Mitochondrial MDM2 Regulates Respiratory Complex I Activity Independently of p53.

Accumulating evidence indicates that the MDM2 oncoprotein promotes tumorigenesis beyond its canonical negative effects on the p53 tumor suppressor, but these p53-independent functions remain poorly understood. Here, we show that a fraction of endogenous MDM2 is actively imported in mitochondria to control respiration and mitochondrial dynamics independently of p53. Mitochondrial MDM2 represses the transcription of NADH-dehydrogenase 6 (MT-ND6) in vitro and in vivo, impinging on respiratory complex I activity and enhancing mitochondrial ROS production. Recruitment of MDM2 to mitochondria increases during oxidative stress and hypoxia. Accordingly, mice lacking MDM2 in skeletal muscles exhibit higher MT-ND6 levels, enhanced complex I activity, and increased muscular endurance in mild hypoxic conditions. Furthermore, increased mitochondrial MDM2 levels enhance the migratory and invasive properties of cancer cells. Collectively, these data uncover a previously unsuspected function of the MDM2 oncoprotein in mitochondria that play critical roles in skeletal muscle physiology and may contribute to tumor progression.

Increased dNTP pools rescue mtDNA depletion in human POLG-deficient fibroblasts.

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.-Blázquez-Bermejo, C., Carreño-Gago, L., Molina-Granada, D., Aguirre, J., Ramón, J., Torres-Torronteras, J., Cabrera-Pérez, R., Martín, M. Á., Domínguez-González, C., de la Cruz, X., Lombès, A., García-Arumí, E., Martí, R., Cámara, Y. Increased dNTP pools rescue mtDNA depletion in human POLG-deficient fibroblasts.

Incidence and predictors of total mortality in 267 adults presenting with mitochondrial diseases.

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.

Use of H2O2 to Cause Oxidative Stress, the Catalase Issue.

Addition of hydrogen peroxide (H2O2) is a method commonly used to trigger cellular oxidative stress. However, the doses used (often hundreds of micromolar) are disproportionally high with regard to physiological oxygen concentration (low micromolar). In this study using polarographic measurement of oxygen concentration in cellular suspensions we show that H2O2 addition results in O2 release as expected from catalase reaction. This reaction is fast enough to, within seconds, decrease drastically H2O2 concentration and to annihilate it within a few minutes. Firstly, this is likely to explain why recording of oxidative damage requires the high concentrations found in the literature. Secondly, it illustrates the potency of intracellular antioxidant (H2O2) defense. Thirdly, it complicates the interpretation of experiments as subsequent observations might result from high/transient H2O2 exposure and/or from the diverse possible consequences of the O2 release.

Drug-Induced Alterations of Mitochondrial DNA Homeostasis in Steatotic and Nonsteatotic HepaRG Cells.

Although mitochondriotoxicity plays a major role in drug-induced hepatotoxicity, alteration of mitochondrial DNA (mtDNA) homeostasis has been described only with a few drugs. Because it requires long drug exposure, this mechanism of toxicity cannot be detected with investigations performed in isolated liver mitochondria or cultured cells exposed to drugs for several hours or a few days. Thus, a first aim of this study was to determine whether a 2-week treatment with nine hepatotoxic drugs could affect mtDNA homeostasis in HepaRG cells. Previous investigations with these drugs showed rapid toxicity on oxidative phosphorylation but did not address the possibility of delayed toxicity secondary to mtDNA homeostasis impairment. The maximal concentration used for each drug induced about 10% cytotoxicity. Two other drugs, zalcitabine and linezolid, were used as positive controls for their respective effects on mtDNA replication and translation. Another goal was to determine whether drug-induced mitochondriotoxicity could be modulated by lipid overload mimicking nonalcoholic fatty liver. Among the nine drugs, imipramine and ritonavir induced mitochondrial effects suggesting alteration of mtDNA translation. Ritonavir toxicity was stronger in nonsteatotic cells. None of the nine drugs decreased mtDNA levels. However, increased mtDNA was observed with five drugs, especially in nonsteatotic cells. The mtDNA levels could not be correlated with the expression of key factors involved in mitochondrial biogenesis, such as peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α), PGC1ß, and AMP-activated protein kinase α-subunit. Hence, drug-induced impairment of mtDNA translation might not be rare, and increased mtDNA levels could be a frequent adaptive response to slight energy shortage. Nevertheless, this adaptation could be impaired by lipid overload.

ATAD3 gene cluster deletions cause cerebellar dysfunction associated with altered mitochondrial DNA and cholesterol metabolism.

