Succinate Dehydrogenase Is a Direct Target of Sirtuin 3 Deacetylase Activity

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

Acute and late-onset optic atrophy due to a novel OPA1 mutation leading to a mitochondrial coupling defect

PurposeAutosomal dominant optic atrophy (ADOA, OMIM 165500), an inherited optic neuropathy that leads to retinal ganglion cell degeneration and reduced visual acuity during the early decades of life, is mainly associated with mutations in the OPA1 gene. Here we report a novel ADOA phenotype associated with a new pathogenic OPA1 gene mutation. Methods The patient, a 62-year-old woman, was referred for acute, painless, and severe visual loss in her right eye. Acute visual loss in her left eye occurred a year after initial presentation. MRI confirmed the diagnosis of isolated atrophic bilateral optic neuropathy. We performed DNA sequencing of the entire coding sequence and the exon/intron junctions of the OPA1 gene, and we searched for the mitochondrial DNA mutations responsible for Leber hereditary optic atrophy by sequencing entirely mitochondrial DNA. Mitochondrial respiratory chain complex activity and mitochondrial morphology were investigated in skin fibroblasts from the patient and controls. Results We identified a novel heterozygous missense mutation (c.2794C>T) in exon 27 of the OPA1 gene, resulting in an amino acid change (p.R932C) in the protein. This mutation, which affects a highly conserved amino acids, has not been previously reported, and was absent in 400 control chromosomes. Mitochondrial DNA sequence analysis did not reveal any mutation associated with Leber hereditary optic neuropathy or any pathogenic mutations. The investigation of skin fibroblasts from the patient revealed a coupling defect of oxidative phosphorylation and a larger proportion of short mitochondria than in controls. Conclusions The presence of an OPA1 mutation indicates that this sporadic, late-onset acute case of optic neuropathy is related to ADOA and to a mitochondrial energetic defect. This suggests that the mutational screening of the OPA1 gene would be justified in atypical cases of optic nerve atrophy with no evident cause

Bioinformatics Tools and Databases to Assess the Pathogenicity of Mitochondrial DNA Variants in the Field of Next Generation Sequencing

The development of next generation sequencing (NGS) has greatly enhanced the diagnosis of mitochondrial disorders, with a systematic analysis of the whole mitochondrial DNA (mtDNA) sequence and better detection sensitivity. However, the exponential growth of sequencing data renders complex the interpretation of the identified variants, thereby posing new challenges for the molecular diagnosis of mitochondrial diseases. Indeed, mtDNA sequencing by NGS requires specific bioinformatics tools and the adaptation of those developed for nuclear DNA, for the detection and quantification of mtDNA variants from sequence alignment to the calling steps, in order to manage the specific features of the mitochondrial genome including heteroplasmy, i.e., coexistence of mutant and wildtype mtDNA copies. The prioritization of mtDNA variants remains difficult, relying on a limited number of specific resources: population and clinical databases, and in silico tools providing a prediction of the variant pathogenicity. An evaluation of the most prominent bioinformatics tools showed that their ability to predict the pathogenicity was highly variable indicating that special efforts should be directed at developing new bioinformatics tools dedicated to the mitochondrial genome. In addition, massive parallel sequencing raised several issues related to the interpretation of very low mtDNA mutational loads, discovery of variants of unknown significance, and mutations unrelated to patient phenotype or the co-occurrence of mtDNA variants. This review provides an overview of the current strategies and bioinformatics tools for accurate annotation, prioritization and reporting of mtDNA variations from NGS data, in order to carry out accurate genetic counseling in individuals with primary mitochondrial diseases

Mutations in the m-AAA proteases AFG3L2 and SPG7 are causing isolated dominant optic atrophy.

