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NADH:ubiquinone oxidoreductase (respiratory complex I) plays a major role in energy metabolism by coupling electron transfer from NADH to quinone with proton translocation across the membrane. Complex I deficiencies were found to be the most common source of human mitochondrial dysfunction that manifest in a wide variety of neurodegenerative diseases. Seven subunits of human complex I are encoded by mitochondrial DNA (mtDNA) that carry an unexpectedly large number of mutations discovered in mitochondria from patients' tissues. However, whether or how these genetic aberrations affect complex I at a molecular level is unknown. Here, we used Escherichia coli as a model system to biochemically characterize two mutations that were found in mtDNA of patients. The V253A MT-ND5 mutation completely disturbed the assembly of complex I, while the mutation D199G MT-ND1 led to the assembly of a stable complex capable to catalyze redox-driven proton translocation. However, the latter mutation perturbs quinone reduction leading to a diminished activity. D199 MT-ND1 is part of a cluster of charged amino acid residues that are suggested to be important for efficient coupling of quinone reduction and proton translocation. A mechanism considering the role of D199 MT-ND1 for energy conservation in complex I is discussed.

Inherited optic neuropathies are the most common mitochondrial diseases, leading to neurodegeneration involving the irreversible loss of retinal ganglion cells, optic nerve degeneration and central visual loss. Importantly, properly regulated mitochondrial dynamics are critical for maintaining cellular homeostasis, and are further regulated by MIEF1 (mitochondrial elongation factor 1) which encodes for MID51 (mitochondrial dynamics protein 51), an outer mitochondrial membrane protein that acts as an adaptor protein to regulate mitochondrial fission. However, dominant mutations in MIEF1 have not been previously linked to any human disease. Using targeted sequencing of genes involved in mitochondrial dynamics, we report the first heterozygous variants in MIEF1 linked to disease, which cause an unusual form of late-onset progressive optic neuropathy characterized by the initial loss of peripheral visual fields. Pathogenic MIEF1 variants linked to optic neuropathy do not disrupt MID51's localization to the outer mitochondrial membrane or its oligomerization, but rather, significantly disrupt mitochondrial network dynamics compared to wild-type MID51 in high spatial and temporal resolution confocal microscopy live imaging studies. Together, our study identifies dominant MIEF1 mutations as a cause for optic neuropathy and further highlights the important role of properly regulated mitochondrial dynamics in neurodegeneration.

Mitochondrial diseases are a clinically heterogeneous group of multisystemic disorders that arise as a result of various mitochondrial dysfunctions. Autosomal recessive aARS deficiencies represent a rapidly growing group of severe rare inherited mitochondrial diseases, involving multiple organs, and currently without curative option. They might be related to defects of mitochondrial aminoacyl t-RNA synthetases (mtARS) that are ubiquitous enzymes involved in mitochondrial aminoacylation and the translation process. Here, using NGS analysis of 281 nuclear genes encoding mitochondrial proteins, we identified 4 variants in different mtARS in three patients from unrelated Tunisian families, with clinical features of mitochondrial disorders. Two homozygous variants were found in KARS (c.683C>T) and AARS2 (c.1150-4C>G), respectively in two patients, while two heterozygous variants in EARS2 (c.486-7C>G) and DARS2 (c.1456C>T) were concomitantly found in the third patient. Bio-informatics investigations predicted their pathogenicity and deleterious effects on pre-mRNA splicing and on protein stability. Thus, our results suggest that mtARS mutations are common in Tunisian patients with mitochondrial diseases.

Pathogenic GFM1 variants have been linked to neurological phenotypes with or without liver involvement, but only a few cases have been reported in the literature. Here, we report clinical, biochemical, and neuroimaging findings from nine unrelated children carrying GFM1 variants, 10 of which were not previously reported. All patients presented with neurological involvement-mainly axial hypotonia and dystonia during the neonatal period-with five diagnosed with West syndrome; two children had liver involvement with cytolysis episodes or hepatic failure. While two patients died in infancy, six exhibited a stable clinical course. Brain magnetic resonance imaging showed the involvement of basal ganglia, brainstem, and periventricular white matter. Mutant EFG1 and OXPHOS proteins were decreased in patient's fibroblasts consistent with impaired mitochondrial translation. Thus, we expand the genetic spectrum of GFM1-linked disease and provide detailed clinical profiles of the patients that will improve the diagnostic success for other patients carrying GFM1 mutations., (© 2019 Wiley Periodicals, Inc.)