Although mitochondrial disorders are clinically heterogeneous, they frequently involve the central nervous system and are among the most common neurogenetic disorders. Identifying the causal genes has benefited enormously from advances in high-throughput sequencing technologies; however, once the defect is known, researchers face the challenge of deciphering the underlying disease mechanism. Here we characterize large biallelic deletions in the region encoding the ATAD3C, ATAD3B and ATAD3A genes. Although high homology complicates genomic analysis of the ATAD3 defects, they can be identified by targeted analysis of standard single nucleotide polymorphism array and whole exome sequencing data. We report deletions that generate chimeric ATAD3B/ATAD3A fusion genes in individuals from four unrelated families with fatal congenital pontocerebellar hypoplasia, whereas a case with genomic rearrangements affecting the ATAD3C/ATAD3B genes on one allele and ATAD3B/ATAD3A genes on the other displays later-onset encephalopathy with cerebellar atrophy, ataxia and dystonia. Fibroblasts from affected individuals display mitochondrial DNA abnormalities, associated with multiple indicators of altered cholesterol metabolism. Moreover, drug-induced perturbations of cholesterol homeostasis cause mitochondrial DNA disorganization in control cells, while mitochondrial DNA aggregation in the genetic cholesterol trafficking disorder Niemann-Pick type C disease further corroborates the interdependence of mitochondrial DNA organization and cholesterol. These data demonstrate the integration of mitochondria in cellular cholesterol homeostasis, in which ATAD3 plays a critical role. The dual problem of perturbed cholesterol metabolism and mitochondrial dysfunction could be widespread in neurological and neurodegenerative diseases.

Unravelling the Effects of the Mutation m.3571insC/MT-ND1 on Respiratory Complexes Structural Organization.

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.

Common data elements for clinical research in mitochondrial disease: a National Institute for Neurological Disorders and Stroke project.

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.

Severe respiratory complex III defect prevents liver adaptation to prolonged fasting.

BACKGROUND & AIMS: Next generation sequencing approaches have tremendously improved the diagnosis of rare genetic diseases. It may however be faced with difficult clinical interpretation of variants. Inherited enzymatic diseases provide an invaluable possibility to evaluate the function of the defective enzyme in human cell biology. This is the case for respiratory complex III, which has 11 structural subunits and requires several assembly factors. An important role of complex III in liver function is suggested by its frequent impairment in human cases of genetic complex III defects. METHODS: We report the case of a child with complex III defect and acute liver dysfunction with lactic acidosis, hypoglycemia, and hyperammonemia. Mitochondrial activities were assessed in liver and fibroblasts using spectrophotometric assays. Genetic analysis was done by exome followed by Sanger sequencing. Functional complementation of defective fibroblasts was performed using lentiviral transduction followed by enzymatic analyses and expression assays. RESULTS: Homozygous, truncating, mutations in LYRM7 and MTO1, two genes encoding essential mitochondrial proteins were found. Functional complementation of the complex III defect in fibroblasts demonstrated the causal role of LYRM7 mutations. Comparison of the patient's clinical history to previously reported patients with complex III defect due to nuclear DNA mutations, some actually followed by us, showed striking similarities allowing us to propose common pathophysiology. CONCLUSIONS: Profound complex III defect in liver does not induce actual liver failure but impedes liver adaptation to prolonged fasting leading to severe lactic acidosis, hypoglycemia, and hyperammonemia, potentially leading to irreversible brain damage. LAY SUMMARY: The diagnosis of rare genetic disease has been tremendously accelerated by the development of high throughput sequencing technology. In this paper we report the investigations that have led to identify LYRM7 mutations causing severe hepatic defect of respiratory complex III. Based on the comparison of the patient's phenotype with other cases of complex III defect, we propose that profound complex III defect in liver does not induce actual liver failure but impedes liver adaptation to prolonged fasting.

Mutations in CYC1, encoding cytochrome c1 subunit of respiratory chain complex III, cause insulin-responsive hyperglycemia.

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.

HIF-1α induction, proliferation and glycolysis of Theileria-infected leukocytes.

Within 2 h of infection by Theileria annulata sporozoites, bovine macrophages display a two- to fourfold increase in transcription of hypoxia inducible factor (HIF-1α). Twenty hours post-invasion sporozoites develop into multi-nucleated macroschizonts that transform the infected macrophage into an immortalized, permanently proliferating, hyper-invasive and disease-causing leukaemia-like cell. Once immortalized Theileria-infected leukocytes can be propagated as cell lines and even though cultivated under normoxic conditions, both infected B cells and macrophages display sustained activation of HIF-1α. Attenuated macrophages used as live vaccines against tropical theileriosis also display HIF-1α activation even though they have lost their tumorigenic phenotype. Here, we review data that ascribes HIF-1α activation to the proliferation status of the infected leukocyte and discuss the possibility that Theileria may have lost its ability to render its host macrophage virulent due to continuous parasite replication in a high Reactive Oxygen Species (ROS) environment. We propose a model where uninfected macrophages have low levels of H2 O2 output, whereas virulent-infected macrophages produce high amounts of H2 O2 . Further increase in H2 O2 output leads to dampening of infected macrophage virulence, a characteristic of disease-resistant macrophages. At the same time exposure to H2 O2 sustains HIF-1α that induces the switch from mitochondrial oxidative phosphorylation to Warburg glycolysis, a metabolic shift that underpins uncontrolled infected macrophage proliferation. We propose that as macroschizonts develop into merozoites and infected macrophage proliferation arrests, HIF-1α levels will decrease and glycolysis will switch back from Warburg to oxidative glycolysis. As Theileria infection transforms its host leukocyte into an aggressive leukaemic-like cell, we propose that manipulating ROS levels, HIF-1α induction and oxidative over Warburg glycolysis could contribute to improved disease control. Finally, as excess amounts of H2 O2 drive virulent Theileria-infected macrophages towards attenuation it highlights how infection-induced pathology and redox balance are intimately linked.