OBJECTIVE: To improve the genetic diagnosis of dominant optic atrophy (DOA), the most frequently inherited optic nerve disease, and infer genotype-phenotype correlations. METHODS: Exonic sequences of 22 genes were screened by new-generation sequencing in patients with DOA who were investigated for ophthalmology, neurology, and brain MRI. RESULTS: We identified 7 and 8 new heterozygous pathogenic variants in SPG7 and AFG3L2. Both genes encode for mitochondrial matricial AAA (m-AAA) proteases, initially involved in recessive hereditary spastic paraplegia type 7 (HSP7) and dominant spinocerebellar ataxia 28 (SCA28), respectively. Notably, variants in AFG3L2 that result in DOA are located in different domains to those reported in SCA28, which likely explains the lack of clinical overlap between these 2 phenotypic manifestations. In comparison, the SPG7 variants identified in DOA are interspersed among those responsible for HSP7 in which optic neuropathy has previously been reported. CONCLUSIONS: Our results position SPG7 and AFG3L2 as candidate genes to be screened in DOA and indicate that regulation of mitochondrial protein homeostasis and maturation by m-AAA proteases are crucial for the maintenance of optic nerve physiology

Mitochondrie et stress énergetique : voies de signalisation et adaptations cellulaires

Mitochondria are integrators of intracellular signaling (adjusting its functioning to cellular energy demand) and initaitors of retrograde pathways (triggering cellular response to variations of functional status of mitochondria). This work focus on oxidative mitochondrial metabolism and signaling pathways, in HepG2 cells, in response of two energetic stresses : mitochondrial uncoupling and glucocorticoids treatement. Mitochondrial uncoupling triggers an increase in oxidative metabolism without any change in glycolysis (notably by a stimulation of nuclear transcription of genes encoding mitonchondrial proteins). Mitochondria are also one of targets of glucocorticoids, homones tht induce short term and long term effects. Rapid effects (modification of respiratory chain complexes I, II and III activities) involve dexamethasone binding on a membrane glucocorticoid receptor. These effects are mediated by calcium dependent activation of p38MAPK. Long term genomic effects (increase in respiratory chain capacity) implicate the classical intracellular glucocorticoid receptor. Modifications of the respiratory chain functioning induced by glucocorticoids involve the gradual recruitement of glucocorticoid binding sites (located in plasma membrane or in cytosol).La mitochondrie est un centre de régulation métabolique à la fois intégrateur de signaux (visant à ajuster son fonctionnement selon les besoins énergétiques cellulaires) et initiateur de voies rétrogrades (permettant une réponse cellulaire à des changements d'états fonctionneles de la mitochondrie). Ce travail s'intéresse plus particulièrement au métabolisme oxydatif mitochondrial et aux voies de signalisation activées, dans les cellules HepG2, lors de deux situations de stress énergétique : le découplage mitochondrial constitue un signal conduisant les cellules à développer leur métabolisme oxydatif sans modifier la glycolyse (notamment par activation de la transcription de gènes codant pour des protéines mitochondriales). La mitochondrie est également une des cibles du traitement par glucocorticoïdes, ces hormones induisant à la fois des effets à court terme et à long terme. les effets rapides (modification de l'activité des complexes I, II et III de la chaîne respiratoire mitochondriale) sont non génomiques et impliquent la fixation de la dexamethasone sur un récepteur membranaire. Ces effets sont médiés par l'activation calcium-dépendante de la protéine p38MAPK. Les effets à long terme (augmentation de la capacité de la chaîne respiratoire) sont transcriptionnels et nécessitent le recrutement du récepteur intracellulaire classique aux glucocorticoïdes. Les modifications du fonctionnement de la chaîne respiratoire mitochondriale par les glucocorticoïdes sont induites par le recrutement graduel de différents sites de liaison aux glucocorticîdes (membranaire et intracellulaire)

Mitochondrie et stress énergétique (voies de signalisation et adaptations cellulaires)

