxidation and OXPHOS. Heart-specificity of the defect may be related to the specific localization of the defect, to the high myocardium dependency on OXPHOS, and/or to interaction between the primary genetic alteration and other factors such as modifier genes. Therapeutic options are limited but standardized diagnostic procedures are mandatory to confirm the OXPHOS defect and to identify its causal mutation, allowing genetic counseling and potential prenatal diagnosis.

Resume Introduction Le deficit multiple en acyl-CoA deshydrogenases (MADD) est une maladie hereditaire rare affectant l’oxydation des acides gras, et pouvant resulter du deficit de l’un des deux transporteurs d’electrons : electron transfer flavoprotein (ETF) ou electron transfer flavoprotein ubiquinone oxydoreductase (ETF-QO). Il se manifeste le plus souvent dans l’enfance ou chez l’adulte jeune, par une atteinte plurisytemique avec encephalopathie ou une atteinte musculaire. Observations Nous rapportons deux observations de patients adultes presentant un deficit en ETF-QO, confirme sur le plan moleculaire (gene ETFDH), revele par une faiblesse musculaire, avec une importante surcharge en lipides a la biopsie musculaire. L’un des patients avait presente un episode d’encephalopathie avec vomissements dix ans avant l’apparition des manifestations musculaires. Le diagnostic fut pose devant la presence d’anomalies caracteristiques du profil des acylcarnitines apres analyse par spectrometrie de masse en tandem. Une amelioration clinique spectaculaire fut obtenue apres instauration d’un traitement par riboflavine et L-carnitine chez les deux patients. Conclusion Le caractere traitable de cette affection doit conduire a envisager ce diagnostic en effectuant une etude du profil des acylcarnitines chez tout patient presentant une faiblesse musculaire inexpliquee.

Introduction Le FEEc reproduit le site de liaison de la Fertiline β presente a la surface du spermatozoide. Cette molecule est impliquee dans les mecanismes d’adhesion et de fusion gametique. La supplementation du milieu de FIV en FEEc permet d’augmenter l’index de fecondation d’ovocytes depellucides chez l’humain [1] . Chez la souris, le peptide equivalent (QDEc) permet d’augmenter de 80 % le taux de fecondation des ovocytes en cumulus intact sans augmenter la polyspermie [2] . L’objectif est de determiner le/les mecanismes d’action du FEEc qui ameliore les capacites fusiogenes des spermatozoides. Materiel et methodes Les parametres du mouvement ont ete etudies avec un CASA sur des echantillons de sperme de 38 hommes avec des parametres spermatiques normaux. La fonction mitochondriale des spermatozoides de sperme de donneurs, incubes en presence ou en absence de FEEc, a ete evalues en mesurant : – la production d’ATP par bioluminescence ; – la respiration avec un oxygraphe ; – le potentiel de membrane mitochondrial (PMM) avec des sondes potentio-metriques et un cytometre de flux. Resultats Le FEEc ameliore significativement les parametres du mouvement (VAP, p = 0,008 ; VSL, p = 0,048 ; VCL, p Conclusion Le peptide pourrait constituer une nouvelle therapeutique non invasive pour ameliorer les resultats en FIV humaine. Le FEEc a ete evalue au cours d’un essai clinique preliminaire randomise dans notre laboratoire de FIV avec des resultats tres encourageants.

Annonacin, a natural lipophilic inhibitor of mitochondrial complex I has been implicated in the etiology of a sporadic neurodegenerative tauopathy in Guadeloupe. We therefore studied further compounds representing the broad biochemical spectrum of complex I inhibitors to which humans are potentially exposed. We determined their lipophilicity, their effect on complex I activity in submitochondrial particles, and their effect on cellular ATP levels, neuronal cell death and somatodendritic redistribution of phosphorylated tau protein (AD2 antibody against pS396/pS404-tau) in primary cultures of fetal rat striatum. The 24 compounds tested were lipophilic (logP range 0.9-8.5; exception: MPP(+) logP=-1.35) and potent complex I inhibitors (IC(50) range 0.9 nM-2.6 mM). They all decreased ATP levels (EC(50) range 1.9 nM-54.2 microM), induced neuronal cell death (EC(50) range 1.1 nM-54.5 microM) and caused the redistribution of AD2(+) tau from axons to the cell body (EC(5) range 0.6 nM-33.3 microM). The potency of the compounds to inhibit complex I correlated with their potency to induce tau redistribution (r=0.80, p

