RESUMO
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.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Canais KATP/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Western Blotting , Encéfalo/metabolismo , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Diazóxido/farmacologia , GTP Fosfo-Hidrolases/genética , Humanos , Imunoprecipitação , Canais KATP/agonistas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismoRESUMO
PURPOSE: Autosomal 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.
Assuntos
DNA Mitocondrial/análise , GTP Fosfo-Hidrolases/genética , Doenças Mitocondriais/complicações , Doenças Mitocondriais/genética , Mutação de Sentido Incorreto , Atrofia Óptica/etiologia , Atrofia Óptica/genética , Eletrorretinografia , Feminino , GTP Fosfo-Hidrolases/biossíntese , Humanos , Imageamento por Ressonância Magnética , Pessoa de Meia-Idade , Doenças Mitocondriais/fisiopatologia , Atrofia Óptica/fisiopatologia , Análise de Sequência de DNA , Baixa VisãoRESUMO
Glucocorticoid treatment is often linked to increased whole-body energy expenditure and hypermetabolism. Glucocorticoids affect mitochondrial energy production, notably in the liver, where they lead to mitochondrial uncoupling reducing the efficacy of oxidative phosphorylation. However, the signaling pathways involved in these phenomena are poorly understood. Here we treated HepG2 cells with dexamethasone for different times and, by using different combinations of inhibitors, we showed that dexamethasone treatment leads to recruitment of two main signaling pathways. The first one involves a G-protein coupled membrane glucocorticoid binding site and rapidly decreases complexes I and II activities while complex III activity is upregulated in a p38MAPK dependent mechanism. The second one implies the classical cytosolic glucocorticoid receptor and triggers long-term transcriptional increases of respiration rates and of complex IV activity and quantity. We concluded that mitochondria are the target of multiple dexamethasone-induced regulatory pathways that are set up gradually after the beginning of hormone exposure and that durably influence mitochondrial oxidative phosphorylation.
Assuntos
Membrana Celular/fisiologia , Citosol/fisiologia , Dexametasona/farmacologia , Mitocôndrias/fisiologia , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Citosol/efeitos dos fármacos , Transporte de Elétrons/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/metabolismo , Mitocôndrias/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Fatores de TempoRESUMO
Here, we show that 3 days of mitochondrial uncoupling, induced by low concentrations of dinitrophenol (10 and 50 microM) in cultured human HepG2 cells, triggers cellular metabolic adaptation towards oxidative metabolism. Chronic respiratory uncoupling of HepG2 cells induced an increase in cellular oxygen consumption, oxidative capacity and cytochrome c oxidase activity. This was associated with an upregulation of COXIV and ANT3 gene expression, two nuclear genes that encode mitochondrial proteins involved in oxidative phosphorylation. Glucose consumption, lactate and pyruvate production and growth rate were unaffected, indicating that metabolic adaptation of HepG2 cells undergoing chronic respiratory uncoupling allows continuous and efficient mitochondrial ATP production without the need to increase glycolytic activity. In contrast, 3 days of dinitrophenol treatment did not change the oxidative capacity of human 143B.TK(-) cells, but it increased glucose consumption, lactate and pyruvate production. Despite a large increase in glycolytic metabolism, the growth rate of 143B.TK(-) cells was significantly reduced by dinitrophenol-induced mitochondrial uncoupling. We propose that chronic respiratory uncoupling may constitute an internal bioenergetic signal, which would initiate a coordinated increase in nuclear respiratory gene expression, which ultimately drives mitochondrial metabolic adaptation within cells.
