RESUMO
Mitochondrial fission and a metabolic switch from oxidative phosphorylation to glycolysis are key features of vascular pathology in pulmonary arterial hypertension (PAH) and are associated with exuberant endothelial proliferation and apoptosis. The underlying mechanisms are poorly understood. We describe the contribution of two intracellular chloride channel proteins, CLIC1 and CLIC4, both highly expressed in PAH and cancer, to mitochondrial dysfunction and energy metabolism in PAH endothelium. Pathological overexpression of CLIC proteins induces mitochondrial fragmentation, inhibits mitochondrial cristae formation, and induces metabolic shift toward glycolysis in human pulmonary artery endothelial cells, consistent with changes observed in patient-derived cells. Interactions of CLIC proteins with structural components of the inner mitochondrial membrane offer mechanistic insights. Endothelial CLIC4 excision and mitofusin 2 supplementation have protective effects in human PAH cells and preclinical PAH. This study is the first to demonstrate the key role of endothelial intracellular chloride channels in the regulation of mitochondrial structure, biogenesis, and metabolic reprogramming in expression of the PAH phenotype.
Assuntos
Hipertensão Pulmonar , Hipertensão Arterial Pulmonar , Humanos , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Células Endoteliais/metabolismo , Hipertensão Pulmonar Primária Familiar/metabolismo , Artéria Pulmonar/patologia , Endotélio/metabolismo , Canais de Cloreto/genética , Canais de Cloreto/metabolismoRESUMO
Heterozygous mutations of the lysosomal enzyme glucocerebrosidase (GBA1) represent the major genetic risk for Parkinson's disease (PD), while homozygous GBA1 mutations cause Gaucher disease, a lysosomal storage disorder, which may involve severe neurodegeneration. We have previously demonstrated impaired autophagy and proteasomal degradation pathways and mitochondrial dysfunction in neurons from GBA1 knockout (gba1-/-) mice. We now show that stimulation with physiological glutamate concentrations causes pathological [Ca2+]c responses and delayed calcium deregulation, collapse of mitochondrial membrane potential and an irreversible fall in the ATP/ADP ratio. Mitochondrial Ca2+ uptake was reduced in gba1-/- cells as was expression of the mitochondrial calcium uniporter. The rate of free radical generation was increased in gba1-/- neurons. Behavior of gba1+/- neurons was similar to gba1-/- in terms of all variables, consistent with a contribution of these mechanisms to the pathogenesis of PD. These data signpost reduced bioenergetic capacity and [Ca2+]c dysregulation as mechanisms driving neurodegeneration.
Assuntos
Cálcio/metabolismo , Metabolismo Energético , Glucosilceramidase/deficiência , Neurônios/patologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Encéfalo/patologia , Radicais Livres/metabolismo , Glucosilceramidase/metabolismo , Ácido Glutâmico/toxicidade , Homeostase/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurônios/efeitos dos fármacos , Receptores de Glutamato/metabolismoRESUMO
Mitochondrial Ca(2+) uptake has key roles in cell life and death. Physiological Ca(2+) signaling regulates aerobic metabolism, whereas pathological Ca(2+) overload triggers cell death. Mitochondrial Ca(2+) uptake is mediated by the Ca(2+) uniporter complex in the inner mitochondrial membrane, which comprises MCU, a Ca(2+)-selective ion channel, and its regulator, MICU1. Here we report mutations of MICU1 in individuals with a disease phenotype characterized by proximal myopathy, learning difficulties and a progressive extrapyramidal movement disorder. In fibroblasts from subjects with MICU1 mutations, agonist-induced mitochondrial Ca(2+) uptake at low cytosolic Ca(2+) concentrations was increased, and cytosolic Ca(2+) signals were reduced. Although resting mitochondrial membrane potential was unchanged in MICU1-deficient cells, the mitochondrial network was severely fragmented. Whereas the pathophysiology of muscular dystrophy and the core myopathies involves abnormal mitochondrial Ca(2+) handling, the phenotype associated with MICU1 deficiency is caused by a primary defect in mitochondrial Ca(2+) signaling, demonstrating the crucial role of mitochondrial Ca(2+) uptake in humans.
Assuntos
Sinalização do Cálcio/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Transporte de Cátions/genética , Deficiências da Aprendizagem/genética , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Transtornos dos Movimentos/genética , Doenças Musculares/genética , Fenótipo , Análise de Variância , Sequência de Bases , Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , DNA Complementar/genética , Exoma/genética , Tratos Extrapiramidais/patologia , Imunofluorescência , Técnicas Histológicas , Humanos , Imuno-Histoquímica , Potencial da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Dados de Sequência Molecular , Linhagem , Polimorfismo de Nucleotídeo Único/genética , Músculo Quadríceps/patologia , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNARESUMO
Increasing evidence indicates that endoplasmic reticulum (ER) stress activates the adaptive unfolded protein response (UPR), but that beyond a certain degree of ER damage, this response triggers apoptotic pathways. The general mechanisms of the UPR and its apoptotic pathways are well characterized. However, the metabolic events that occur during the adaptive phase of ER stress, before the cell death response, remain unknown. Here, we show that, during the onset of ER stress, the reticular and mitochondrial networks are redistributed towards the perinuclear area and their points of connection are increased in a microtubule-dependent fashion. A localized increase in mitochondrial transmembrane potential is observed only in redistributed mitochondria, whereas mitochondria that remain in other subcellular zones display no significant changes. Spatial re-organization of these organelles correlates with an increase in ATP levels, oxygen consumption, reductive power and increased mitochondrial Ca²âº uptake. Accordingly, uncoupling of the organelles or blocking Ca²âº transfer impaired the metabolic response, rendering cells more vulnerable to ER stress. Overall, these data indicate that ER stress induces an early increase in mitochondrial metabolism that depends crucially upon organelle coupling and Ca²âº transfer, which, by enhancing cellular bioenergetics, establishes the metabolic basis for the adaptation to this response.