BACKGROUND In patients coinfected with hepatitis B virus (HBV) and human immunodeficiency virus (HIV), evolution toward cirrhosis and its complications is more rapid and severe than in patients infected with HBV alone. The outcome of liver transplantation in HBV-HIV-coinfected patients is poorly understood in terms of survival rate, HBV reactivation and mitochondrial toxicity on the liver graft. PATIENTS AND METHODS Between November 2002 and June 2007, 13 HIV-positive patients underwent liver transplantation because of end-stage liver disease due to HBV with or without coinfection with hepatitis D or C virus. These patients were prospectively followed for an average of 32 +/- 5.2 months (range 10-63 months). RESULTS All patients were alive at the end of the follow-up period and had normal liver function. Their HBV viral load was undetectable, no cccDNA was found in the liver graft and HIV infection was nonprogressive under antiretroviral therapy. Moreover, no mitochondrial toxicity was noted in the liver graft, as assessed by the spectrophotometric analysis of respiratory chain activities and by quantifying the mitochondrial DNA copy number. CONCLUSION HBV-HIV-coinfected patients can successfully undergo liver transplantation with excellent results in terms of survival, control of HBV replication after transplantation and mitochondrial toxicity.

Very Long-Chain Acyl-CoA dehydrogenase (VLCAD) deficiency is an inborn error of mitochondrial long-chain fatty acid oxidation (FAO) most often occurring in childhood with cardiac or liver involvement, but rhabdomyolysis attacks have also been reported in adults. We report in this study the clinical, biochemical and molecular studies in 13 adult patients from 10 different families with VLCAD deficiency. The enzyme defect was demonstrated in cultured skin fibroblasts or lymphocytes. All patients exhibited exercise intolerance and recurrent rhabdomyolysis episodes, which were generally triggered by strenuous exercise, fasting, cold or fever (mean age at onset: 10 years). Inaugural life-threatening general manifestations also occurred before the age of 3 years in four patients. Increased levels of long-chain acylcarnitines with tetradecenoylcarnitine (C14:1) as the most prominent species were observed in all patients. Muscle biopsies showed a mild lipidosis in four patients. For all patients but two, molecular analysis showed homozygous (4 patients) or compound heterozygous genotype (7 patients). For the two remaining patients, only one mutation in a heterozygous state was detected. This study confirms that VLCAD deficiency, although being less frequent than CPT II deficiency, should be systematically considered in the differential diagnosis of exercise-induced rhabdomyolysis. Measurement of fasting blood acylcarnitines by tandem mass spectrometry allows accurate biochemical diagnosis and should therefore be performed in all patients presenting with unexplained muscle exercise intolerance or rhabdomyolysis.

Resume Objectifs Les deficits de la chaine respiratoire mitochondriale sont connus pour leur grande variabilite phenotypique. Leur prevalence est probablement sous-estimee en raison de la difficulte a porter le diagnostic. Nous rapportons les donnees cliniques de notre serie de cytopathies mitochondriales en fonction de l’âge de debut des symptomes et l’interet des biopsies tissulaires, en particulier hepatique, dans l’aide au diagnostic des cytopathies mitochondriales. Methodes Notre serie est constituee de 18 cas pediatriques de deficit de la chaine respiratoire diagnostiques au CHU de Tours au cours des 10 dernieres annees et caracterises au plan clinique et paraclinique. Resultats Trois profils cliniques se distinguent en fonction de l’âge de survenue des premiers symptomes : neonatal pour 4 cas, entre 1 mois et 2 ans pour 10 cas ou apres 10 ans pour 4 cas. En revanche, aucun signe clinique n’apparait specifique d’un âge donne. La 2nde donnee importante concerne les explorations paracliniques : l’etude de la chaine respiratoire sur biopsie hepatique s’avere tres informative pour 100 % de nos patients, quels que soient leur âge ou leurs phenotypes, meme en presence d’une symptomatologie purement neurologique ou la biopsie musculaire demeure parfois non contributive. Commentaires Ces explorations biochimiques permettent de conforter le diagnostic de cytopathie mitochondriale en vue d’une etude moleculaire qui reste neanmoins souvent infructueuse aujourd’hui. Ces investigations devraient beneficier, a l’avenir, de l’apport des nouvelles techniques de criblage moleculaire basees sur l’utilisation des puces a ADN pour preciser les mutations responsables de ces affections graves et relativement frequentes.