La mitochondrie est un centre de régulation métabolique à la fois intégrateur de signaux (visant à ajuster son fonctionnement selon les besoins énergétiques cellulaires) et initiateur de voies rétrogrades (permettant une réponse cellulaire à des changements d'états fonctionneles de la mitochondrie). Ce travail s'intéresse plus particulièrement au métabolisme oxydatif mitochondrial et aux voies de signalisation activées, dans les cellules HepG2, lors de deux situations de stress énergétique : le découplage mitochondrial constitue un signal conduisant les cellules à développer leur métabolisme oxydatif sans modifier la glycolyse (notamment par activation de la transcription de gènes codant pour des protéines mitochondriales). La mitochondrie est également une des cibles du traitement par glucocorticoïdes, ces hormones induisant à la fois des effets à court terme et à long terme. les effets rapides (modification de l'activité des complexes I, II et III de la chaîne respiratoire mitochondriale) sont non génomiques et impliquent la fixation de la dexamethasone sur un récepteur membranaire. Ces effets sont médiés par l'activation calcium-dépendante de la protéine p38MAPK. Les effets à long terme (augmentation de la capacité de la chaîne respiratoire) sont transcriptionnels et nécessitent le recrutement du récepteur intracellulaire classique aux glucocorticoïdes. Les modifications du fonctionnement de la chaîne respiratoire mitochondriale par les glucocorticoïdes sont induites par le recrutement graduel de différents sites de liaison aux glucocorticîdes (membranaire et intracellulaire).Mitochondria are integrators of intracellular signaling (adjusting its functioning to cellular energy demand) and initaitors of retrograde pathways (triggering cellular response to variations of functional status of mitochondria). This work focus on oxidative mitochondrial metabolism and signaling pathways, in HepG2 cells, in response of two energetic stresses : mitochondrial uncoupling and glucocorticoids treatement. Mitochondrial uncoupling triggers an increase in oxidative metabolism without any change in glycolysis (notably by a stimulation of nuclear transcription of genes encoding mitonchondrial proteins). Mitochondria are also one of targets of glucocorticoids, homones tht induce short term and long term effects. Rapid effects (modification of respiratory chain complexes I, II and III activities) involve dexamethasone binding on a membrane glucocorticoid receptor. These effects are mediated by calcium dependent activation of p38MAPK. Long term genomic effects (increase in respiratory chain capacity) implicate the classical intracellular glucocorticoid receptor. Modifications of the respiratory chain functioning induced by glucocorticoids involve the gradual recruitement of glucocorticoid binding sites (located in plasma membrane or in cytosol).ANGERS-BU Médecine-Pharmacie (490072105) / SudocSudocFranceF

Bioinformatics Tools and Databases to Assess the Pathogenicity of Mitochondrial DNA Variants in the Field of Next Generation Sequencing

International audienceThe development of next generation sequencing (NGS) has greatly enhanced the diagnosis of mitochondrial disorders, with a systematic analysis of the whole mitochondrial DNA (mtDNA) sequence and better detection sensitivity. However, the exponential growth of sequencing data renders complex the interpretation of the identified variants, thereby posing new challenges for the molecular diagnosis of mitochondrial diseases. Indeed, mtDNA sequencing by NGS requires specific bioinformatics tools and the adaptation of those developed for nuclear DNA, for the detection and quantification of mtDNA variants from sequence alignment to the calling steps, in order to manage the specific features of the mitochondrial genome including heteroplasmy, i.e., coexistence of mutant and wildtype mtDNA copies. The prioritization of mtDNA variants remains difficult, relying on a limited number of specific resources: population and clinical databases, and in silico tools providing a prediction of the variant pathogenicity. An evaluation of the most prominent bioinformatics tools showed that their ability to predict the pathogenicity was highly variable indicating that special efforts should be directed at developing new bioinformatics tools dedicated to the mitochondrial genome. In addition, massive parallel sequencing raised several issues related to the interpretation of very low mtDNA mutational loads, discovery of variants of unknown significance, and mutations unrelated to patient phenotype or the co-occurrence of mtDNA variants. This review provides an overview of the current strategies and bioinformatics tools for accurate annotation, prioritization and reporting of mtDNA variations from NGS data, in order to carry out accurate genetic counseling in individuals with primary mitochondrial diseases