Assuntos
2,4-Dinitrofenol/farmacologia , Translocador 3 do Nucleotídeo Adenina/genética , Respiração Celular/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Mitocôndrias/efeitos dos fármacos , Desacopladores/farmacologia , Adaptação Fisiológica/efeitos dos fármacos , Respiração Celular/efeitos dos fármacos , Células Cultivadas , Expressão Gênica , Glucose/metabolismo , Humanos , Ácido Láctico/biossíntese , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Fator 1 Nuclear Respiratório/genética , Fosforilação Oxidativa/efeitos dos fármacos , Ácido Pirúvico/metabolismo , Regulação para CimaRESUMO
Heme oxygenase 1 is induced by hemodynamic forces in vascular smooth muscle and endothelial cells. We investigated the involvement of heme oxygenase 1 in flow (shear stress)-dependent remodeling. Two or 14 days after ligation of mesenteric resistance arteries, vessels were isolated. In rats, at 14 days, diameter increased by 23% in high-flow arteries and decreased by 22% in low-flow arteries compared with normal flow vessels. Heme oxygenase activity inhibition using Tin-protoporphyrin abolished diameter enlargement in high-flow arteries and accentuated arterial narrowing in low-flow arteries (32% diameter decrease versus 22% in control). Two days after ligation, heme oxygenase 1 expression increased in high-flow and low-flow vessels, in association with a reduced mitochondrial aconitase activity (marker of oxidative stress) in high-flow arteries only. Inhibition of macrophage infiltration (clodronate) decreased heme oxygenase 1 induction in low-flow but not in high-flow arteries. Similarly, inhibition of NADPH oxidase activity (apocynin) decreased heme oxygenase 1 induction in low-flow but not high-flow arteries. However, dihydroethidium staining was higher in high-flow and low-flow compared with normal flow arteries. In arteries cannulated in an arteriograph, heme oxygenase 1 mRNA increased in a flow-dependent manner and was abolished by N(G)-nitro-l-arginine methyl ester, catalase, or mitochondrial electron transport chain inhibition. Furthermore, heme oxygenase 1 induction using cobalt-protoporphyrin restored altered high-flow remodeling in endothelial NO synthase knockout mice. Thus, in high-flow remodeling, heme oxygenase 1 induction depends on shear stress-generated NO and mitochondria-derived hydrogen peroxide. In low-flow remodeling, heme oxygenase 1 induction requires macrophage infiltration and is mediated by NADPH oxidase-derived superoxide.
Assuntos
Heme Oxigenase-1/metabolismo , Inflamação/metabolismo , Artérias Mesentéricas/metabolismo , Óxido Nítrico/metabolismo , Estresse Oxidativo/fisiologia , Fluxo Sanguíneo Regional/fisiologia , Acetofenonas/farmacologia , Animais , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Hemodinâmica/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Artérias Mesentéricas/efeitos dos fármacos , Metaloporfirinas/farmacologia , Camundongos , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Protoporfirinas/farmacologia , Ratos , Ratos Wistar , Fluxo Sanguíneo Regional/efeitos dos fármacos , Resistência Vascular/efeitos dos fármacos , Vasodilatação/efeitos dos fármacosRESUMO
Hereditary spastic paraplegia refers to a genetically heterogeneous syndrome. We identified five members of a family suffering from a late-onset spastic paraplegia-like disorder, carrying the homoplasmic m.9176 T>C mutation in the mitochondrial ATP6 gene. The clinical severity of the disease observed in the family was correlated with the biochemical and assembly defects of the ATP synthase. The m.9176 T>C mutation has been previously associated to Leigh syndrome or familial bilateral striatal necrosis. Other factors such as modifying genes may be involved in the phenotypic expression of the disease. The family belongs to the mitochondrial haplogroup J, previously shown to play a role in modulating the phenotype of mitochondrial diseases and be associated with longevity. Moreover other nuclear modifying genes or environmental factors may contribute to the disease phenotype. This finding extends the genetic heterogeneity of the hereditary spastic paraplegia together with the clinical spectrum of mutations of the ATP6 gene.
