RESUMO
The Parkinson's disease-associated protein kinase PINK1 and ubiquitin ligase Parkin coordinate the ubiquitination of mitochondrial proteins, which marks mitochondria for degradation. Miro1, an atypical GTPase involved in mitochondrial trafficking, is one of the substrates tagged by Parkin after mitochondrial damage. Here, we demonstrate that a small pool of Parkin interacts with Miro1 before mitochondrial damage occurs. This interaction does not require PINK1, does not involve ubiquitination of Miro1 and also does not disturb Miro1 function. However, following mitochondrial damage and PINK1 accumulation, this initial pool of Parkin becomes activated, leading to the ubiquitination and degradation of Miro1. Knockdown of Miro proteins reduces Parkin translocation to mitochondria and suppresses mitophagic removal of mitochondria. Moreover, we demonstrate that Miro1 EF-hand domains control Miro1's ubiquitination and Parkin recruitment to damaged mitochondria, and they protect neurons from glutamate-induced mitophagy. Together, our results suggest that Miro1 functions as a calcium-sensitive docking site for Parkin on mitochondria.
Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Mitofagia , Domínios Proteicos , Transporte Proteico , Proteólise , Ratos , Ubiquitinação , Proteínas rho de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/genéticaRESUMO
During early development, neurons undergo complex morphological rearrangements to assemble into neuronal circuits and propagate signals. Rapid growth requires a large quantity of building materials, efficient intracellular transport and also a considerable amount of energy. To produce this energy, the neuron should first generate new mitochondria because the pre-existing mitochondria are unlikely to provide a sufficient acceleration in ATP production. Here, we demonstrate that mitochondrial biogenesis and ATP production are required for axonal growth and neuronal development in cultured rat cortical neurons. We also demonstrate that growth signals activating the CaMKKß, LKB1-STRAD or TAK1 pathways also co-activate the AMPK-PGC-1α-NRF1 axis leading to the generation of new mitochondria to ensure energy for upcoming growth. In conclusion, our results suggest that neurons are capable of signalling for upcoming energy requirements. Earlier activation of mitochondrial biogenesis through these pathways will accelerate the generation of new mitochondria, thereby ensuring energy-producing capability for when other factors for axonal growth are synthesized.
Assuntos
Axônios/metabolismo , Biogênese de Organelas , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Adenilato Quinase/metabolismo , Animais , Animais Recém-Nascidos , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Proliferação de Células , Células Cultivadas , Córtex Cerebral/citologia , Metabolismo Energético , MAP Quinase Quinase Quinases/metabolismo , Mitocôndrias/metabolismo , Modelos Biológicos , Neurogênese , Fator 1 Nuclear Respiratório/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Ratos Wistar , Fator de Crescimento Transformador beta/metabolismoRESUMO
Deficiency of the protein Wolfram syndrome 1 (WFS1) is associated with multiple neurological and psychiatric abnormalities similar to those observed in pathologies showing alterations in mitochondrial dynamics. The aim of this study was to examine the hypothesis that WFS1 deficiency affects neuronal function via mitochondrial abnormalities. We show that down-regulation of WFS1 in neurons leads to dramatic changes in mitochondrial dynamics (inhibited mitochondrial fusion, altered mitochondrial trafficking, and augmented mitophagy), delaying neuronal development. WFS1 deficiency induces endoplasmic reticulum (ER) stress, leading to inositol 1,4,5-trisphosphate receptor (IP3R) dysfunction and disturbed cytosolic Ca2+ homeostasis, which, in turn, alters mitochondrial dynamics. Importantly, ER stress, impaired Ca2+ homeostasis, altered mitochondrial dynamics, and delayed neuronal development are causatively related events because interventions at all these levels improved the downstream processes. Our data shed light on the mechanisms of neuronal abnormalities in Wolfram syndrome and point out potential therapeutic targets. This work may have broader implications for understanding the role of mitochondrial dynamics in neuropsychiatric diseases.