Importance: Neurologic disorders with isolated symptoms or complex syndromes are relatively frequent among mitochondrial inherited diseases. Recessive RTN4IP1 gene mutations have been shown to cause isolated and syndromic optic neuropathies., Objective: To define the spectrum of clinical phenotypes associated with mutations in RTN4IP1 encoding a mitochondrial quinone oxidoreductase., Design, Setting, and Participants: This study involved 12 individuals from 11 families with severe central nervous system diseases and optic atrophy. Targeted and whole-exome sequencing were performed-at Hospital Angers (France), Institute of Neurology Milan (Italy), Imagine Institute Paris (France), Helmoltz Zentrum of Munich (Germany), and Beijing Genomics Institute (China)-to clarify the molecular diagnosis of patients. Each patient's neurologic, ophthalmologic, magnetic resonance imaging, and biochemical features were investigated. This study was conducted from May 1, 2014, to June 30, 2016., Main Outcomes and Measures: Recessive mutations in RTN4IP1 were identified. Clinical presentations ranged from isolated optic atrophy to severe encephalopathies., Results: Of the 12 individuals in the study, 6 (50%) were male and 6 (50%) were female. They ranged in age from 5 months to 32 years. Of the 11 families, 6 (5 of whom were consanguineous) had a member or members who presented isolated optic atrophy with the already reported p.Arg103His or the novel p.Ile362Phe, p.Met43Ile, and p.Tyr51Cys amino acid changes. The 5 other families had a member or members who presented severe neurologic syndromes with a common core of symptoms, including optic atrophy, seizure, intellectual disability, growth retardation, and elevated lactate levels. Additional clinical features of those affected were deafness, abnormalities on magnetic resonance images of the brain, stridor, and abnormal electroencephalographic patterns, all of which eventually led to death before age 3 years. In these patients, novel and very rare homozygous and compound heterozygous mutations were identified that led to the absence of the protein and complex I disassembly as well as mild mitochondrial network fragmentation., Conclusions and Relevance: A broad clinical spectrum of neurologic features, ranging from isolated optic atrophy to severe early-onset encephalopathies, is associated with RTN4IP1 biallelic mutations and should prompt RTN4IP1 screening in both syndromic neurologic presentations and nonsyndromic recessive optic neuropathies.

Dominant optic atrophy is a blinding disease due to the degeneration of the retinal ganglion cells, the axons of which form the optic nerves. In most cases, the disease is caused by mutations in OPA1, a gene encoding a mitochondrial large GTPase involved in cristae structure and mitochondrial network fusion. Using exome sequencing, we identified dominant mutations in DNM1L on chromosome 12p11.21 in three large families with isolated optic atrophy, including the two families that defined the OPA5 locus on chromosome 19q12.1-13.1, the existence of which is denied by the present study. Analyses of patient fibroblasts revealed physiological abundance and homo-polymerization of DNM1L, forming aggregates in the cytoplasm and on highly tubulated mitochondrial network, whereas neither structural difference of the peroxisome network, nor alteration of the respiratory machinery was noticed. Fluorescence microscopy of wild-type mouse retina disclosed a strong DNM1L expression in the ganglion cell layer and axons, and comparison between 3-month-old wild-type and Dnm1l+/- mice revealed increased mitochondrial length in retinal ganglion cell soma and axon, but no degeneration. Thus, our results disclose that in addition to OPA1, OPA3, MFN2, AFG3L2 and SPG7, dominant mutations in DNM1L jeopardize the integrity of the optic nerve, suggesting that alterations of the opposing forces governing mitochondrial fusion and fission, similarly affect retinal ganglion cell survival., (© The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