Complex I or reduced nicotinamide adenine dinucleotide (NADH): ubiquinone oxydoreductase deficiency is the most common cause of respiratory chain defects. Molecular bases of complex I deficiencies are rarely identified because of the dual genetic origin of this multi-enzymatic complex (nuclear DNA and mitochondrial DNA) and the lack of phenotype-genotype correlation. We used a rapid method to screen patients with isolated complex I deficiencies for nuclear genes mutations by Surveyor nuclease digestion of cDNAs. Eight complex I nuclear genes, among the most frequently mutated (NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS7, NDUFS8, NDUFV1 and NDUFV2), were studied in 22 cDNA fragments spanning their coding sequences in 8 patients with a biochemically proved complex I deficiency. Single nucleotide polymorphisms and missense mutations were detected in 18.7% of the cDNA fragments by Surveyor nuclease treatment. Molecular defects were detected in 3 patients. Surveyor nuclease screening is a reliable method for genotyping nuclear complex I deficiencies, easy to interpret, and limits the number of sequence reactions. Its use will enhance the possibility of prenatal diagnosis and help us for a better understanding of complex I molecular defects.

Mitochondria are dynamic organelles with continuous fusion and fission, the equilibrium of which results in mitochondrial morphology. Evidence points to there being an intricate relationship between mitochondrial dynamics and oxidative phosphorylation. We investigated the bioenergetics modulation of mitochondrial morphology in five control cultured primary skin fibroblasts and seven with genetic alterations of oxidative phosphorylation. Under basal conditions, control fibroblasts had essentially filamentous mitochondria. Oxidative phosphorylation inhibition with drugs targeting complex I, III, IV or V induced partial but significant mitochondrial fragmentation, whereas dissipation of mitochondrial membrane potential (DΨm) provoked complete fragmentation, and glycolysis inhibition had no effect. Oxidative phosphorylation defective fibroblasts had essentially normal filamentous mitochondria under basal conditions, although when challenged some of them presented with mild alteration of fission or fusion efficacy. Severely defective cells disclosed complete mitochondrial fragmentation under glycolysis inhibition. In conclusion, mitochondrial morphology is modulated by DΨm but loosely linked to mitochondrial oxidative phosphorylation. Its alteration by glycolysis inhibition points to a severe oxidative phosphorylation defect.

ObjectiveTo examine the reversibility of adipose tissue alterations in HIV-infected patients after a 6-month interruption of antiretroviral therapy (ART).MethodsForty HIV-infected patients on stable effective ART were enrolled, 33 of them completed the study. Patients underwent a physical examination, laboratory tests and needle biopsy of subcutaneous abdominal adipose tissue both at inclusion and at month 6. Changes in fat morphology, mitochondrial DNA (mtDNA) content and gene expression were examined in 29, 23 and 20 patients, respectively.ResultsBody fat distribution was not clearly modified at month 6. Adipose tissue inflammation improved markedly, with fewer infiltrating macrophages and fewer tumour necrosis factor a (TNFα)- and interleukin 6 (IL6)-expressing cells. Expression of peroxisome proliferator-activated receptor γ (PPAR-γ) and of markers of mitochondrial function and biogenesis (cytochrome oxidase 2 and PPAR-γ coreceptor 1α) improved after protease inhibitor (PI) withdrawal. In patients who stopped taking stavudine or zidovudine, the number of TNFα- and IL6-expressing cells was lower at month 6 than at month 0, and so was CD68 expression, a macrophage marker. Adipocyte mitochondrial status also improved, with lower mitochondrial density and cytochrome oxidase 4 mRNA levels, and higher mtDNA content. Sterol regulatory element binding protein 1 mRNA levels increased, reflecting better adipocyte differentiation.ConclusionsA 6-month ART interruption markedly improved adipose tissue functions, although fat distribution did not visibly change. Stavudine and zidovudine were associated with marked inflammation, which improved when these drugs were withdrawn; they also had a negative effect on differentiation and mitochondrial status. PIs were also associated with altered adipocyte differentiation and mitochondrial status. These data clearly show the detrimental effect of antiretroviral drugs, in particular thymidine analogues, on adipose tissue and argue for switch strategies sparing these drugs.