Long-term cardiac prognosis and risk stratification in 260 adults presenting with mitochondrial diseases.

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.

Functional interplay between Parkin and Drp1 in mitochondrial fission and clearance.

Autosomal recessive early-onset Parkinson's disease is most often caused by mutations in the genes encoding the cytosolic E3 ubiquitin ligase Parkin and the mitochondrial serine/threonine kinase PINK1. Studies in Drosophila models and mammalian cells have demonstrated that these proteins regulate various aspects of mitochondrial physiology, including organelle transport, dynamics and turnover. How PINK1 and Parkin orchestrate these processes, and whether they always do so within a common pathway remain to be clarified. We have revisited the role of PINK1 and Parkin in mitochondrial dynamics, and explored its relation to the mitochondrial clearance program controlled by these proteins. We show that PINK1 and Parkin promote Drp1-dependent mitochondrial fission by mechanisms that are at least in part independent. Parkin-mediated mitochondrial fragmentation was abolished by treatments interfering with the calcium/calmodulin/calcineurin signaling pathway, suggesting that it requires dephosphorylation of serine 637 of Drp1. Parkinson's disease-causing mutations with differential impact on mitochondrial morphology and organelle degradation demonstrated that the pro-fission effect of Parkin is not required for efficient mitochondrial clearance. In contrast, the use of Förster energy transfer imaging microscopy revealed that Drp1 and Parkin are co-recruited to mitochondria in proximity of PINK1 following mitochondrial depolarization, indicating spatial coordination between these events in mitochondrial degradation. Our results also hint at a major role of the outer mitochondrial adaptor MiD51 in Drp1 recruitment and Parkin-dependent mitophagy. Altogether, our observations provide new insight into the mechanisms underlying the regulation of mitochondrial dynamics by Parkin and its relation to the mitochondrial clearance program mediated by the PINK1/Parkin pathway.

Mutations in C12orf62, a factor that couples COX I synthesis with cytochrome c oxidase assembly, cause fatal neonatal lactic acidosis.

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.

A diagnostic flow chart for POLG-related diseases based on signs sensitivity and specificity.

OBJECTIVE: Diseases due to mutations of POLG gene, encoding the mitochondrial DNA polymerase, are reputed to have very diverse clinical presentations and have been proposed to cause up to 25% adult mitochondrial diseases. Our objective was the evaluation of the specificity and sensitivity of the signs encountered with POLG mutations. DESIGN: Forty-four patients out of 154 with sequenced POLG gene had mutations affecting either one (POLG(+/-) group) or two POLG alleles (POLG(+/+) group). Phenotyping included clinical signs, electroneuromyography and brain imaging while mitochondrial investigations encompassed muscle histochemistry, respiratory chain assays and search for multiple mitochondrial deletions. The specificity and sensitivity of the signs associated with POLG mutations were analysed by comparison between POLG(+/+) and patients without POLG mutation. RESULTS: High sensitivity but low specificity was observed with single signs such as axonal sensory neuropathy, cerebellar syndrome, movement disorders and weakness involving ocular, pharyngeal, axial and/or limb muscles. Specificity was increased with combination of previous signs plus psychiatric symptoms, cognitive impairment and epilepsy. High specificity and sensitivity was only obtained with sensory neuronopathy associated with one of the following signs: weakness of ocular, pharyngeal, axial and/or limb muscles. Mitochondrial investigations did not suffice for diagnosis. The widespread neuromuscular signs were often present since disease onset and were the rule above 50 years of age leading to a very low probability of POLG mutations in patients with less than three signs and absent sensory neuropathy. CONCLUSIONS: Phenotypes associated with POLG mutations follow a reproducible pattern, which allows establishing a diagnostic flow chart.

Mitochondrial targeting of recombinant RNAs modulates the level of a heteroplasmic mutation in human mitochondrial DNA associated with Kearns Sayre Syndrome.

Mitochondrial mutations, an important cause of incurable human neuromuscular diseases, are mostly heteroplasmic: mutated mitochondrial DNA is present in cells simultaneously with wild-type genomes, the pathogenic threshold being generally >70% of mutant mtDNA. We studied whether heteroplasmy level could be decreased by specifically designed oligoribonucleotides, targeted into mitochondria by the pathway delivering RNA molecules in vivo. Using mitochondrially imported RNAs as vectors, we demonstrated that oligoribonucleotides complementary to mutant mtDNA region can specifically reduce the proportion of mtDNA bearing a large deletion associated with the Kearns Sayre Syndrome in cultured transmitochondrial cybrid cells. These findings may be relevant to developing of a new tool for therapy of mtDNA associated diseases.

Oxidation of hydrogen sulfide by human liver mitochondria.

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.

Defective mitochondrial fusion, altered respiratory function, and distorted cristae structure in skin fibroblasts with heterozygous OPA1 mutations.

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.