To identify nuclear DNA (nDNA) oxidative phosphorylation (OXPHOS) gene mutations using cultured cells, we have developed a complementation system based on retroviral transduction with a full length cDNA expression library and selection for OXHOS function by growth in galactose. We have used this system to transduce the Chinese hamster V79-G7 OXPHOS mutant cell line with a defect in mitochondrial protein synthesis. The complemented cells were found to have acquired the cDNA for the bS6m polypeptide of the small subunit of the mitochondrial ribosome. bS6m is a 14 kDa polypeptide located on the outside of the mitochondrial 28S ribosomal subunit and interacts with the rRNA. The V79-G7 mutant protein was found to harbor a methionine to threonine missense mutation at codon 13. The hamster bS6m null mutant could also be complemented by its orthologs from either mouse or human. bS6m protein tagged at its C-terminus by HA, His or GFP localized to the mitochondrion and was fully functional. Through site-directed mutagenesis we identified the probable RNA interacting residues of the bS6m peptide and tested the functional significance of mammalian specific C-terminal region. The N-terminus of the bS6m polypeptide functionally corresponds to that of the prokaryotic small ribosomal subunit, but deletion of C-terminal residues along with the zinc ion coordinating cysteine had no functional effect. Since mitochondrial diseases can result from hundreds to thousands of different nDNA gene mutations, this one step viral complementation cloning may facilitate the molecular diagnosis of a range of nDNA mitochondrial disease mutations. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi., (Copyright © 2016 Elsevier B.V. All rights reserved.)

MFN2 and OPA1 genes encode two dynamin-like GTPase proteins involved in the fusion of the mitochondrial membrane. They have been associated with Charcot-Marie-Tooth disease type 2A and autosomal dominant optic atrophy, respectively. We report a large family with optic atrophy beginning in early childhood, associated with axonal neuropathy and mitochondrial myopathy in adult life. The clinical presentation looks like the autosomal dominant optic atrophy 'plus' phenotype linked to OPA1 mutations but is associated with a novel MFN2 missense mutation (c.629A>T, p.D210V). Multiple mitochondrial DNA deletions were found in skeletal muscle and this observation makes MFN2 a novel gene associated with 'mitochondrial DNA breakage' syndrome. Contrary to previous studies in patients with Charcot-Marie-Tooth disease type 2A, fibroblasts carrying the MFN2 mutation present with a respiratory chain deficiency, a fragmentation of the mitochondrial network and a significant reduction of MFN2 protein expression. Furthermore, we show for the first time that impaired mitochondrial fusion is responsible for a deficiency to repair stress-induced mitochondrial DNA damage. It is likely that defect in mitochondrial DNA repair is due to variability in repair protein content across the mitochondrial population and is at least partially responsible for mitochondrial DNA instability.

Charcot-Marie-Tooth disease type 2A (CMT2A) is an autosomal dominant axonal form of peripheral neuropathy caused by mutations in the mitofusin 2 gene (MFN2), which encodes a mitochondrial outer membrane protein that promotes mitochondrial fusion. Emerging evidence also points to a role of MFN2 in the regulation of mitochondrial metabolism. To examine whether mitochondrial dysfunction is a feature of CMT2A, we used a transgenic mouse model expressing in neurons a mutated R94Q form of human MFN2 shown to induce a CMT2A phenotype. Oxygraphic and enzymatic measurements both revealed a combined defect of mitochondrial complexes II and V (40 and 30% decrease, respectively) in the brain of Tg-R94 mice, leading to a drastic decrease of ATP synthesis. These deficiencies were reversed by the mitochondrial ATP-sensitive potassium channel (mK(ATP)) inhibitor 5-hydroxydecanoate. Conversely, in controls and wild-type human MFN2 mice, the mK(ATP) activator diazoxide mimicked the deficiency observed with the R94Q mutation. The physical links between complexes II and V, previously proposed as part of mK(ATP), were reinforced in Tg-R94Q mice. Our results show that the R94Q MFN2 mutation induces a combined defect of complexes II and V linked to the opening of mK(ATP), which could participate in the pathophysiology of the disease.