Genetic susceptibility to optic neuropathy in patients with alcohol use disorder

International audienceBackground: The pathophysiology of toxico-nutritional optic neuropathies remains debated, with no clear understanding of the respective roles played by the direct alcohol toxicity, smoking and the often associated vitamin deficiencies, which are risk factors for optic neuropathy. Our aim was to investigate genetic susceptibility in patients with bilateral infraclinical optic neuropathy associated with chronic alcohol use disorder.Methods: This retrospective cohort study included 102 visually asymptomatic patients with documented alcohol use disorder from a French reference center. Optic neuropathy was identified with optical coherence tomography (OCT), after which genetic susceptibility in the group of affected patients was investigated. Genetic testing was performed using panel sequencing of 87 nuclear genes and complete mitochondrial DNA sequencing.Results: Optic neuropathy was detected in 36% (37/102) of the included patients. Genetic testing of affected patients disclosed two patients (2/30, 6.7%) with optic neuropathy associated with pathogenic variants affecting the SPG7 gene and five patients (5/30, 16.7%) who harbored variants of uncertain significance close to probable pathogenicity in the genes WFS1, LOXL1, MMP19, NR2F1 and PMPCA. No pathogenic mitochondrial DNA variants were found in this group.Conclusions: OCT can detect presence of asymptomatic optic neuropathy in patients with chronic alcohol use disorder. Furthermore, genetic susceptibility to optic neuropathy in this setting is found in almost a quarter of affected patients. Further studies may clarify the role of preventative measures in patients who might be predisposed to avoidable visual loss and blindness

Metabolically induced heteroplasmy shifting and l-arginine treatment reduce the energetic defect in a neuronal-like model of MELAS

International audienceThe m.3243A&gt;G variant in the mitochondrial tRNA(Leu(UUR)) gene is a common mitochondrial DNA (mtDNA) mutation. Phenotypic manifestations depend mainly on the heteroplasmy, i.e. the ratio of mutant to normal mtDNA copies. A high percentage of mutant mtDNA is associated with a severe, life-threatening neurological syndrome known as MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes). MELAS is described as a neurovascular disorder primarily affecting the brain and blood vessels, but the pathophysiology of the disease is poorly understood. We developed a series of cybrid cell lines at two different mutant loads: 70% and 100% in the nuclear background of a neuroblastoma cell line (SH-SY5Y). We investigated the impact of the mutation on the metabolism and mitochondrial respiratory chain activity of the cybrids. The m.3243A&gt;G mitochondrial mutation induced a metabolic switch towards glycolysis in the neuronal cells and produced severe defects in respiratory chain assembly and activity. We used two strategies to compensate for the biochemical defects in the mutant cells: one consisted of lowering the glucose content in the culture medium, and the other involved the addition of l-arginine. The reduction of glucose significantly shifted the 100% mutant cells towards the wild-type, reaching a 90% mutant level and restoring respiratory chain complex assembly. The addition of l-arginine, a nitric oxide (NO) donor, improved complex I activity in the mutant cells in which the defective NO metabolism had led to a relative shortage of NO. Thus, metabolically induced heteroplasmy shifting and l-arginine therapy may constitute promising therapeutic strategies against MELAS.</p

Doxorubicin-Induced Autophagolysosome Formation Is Partly Prevented by Mitochondrial ROS Elimination in DOX-Resistant Breast Cancer Cells

International audienceSince its discovery, mitophagy has been viewed as a protective mechanism used by cancer cells to prevent the induction of mitochondrial apoptosis. Most cancer treatments directly or indirectly cause mitochondrial dysfunction in order to trigger signals for cell death. Elimination of these dysfunctional mitochondria by mitophagy could thus prevent the initiation of the apoptotic cascade. In breast cancer patients, resistance to doxorubicin (DOX), one of the most widely used cancer drugs, is an important cause of poor clinical outcomes. However, the role played by mitophagy in the context of DOX resistance in breast cancer cells is not well understood. We therefore tried to determine whether an increase in mitophagic flux was associated with the resistance of breast cancer cells to DOX. Our first objective was to explore whether DOX-resistant breast cancer cells were characterized by conditions that favor mitophagy induction. We next tried to determine whether mitophagic flux was increased in DOX-resistant cells in response to DOX treatment. For this purpose, the parental (MCF-7) and DOX-resistant (MCF-7dox) breast cancer cell lines were used. Our results show that mitochondrial reactive oxygen species (ROS) production and hypoxia-inducible factor-1 alpha (HIF-1 alpha) expression are higher in MCF-7dox in a basal condition compared to MCF-7, suggesting DOX-resistant breast cancer cells are prone to stimuli to induce a mitophagy-related event. Our results also showed that, in response to DOX, autophagolysosome formation is induced in DOX-resistant breast cancer cells. This mitophagic step following DOX treatment seems to be partly due to mitochondrial ROS production as autophagolysosome formation is moderately decreased by the mitochondrial antioxidant mitoTEMP