We describe a young woman who presented with a progressive myopathy since the age of 9. Spectrophotometric analysis of the respiratory chain in muscle tissue revealed combined and profound complex I, III, II + III, and IV deficiency ranging from 60% to 95% associated with morphological and histochemical abnormalities of the muscle. An exhaustive screening of mitochondrial transfer and ribosomal RNAs showed a novel G > A substitution at nucleotide position 3090 which was detected only in urine sediment and muscle of the patient and was not found in her mother’s blood cells and urine sample. We suggest that this novel de novo mutation in the 16S ribosomal RNA, a nucleotide which is highly conserved in different species, would impair mitochondrial protein synthesis and would cause a severe myopathy.

Skin fibroblasts are essential tools for biochemical, genetic and physiopathological investigations of mitochondrial diseases. Their immortalization has been previously performed to overcome the limited number of divisions of these primary cells but it has never been systematically evaluated with respect to efficacy and impact on the oxidative phosphorylation (OXPHOS) characteristics of the cells. We successfully immortalized with the human telomerase gene 15 human fibroblasts populations, 4 derived from controls and 11 from patients with diverse respiratory chain defects. Immortalization induced significant but mild modification of the OXPHOS characteristics of the cells with lower rates of oxygen consumption and ATP synthesis associated with their loose coupling. However, it never significantly altered the type and severity of any genetic OXPHOS defect present prior to immortalization. Furthermore, it did not significantly modify the cells' dependence on glucose and sensitivity to galactose thus showing that immortalized cells could be screened by their nutritional requirement. Immortalized skin fibroblasts with significant OXPHOS defect provide reliable tools for the diagnosis and research of the genetic cause of mitochondrial defects. They also represent precious material to investigate the cellular responses to these defects, even though these should afterwards be verified in unmodified primary cells.

Aims The aim of this study is to assess the long-term cardiac prognosis of adults with mitochondrial diseases. Methods and results Between January 2000 and May 2014, we retrospectively included in this study 260 consecutive patients (60% women) ≥18 years (interquartile range 31–54), with genetically proven mitochondrial diseases, including 109 with mitochondrial DNA (mtDNA) single large-scale deletions, 64 with the m.3243A>G mutation in MT-TL1 , 51 with other mtDNA point mutations, and 36 patients with nuclear gene mutations. Cardiac involvement was present at baseline in 81 patients (30%). Single and multiple variable analyses were performed in search of predictors of major adverse cardiac events (MACEs), and hazard ratios (HRs) and 95% confidence intervals (CI) were calculated. Over a median follow-up of 7 years (3.6–11.7), 27 patients (10%) suffered a MACE, defined as sudden death, death due to heart failure (HF), resuscitated cardiac arrest, third-degree atrioventricular block, sinus node dysfunction, cardiac transplantation, or hospitalization for management of HF. Patients with single large-scale mtDNA deletions or m.3243A>G mutations had the highest incidence of MACE. By multiple variable analysis, intraventricular conduction block (HR = 16.9; 95% CI: 7.2–39.4), diabetes (HR = 7.0; 95% CI: 2.9–16.7), premature ventricular complexes (HR = 3.6; 95% CI: 1.4–9.2), and left ventricular (LV) hypertrophy (HR = 2.5; 95% CI: 1.1–5.8) were independent predictors of MACEs. In patients with zero, one, and two or more risk factors, the incidences of MACE were 1.7, 15 and 42%, respectively. Conclusion Patients with mitochondrial diseases are at high risk of MACE, independently predicted by intraventricular conduction block, diabetes, ventricular prematurity, and LV hypertrophy.