Background: Sirtuins (SIRT1-7) are a family of NAD-dependent deacetylases and/or ADP-ribosyltransferases that are involved in metabolism, stress responses and longevity. SIRT3 is localized to mitochondria, where it deacetylates and activates a number of enzymes involved in fuel oxidation and energy production., Methodology/principal Findings: In this study, we performed a proteomic screen to identify SIRT3 interacting proteins and identified several subunits of complex II and V of the electron transport chain. Two subunits of complex II (also known as succinate dehydrogenase, or SDH), SDHA and SDHB, interacted specifically with SIRT3. Using mass spectrometry, we identified 13 acetylation sites on SDHA, including six novel acetylated residues. SDHA is hyperacetylated in SIRT3 KO mice and SIRT3 directly deacetylates SDHA in a NAD-dependent manner. Finally, we found that SIRT3 regulates SDH activity both in cells and in murine brown adipose tissue., Conclusions/significance: Our study identifies SDHA as a binding partner and substrate for SIRT3 deacetylase activity. SIRT3 loss results in decreased SDH enzyme activity, suggesting that SIRT3 may be an important physiological regulator of SDH activity.

Charcot-Marie-Tooth type 2A disease (CMT2A), a dominantly inherited peripheral neuropathy, is caused by mutations in MFN2, a mitochondrial fusion protein. Having previously demonstrated a mitochondrial coupling defect in CMT2A patients' fibroblasts, we here investigate mitochondrial oxygen consumption and the expression of adenine nucleotide translocase (ANT) and uncoupling proteins from eight other patients with the disease. The mitochondrial uncoupling was associated with a higher respiratory rate, essentially involving complex II proteins. Furthermore, a twofold increase in the expression of ANT led to the reduced efficiency of oxidative phosphorylation in CMT2A cells, suggesting that MFN2 plays a role in controlling ATP/ADP exchanges.

Dominant Optic Atrophy (DOA) is one of the most common inherited mitochondrial diseases, leading to blindness. It is caused by the chronic degeneration of the retinal ganglion cells (RGCs) and their axons forming the optic nerve. Until now, DOA has been mainly associated with genes encoding proteins involved in mitochondrial network dynamics. Using next-generation and exome sequencing, we identified for the first time heterozygous PMPCA variants having a causative role in the pathology of late-onset primary DOA in five patients. PMPCA encodes an α subunit of the mitochondrial peptidase (MPP), responsible for the cleavage and maturation of the mitochondrial precursor proteins imported from the cytoplasm into mitochondria. Recently, PMPCA has been identified as the gene responsible for Autosomal Recessive Cerebellar Ataxia type 2 (SCAR2) and another severe recessive mitochondrial disease. In this study, four PMPCA variants were identified, two are frameshifts (c.309delA and c.820delG) classified as pathogenic and two are missenses (c.1363G>A and c.1547G>A) classified with uncertain pathological significance. Functional assays on patients' fibroblasts show a hyperconnection of the mitochondrial network and revealed that frameshift variants reduced α-MPP levels, while not significantly affecting the respiratory machinery. These results suggest that alterations in mitochondrial peptidase function can affect the fusion-fission balance, a key element in maintaining the physiology of retinal ganglion cells, and consequently lead to their progressive degeneration. [ABSTRACT FROM AUTHOR]

Background A recent article in Circulation Research suggests that the protein ABCC6, which when defective is responsible for pseudoxanthoma elasticum, an inherited condition with skin, eye and cardiovascular manifestations, is associated with dysfunction in mitochondria - Martin et al.: ABCC6 Localizes to the Mitochondria-Associated Membrane. Circ Res 2012, 111:516-520. We present complementary information based on a bioinformatics analysis, which was not performed in the article cited, to examine the suggestion that ABCC6 is localized to mitochondria. Results All the computational strategies and integrative approaches that constitute references in the field indicate that ABCC6 is localized outside of mitochondria. Conclusion Our computational and integrative results, including both experimental and predictive data, show that there is no support in favor of the localization of ABCC6 in mitochondria. [ABSTRACT FROM AUTHOR]