Assuntos
Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Neurogênese , Neurônios/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Encéfalo/metabolismo , Cálcio/metabolismo , Células Cultivadas , Estresse do Retículo Endoplasmático/genética , Transferência Ressonante de Energia de Fluorescência , Homeostase , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Potencial da Membrana Mitocondrial/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Knockout , Microscopia Confocal , Mitocôndrias/genética , Mitofagia/genética , Neurônios/citologia , Células PC12 , Interferência de RNA , Ratos , Ratos Wistar , Imagem com Lapso de Tempo/métodos , Síndrome de Wolfram/genética , Síndrome de Wolfram/metabolismoRESUMO
Mitochondrial dysfunction is a significant factor in human disease, ranging from systemic disorders of childhood to cardiomyopathy, ischaemia and neurodegeneration. Cytochrome oxidase, the terminal enzyme of the mitochondrial respiratory chain, is a frequent target. Lower eukaryotes possess alternative respiratory-chain enzymes that provide non-proton-translocating bypasses for respiratory complexes I (single-subunit reduced nicotinamide adenine dinucleotide dehydrogenases, e.g. Ndi1 from yeast) or III + IV [alternative oxidase (AOX)], under conditions of respiratory stress or overload. In previous studies, it was shown that transfer of yeast Ndi1 or Ciona intestinalis AOX to Drosophila was able to overcome the lethality produced by toxins or partial knockdown of complex I or IV. Here, we show that AOX can provide a complete or substantial rescue of a range of phenotypes induced by global or tissue-specific knockdown of different cIV subunits, including integral subunits required for catalysis, as well as peripheral subunits required for multimerization and assembly. AOX was also able to overcome the pupal lethality produced by muscle-specific knockdown of subunit CoVb, although the rescued flies were short lived and had a motility defect. cIV knockdown in neurons was not lethal during development but produced a rapidly progressing locomotor and seizure-sensitivity phenotype, which was substantially alleviated by AOX. Expression of Ndi1 exacerbated the neuronal phenotype produced by cIV knockdown. Ndi1 expressed in place of essential cI subunits produced a distinct residual phenotype of delayed development, bang sensitivity and male sterility. These findings confirm the potential utility of alternative respiratory chain enzymes as tools to combat mitochondrial disease, while indicating important limitations thereof.
Assuntos
Animais Geneticamente Modificados/metabolismo , Deficiência de Citocromo-c Oxidase/complicações , Deficiências do Desenvolvimento/prevenção & controle , Drosophila melanogaster/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Infertilidade Masculina/prevenção & controle , Proteínas Mitocondriais/metabolismo , Doenças Neurodegenerativas/prevenção & controle , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Western Blotting , Células Cultivadas , Deficiência de Citocromo-c Oxidase/genética , Deficiência de Citocromo-c Oxidase/metabolismo , Deficiências do Desenvolvimento/etiologia , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo IV da Cadeia de Transporte de Elétrons/genética , Feminino , Humanos , Técnicas Imunoenzimáticas , Infertilidade Masculina/etiologia , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Doenças Neurodegenerativas/etiologia , Oxirredutases/genética , Fenótipo , Proteínas de Plantas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Wolfram syndrome is a rare genetic disease caused by mutations in the WFS1 or CISD2 gene. A primary defect in Wolfram syndrome involves poor ER Ca2+ handling, but how this disturbance leads to the disease is not known. The current study, performed in primary neurons, the most affected and disease-relevant cells, involving both Wolfram syndrome genes, explains how the disturbed ER Ca2+ handling compromises mitochondrial function and affects neuronal health. Loss of ER Ca2+ content and impaired ER-mitochondrial contact sites in the WFS1- or CISD2-deficient neurons is associated with lower IP3R-mediated Ca2+ transfer from ER to mitochondria and decreased mitochondrial Ca2+ uptake. In turn, reduced mitochondrial Ca2+ content inhibits mitochondrial ATP production leading to an increased NADH/NAD+ ratio. The resulting bioenergetic deficit and reductive stress compromise the health of the neurons. Our work also identifies pharmacological targets and compounds that restore Ca2+ homeostasis, enhance mitochondrial function and improve neuronal health.