Neuroferritinopathy is a progressive potentially treatable adult-onset movement disorder caused by mutations in the ferritin light chain gene (FTL1). Features overlap with common extrapyramidal disorders: idiopathic torsion dystonia, idiopathic Parkinson's disease and Huntington's disease, but the phenotype and natural history have not been defined. We studied a genetically homogeneous group of 41 subjects with the 460InsA mutation in FTL1, documenting the presentation, clinical course, biochemistry and neuroimaging. The mean age of onset was 39.4 years (SD = 13.3, range 13-63), beginning with chorea in 50%, focal lower limb dystonia in 42.5% and parkinsonism in 7.5%. The majority reported a family history of a movement disorder often misdiagnosed as Huntington's disease. The disease progressed relentlessly, becoming generalized over a 5-10 year period, eventually leading to aphonia, dysphagia and severe motor disability with subcortical/frontal cognitive dysfunction as a late feature. A characteristic action-specific facial dystonia was common (65%), and in 63% there was asymmetry throughout the disease course. Serum ferritin levels were low in the majority of males and post-menopausal females, but within normal limits for pre-menopausal females. MR brain imaging was abnormal on all affected individuals and one presymptomatic carrier. In conclusion, isolated parkinsonism is unusual in neuroferritinopathy, and unlike Huntington's disease, cognitive changes are absent or subtle in the early stages. Depressed serum ferritin is common and provides a useful screening test in routine practice, and gradient echo brain MRI will identify all symptomatic cases.

Guidelines in the diagnosis and current dietary treatment of long-chain fatty acid (LCFA) defects have been collected according to evidence-based medicine. Since the identification of carnitine and carnitine palmitoyltransferase deficiency more than 25 years ago, nearly every enzymatic step required for beta-oxidation has been associated with an inherited metabolic disorder. These disorders effectively preclude the use of body fat as an energy source. Clinical consequences can range from no symptoms to severe manifestations including cardiomyopathy, hypoglycaemia, peripheral neuropathy and sudden death. A diet high in carbohydrates, diet with medium-chain triglycerides and reduced amount of LCFA has a beneficial effect (class IV evidence) and in appropriate deficiency states carnitine and riboflavin are used (good practice points).

Eukaryotic cells contain numerous copies of the mitochondrial genome (from 50 to 100 copies in the budding yeast to some thousands in humans) that localize to numerous intramitochondrial nucleoprotein complexes called nucleoids. The transmission of mitochondrial DNA differs significantly from that of nuclear genomes and depends on the number, molecular composition and dynamic properties of nucleoids and on the organization and dynamics of the mitochondrial compartment. While the localization, dynamics and protein composition of mitochondrial DNA nucleoids begin to be described, we are far from knowing all mechanisms and molecules mediating and/or regulating these processes. Here, we review our current knowledge on vertebrate nucleoids and discuss similarities and differences to nucleoids of other eukaryots.

Increased susceptibility to apoptosis has been shown in many models of mitochondrial defects but its relevance to human diseases is still discussed. We addressed the presence of apoptosis in muscle from patients with mitochondrial DNA (mtDNA) disorders. Taking advantage of the mosaic pattern of muscle morphological anomalies associated with heteroplasmic mtDNA alterations, we have used an in situ approach to address the relationship between apoptosis and respiratory defect, mitochondrial proliferation and mutation load. Different patterns of mitochondrial morphological alterations were provided by the analysis of muscles with large mtDNA deletion (16 cases) or with the MELAS mutation (4 cases). The patient's age at biopsy ranged from 0.4 to 66 years and the muscle mutant mtDNA proportion from 32 to 82%. Apoptotic muscle fibres were observed in a small proportion of muscle fibres of 16 out of the 20 biopsies by three different detection methods for different steps of apoptosis: caspase 3 activation, fragmentation of nuclear DNA [terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay] or overexpression of the pro-apoptotic factor Bax. Analysis of apoptotic features in parallel to cytochrome c oxidase (COX) and succinate dehydrogenase activity of more than 34,000 individual muscle fibres showed that apoptosis occurred only in muscle fibres with mitochondrial proliferation (ragged red fibres, RRF) irrespective of their COX activity. Molecular analyses of single muscle fibres evidenced that, as expected, the presence of COX defect was associated with higher proportion of mutant mtDNA and lower amount of normal mtDNA. Within COX-defective fibres, the presence of mitochondrial proliferation was associated with increase of the mtDNA content but without change in the ratio between normal and mutant mtDNA molecules, thus showing that mitochondrial proliferation was accompanied by similar amplification of normal and mutant mtDNA molecules. Within RRF, apoptosis was associated with higher mutation proportion, suggesting that it was provoked by severe respiratory defect in the same time as increased mitochondrial mass. In conclusion, apoptosis most probably contributes to mitochondrial pathology. It is tightly linked to mitochondrial proliferation and high mutation load. When considering training therapeutics, one will have to take into account the possibility to induce apoptosis in parallel to mitochondrial proliferation.