Assuntos
Genes Mitocondriais , Mitocôndrias/enzimologia , ATPases Mitocondriais Próton-Translocadoras/genética , Mutação Puntual , Paraplegia Espástica Hereditária/genética , Adulto , DNA Mitocondrial/análise , DNA Mitocondrial/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , FenótipoRESUMO
Charcot-Marie-Tooth (CMT) disease represents a large group of clinically and genetically heterogeneous disorders leading to inherited peripheral neuropathies affecting motor and sensory neurons. Mutations in the ganglioside-induced differentiation-associated-protein 1 gene (GDAP1), which encodes a protein anchored to the mitochondrial outer membrane, are usually associated with the recessive forms of CMT disease and only rarely with the autosomal dominant forms. The function of GDAP1 is not fully understood but it plays a role in mitochondrial dynamics by promoting fission events. We present an overview of GDAP1 and the corresponding protein together with the complete spectrum of the 41 gene mutations described so far. We examine the relationship between the genotype and the phenotype in the various forms of CMT disease related to GDAP1 mutations, and discuss the pathophysiological hypotheses that link peripheral neuropathies to mitochondrial dysfunction and GDAP1 mutations. The meta-analysis of the literature reveals the great heterogeneity of phenotypic presentations and shows that the recessive forms of CMT disease, i.e. CMT4A and AR-CMT2, are far more severe than the dominant form, i.e. CMT2K. Among patients with recessive forms of the disease, those carrying truncating mutations are more seriously affected, often becoming wheelchair-bound before the end of the third decade. At the neuronal level, GDAP1 mutations may lead to perturbed axonal transport and impaired energy production as in other neurodegenerative diseases due to mutations in genes involved in mitochondrial dynamics.
Assuntos
Doença de Charcot-Marie-Tooth/complicações , Doença de Charcot-Marie-Tooth/genética , Doenças Mitocondriais/complicações , Doenças Mitocondriais/genética , Mutação , Proteínas do Tecido Nervoso/genética , Humanos , Metanálise como Assunto , Modelos Biológicos , Proteínas do Tecido Nervoso/metabolismoRESUMO
BACKGROUND: Aging is associated with reduced structural and functional adaptation to chronic changes in blood flow (shear stress) in small arteries. As heme oxygenase-1 (HO-1) is induced by hemodynamic forces in vascular smooth muscle and endothelial cells, we hypothesized that it might improve flow-dependent remodeling in aging. METHOD: First-order mesenteric arteries from 3 and 16-month-old rats were exposed to high, low, or normal flow by alternate ligation in vivo. Rats were treated with the HO-1 inducer, cobalt protoporphyrin (CoPP, 5 mg/kg) or vehicle. 14 days later, local blood flow was measured in vivo, and arteries were studied in vitro. RESULTS: Despite an equivalent change in blood flow, diameter enlargement in the high-flow arteries was blunted in old compared to young rats and was associated with decreased endothelium-dependent relaxation to acetylcholine. In old rats, HO-1 induction with CoPP restored outward remodeling, via a paradoxical reactive oxygen species-dependent mechanism, and was associated with a Mn-superoxide dismutase (SOD) overexpression, as well as a significant reduction of mitochondrial aconitase activity, used as a biomarker for oxidative stress. The heme oxygenase activity inhibitor, Sn-protoporphyrin, and the SOD-mimetic, TEMPOL, prevented the effect of CoPP on remodeling and oxidative status in old rats. Furthermore, HO-1 induction improved endothelial function, in association with increased endothelial nitric oxide synthase protein expression and phosphorylation (Ser-1177). In low-flow arteries, inward remodeling was unaffected by aging or by CoPP. Thus, in old rats, CoPP-induced up-regulation of HO-1 restored high-flow-dependent remodeling (diameter enlargement) and improved endothelial function in mesenteric arteries. CONCLUSION: This opens new perspectives in the treatment of ischemic diseases in aging.
Assuntos
Fatores Etários , Circulação Sanguínea , Endotélio Vascular/fisiologia , Heme Oxigenase (Desciclizante)/biossíntese , Artérias Mesentéricas/fisiologia , Animais , Indução Enzimática , Protoporfirinas/farmacologia , Ratos , Ratos Wistar , VasodilataçãoRESUMO
Ethambutol (EMB), widely used in the treatment of tuberculosis, has been reported to cause Leber's hereditary optic neuropathy in patients carrying mitochondrial DNA mutations. We study the effect of EMB on mitochondrial metabolism in fibroblasts from controls and from a man carrying an OPA1 mutation, in whom the drug induced the development of autosomal dominant optic atrophy (ADOA). EMB produced a mitochondrial coupling defect together with a 25% reduction in complex IV activity. EMB induced the formation of vacuoles associated with decreased mitochondrial membrane potential and increased fragmentation of the mitochondrial network. Mitochondrial genetic variations may therefore be predisposing factors in EMB-induced ocular injury.