Assuntos
Cálcio , Retículo Endoplasmático , Proteínas de Membrana , Mitocôndrias , Neurônios , Síndrome de Wolfram , Síndrome de Wolfram/metabolismo , Síndrome de Wolfram/genética , Cálcio/metabolismo , Mitocôndrias/metabolismo , Retículo Endoplasmático/metabolismo , Animais , Neurônios/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Camundongos , Humanos , Trifosfato de Adenosina/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Camundongos Knockout , NAD/metabolismo , Sinalização do CálcioRESUMO
Mutations in mitochondrial oxidative phosphorylation complex I are associated with multiple pathologies, and complex I has been proposed as a crucial regulator of animal longevity. In yeast, the single-subunit NADH dehydrogenase Ndi1 serves as a non-proton-translocating alternative enzyme that replaces complex I, bringing about the reoxidation of intramitochondrial NADH. We have created transgenic strains of Drosophila that express yeast NDI1 ubiquitously. Mitochondrial extracts from NDI1-expressing flies displayed a rotenone-insensitive NADH dehydrogenase activity, and functionality of the enzyme in vivo was confirmed by the rescue of lethality resulting from RNAi knockdown of complex I. NDI1 expression increased median, mean, and maximum lifespan independently of dietary restriction, and with no change in sirtuin activity. NDI1 expression mitigated the aging associated decline in respiratory capacity and the accompanying increase in mitochondrial reactive oxygen species production, and resulted in decreased accumulation of markers of oxidative damage in aged flies. Our results support a central role of mitochondrial oxidative phosphorylation complex I in influencing longevity via oxidative stress, independently of pathways connected to nutrition and growth signaling.
Assuntos
Envelhecimento/metabolismo , Drosophila melanogaster/fisiologia , Complexo I de Transporte de Elétrons/metabolismo , Longevidade/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Animais , Western Blotting , Restrição Calórica , Drosophila melanogaster/enzimologia , Complexo I de Transporte de Elétrons/genética , Histocitoquímica , Longevidade/genética , Mitocôndrias/metabolismo , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
If cellular reactive oxygen species (ROS) production surpasses the intracellular antioxidant capacity, thus altering the ROS homeostasis, the cell needs to eradicate faulty mitochondria responsible for these excessive ROS. We have shown that even moderate ROS production breaks the KEAP1-PGAM5 complex, inhibiting the proteasomal removal of PGAM5. This leads to an accumulation of PGAM5 interfering with PINK1 processing that sensitizes mitochondria to autophagic removal. We propose that such a negative feedback system maintains cell ROS homeostasis.
Assuntos
Proteínas Mitocondriais , Mitofagia , Autofagia , Retroalimentação , Homeostase , Proteína 1 Associada a ECH Semelhante a Kelch , Proteínas Mitocondriais/metabolismo , Fator 2 Relacionado a NF-E2 , Fosfoproteínas Fosfatases/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Mitochondria in the cell are the center for energy production, essential biomolecule synthesis, and cell fate determination. Moreover, the mitochondrial functional versatility enables cells to adapt to the changes in cellular environment and various stresses. In the process of discharging its cellular duties, mitochondria face multiple types of challenges, such as oxidative stress, protein-related challenges (import, folding, and degradation) and mitochondrial DNA damage. They mitigate all these challenges with robust quality control mechanisms which include antioxidant defenses, proteostasis systems (chaperones and proteases) and mitochondrial biogenesis. Failure of these quality control mechanisms leaves mitochondria as terminally damaged, which then have to be promptly cleared from the cells before they become a threat to cell survival. Such damaged mitochondria are degraded by a selective form of autophagy called mitophagy. Rigorous research in the field has identified multiple types of mitophagy processes based on targeting signals on damaged or superfluous mitochondria. In this review, we provide an in-depth overview of mammalian mitophagy and its importance in human health and diseases. We also attempted to highlight the future area of investigation in the field of mitophagy.
Assuntos
Mamíferos/metabolismo , Animais , Humanos , Mitofagia/genética , Modelos Biológicos , Biogênese de Organelas , Receptores de Superfície Celular/metabolismo , Ubiquitina/metabolismoRESUMO
When ROS production exceeds the cellular antioxidant capacity, the cell needs to eliminate the defective mitochondria responsible for excessive ROS production. It has been proposed that the removal of these defective mitochondria involves mitophagy, but the mechanism of this regulation remains unclear. Here, we demonstrate that moderate mitochondrial superoxide and hydrogen peroxide production oxidates KEAP1, thus breaking the interaction between this protein and PGAM5, leading to the inhibition of its proteasomal degradation. Accumulated PGAM5 interferes with the processing of the PINK1 in the mitochondria leading to the accumulation of PINK1 on the outer mitochondrial membrane. In turn, PINK1 promotes Parkin recruitment to mitochondria and sensitizes mitochondria for autophagic removal. We also demonstrate that inhibitors of the KEAP1-PGAM5 protein-protein interaction (including CPUY192018) mimic the effect of mitochondrial ROS and sensitize mitophagy machinery, suggesting that these inhibitors could be used as pharmacological regulators of mitophagy. Together, our results show that KEAP1/PGAM5 complex senses mitochondrially generated superoxide/hydrogen peroxide to induce mitophagy.