Resume Introduction La confusion diagnostique entre myasthenie et myopathie mitochondriale est frequente du fait du caractere voisin de la symptomatologie dans les deux affections (ophtalmoplegie, fatigabilite). Methodes Nous avons revu l’histoire clinique et le resultat des explorations complementaires de 12 patients (7F/5H, d’âge moyen 47 ± 14 ans) presentant une myopathie mitochondriale pour lesquels un premier diagnostic errone de myasthenie auto-immune avait conduit a une thymectomie. Resultats Les symptomes les plus frequents etaient la paralysie oculomotrice avec ptosis, les troubles bulbaires et la fatigabilite musculaire des membres. Les symptomes etaient moderement fluctuants, mais sans remission. Les elements qui avaient conduit au diagnostic de myasthenie etaient les suivants : un bloc neuromusculaire (2 cas) ; une reponse consideree comme positive aux anticholinesterasiques injectables (3 cas), oraux (2 cas) ; un taux d’anticorps anti-RACh limite considere comme positif (1 cas). Une atteinte plurisystemique etait relevee chez 5 patients. Le diagnostic de myopathie mitochondriale a ete etabli (a un âge moyen de 38 ± 12 ans) sur les donnees de la biopsie musculaire et la presence d’une deletion de l’ADN mitochondrial. Conclusions Chez un patient presentant une atteinte oculomotrice et une fatigue musculaire, l’evolution progressive et la presence d’une atteinte plurisystemique sont des arguments majeurs en faveur d’une pathologie mitochondriale. En l’absence d’elements convaincants en faveur d’une myasthenie, la biopsie musculaire a la recherche d’une mitochondriopathie doit etre pratiquee prealablement a l’indication d’une thymectomie.

Mitochondrial diseases are relatively common inherited metabolic diseases due to mitochondrial respiratory chain dysfunction. Their clinical presentation is extremely diverse, multisystemic or confined to a single tissue, sporadic or transmitted, by maternal or mendelian inheritance. The diagnosis of mitochondrial disorders is difficult. It is based upon several types of clues both clinical (family history, type of symptoms but also their association in syndromic presentation,...) and biological (alteration of the lactate metabolism, brain imaging, morphological alterations especially of muscle tissue). The diagnosis relies upon the demonstration of a defect of the respiratory chain activities and/or upon the identification of the underlying genetic alteration. Molecular diagnosis remains quite difficult and up to-date concerns essentially mitochondrial DNA mutations. On one hand, clinical and biological presentations as well as enzymatic defects lack specificity. On the other hand, candidate genes are very numerous and part of them are probably still unknown.

The mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) syndrome is characterized by the association of gastrointestinal and neurological symptoms. It is a rare autosomal recessive mitochondrial disorder with multiple mitochondrial DNA deletions and/or depletion. It is caused by thymidine phosphorylase (TP) gene mutations resulting in a complete abolition of TP activity. We tested 31 unrelated patients presenting either with a complete MNGIE syndrome (8 patients), a severe intestinal pseudo-obstruction (10 patients), and multiple deletions and/or depletion of mitochondrial DNA (13 patients). All the tested patients presenting with a complete MNGIE had increased thymidine levels in plasma and urine, and no TP activity. The group with pseudo-obstruction syndrome had normal or partial reduction of TP activity. We found pathogenic mutations on TP gene only in the MNGIE syndrome group: all the MNGIE patients were compound heterozygous or homozygous for mutations in the TP gene. Eight of these mutations are yet unreported, confirming the lack of genotype/phenotype correlation in this syndrome. Enzymatic activity and thymidine level are thus rapid diagnosis tests to detect MNGIE affected patients prior to genetic testing for patients with gastrointestinal symptoms.