RESUMO
An increase in plasma high glucose promotes endothelial dysfunction mainly through increasing mitochondrial ROS production. High glucose ROS-induced has been implicated in the fragmentation of the mitochondrial network, mainly by an unbalance expression of mitochondrial fusion and fission proteins. Mitochondrial dynamics alterations affect cellular bioenergetics. Here, we assessed the effect of PDGF-C on mitochondrial dynamics and glycolytic and mitochondrial metabolism in a model of endothelial dysfunction induced by high glucose. High glucose induced a fragmented mitochondrial phenotype associated with the reduced expression of OPA1 protein, high DRP1pSer616 levels and reduced basal respiration, maximal respiration, spare respiratory capacity, non-mitochondrial oxygen consumption and ATP production, regarding normal glucose. In these conditions, PDGF-C significantly increased the expression of OPA1 fusion protein, diminished DRP1pSer616 levels and restored the mitochondrial network. On mitochondrial function, PDGF-C increased the non-mitochondrial oxygen consumption diminished by high glucose conditions. These results suggest that PDGF-C modulates the damage induced by HG on the mitochondrial network and morphology of human aortic endothelial cells; additionally, it compensates for the alteration in the energetic phenotype induced by HG.
Assuntos
Dinaminas , Doenças Vasculares , Humanos , Dinaminas/genética , Células Endoteliais/metabolismo , Glucose/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Proteínas Mitocondriais/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Doenças Vasculares/metabolismoRESUMO
Nek4 is a serine/threonine kinase which has been implicated in primary cilia stabilization, DNA damage response, autophagy and epithelial-to-mesenchymal transition. The role of Nek4 in cancer cell survival and chemotherapy resistance has also been shown. However, the precise mechanisms by which Nek4 operates remain to be elucidated. Here, we show that Nek4 overexpression activates mitochondrial respiration coupled to ATP production, which is paralleled by increased mitochondrial membrane potential, and resistance to mitochondrial DNA damage. Congruently, Nek4 depletion reduced mitochondrial respiration and mtDNA integrity. Nek4 deficiency caused mitochondrial elongation, probably via reduced activity of the fission protein DRP1. In Nek4 overexpressing cells, the increase in mitochondrial fission was concomitant to enhanced phosphorylation of DRP1 and Erk1/2 proteins, and the effects on mitochondrial respiration were abolished in the presence of a DRP1 inhibitor. This study shows Nek4 as a novel regulator of mitochondrial function that may explain the joint appearance of high mitochondrial respiration and mitochondrial fragmentation.
Assuntos
Dinaminas , Dinâmica Mitocondrial , DNA Mitocondrial/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Fosforilação , RespiraçãoRESUMO
The Rad, Rem, Rem2, and Gem/Kir (RGK) sub-family of small GTP-binding proteins are crucial in regulating high voltage-activated (HVA) calcium channels. RGK proteins inhibit calcium current by either promoting endocytosis or reducing channel activity. They all can associate directly with Ca2+ channel ß subunit (CaVß), and the binding between CaVα1/CaVß appears essential for the endocytic promotion of CaV1.X, CaV2.1, and CaV2.2 channels. In this study, we investigated the inhibition of CaV2.3 channels by RGK proteins in the absence of CaVß. To this end, Xenopus laevis oocytes expressing CaV2.3 channels devoid of auxiliary subunit were injected with purified Gem and Rem and found that only Gem had an effect. Ca currents and charge movements were reduced by injection of Gem, pointing to a reduction in the number of channels in the plasma membrane. Since this reduction was ablated by co-expression of the dominant-negative mutant of dynamin K44A, enhanced endocytosis appears to mediate this reduction in the number of channels. Thus, Gem inhibition of CaV2.3 channels would be the only example of a CaVß independent promotion of dynamin-dependent endocytosis.
Assuntos
Potenciais de Ação/fisiologia , Canais de Cálcio Tipo R/genética , Proteínas de Transporte de Cátions/genética , Dinaminas/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Substituição de Aminoácidos , Animais , Canais de Cálcio Tipo R/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Dinaminas/metabolismo , Endocitose/genética , Feminino , Expressão Gênica , Humanos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mutação , Oócitos/citologia , Oócitos/metabolismo , Técnicas de Patch-Clamp , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção , Transgenes , Xenopus laevisRESUMO
Mitochondrial impairments in dynamic behavior (fusion/fission balance) associated with mitochondrial dysfunction play a key role in cell lipotoxicity and lipid-induced metabolic diseases. The present work aimed to evaluate dose- and time-dependent effects of the monounsaturated fatty acid oleate on mitochondrial fusion/fission proteins in comparison with the saturated fatty acid palmitate in hepatic cells. To this end, HepG-2 cells were treated with 0, 10 µM, 50 µM, 100 µM, 250 µM or 500 µM of either oleate or palmitate for 8 or 24 h. Cell viability and lipid accumulation were evaluated to assess lipotoxicity. Mitochondrial markers of fusion (mitofusin 2, MFN2) and fission (dynamin-related protein 1, DRP1) processes were evaluated by Western blot analysis. After 8 h, the highest dose of oleate induced a decrease in DRP1 content without changes in MFN2 content in association with cell viability maintenance, whereas palmitate induced a decrease in cell viability associated with a decrease mainly in MFN2 content. After 24 h, oleate induced MFN2 increase, whereas palmitate induced DRP1 increase associated with a higher decrease in cell viability with high doses compared to oleate. This finding could be useful to understand the role of mitochondria in the protective effects of oleate as a bioactive compound.
Assuntos
Dinaminas/genética , GTP Fosfo-Hidrolases/genética , Doenças Metabólicas/genética , Dinâmica Mitocondrial/efeitos dos fármacos , Proteínas Mitocondriais/genética , Ácido Oleico/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ácidos Graxos Monoinsaturados/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/toxicidade , Doenças Metabólicas/etiologia , Doenças Metabólicas/metabolismo , Doenças Metabólicas/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/patologia , Dinâmica Mitocondrial/genética , Ácido Oleico/farmacologia , Palmitatos/metabolismo , Palmitatos/farmacologiaRESUMO
Reactive oxygen species (ROS) production has been associated with neuronal death. ROS are also involved in mitochondrial fission, which is mediated by Dynamin-related protein 1 (Drp1). The regulation of mitochondrial fragmentation mediated by Drp1 and its relationship to mitochondrial ROS (mtROS) in neuronal death have not been completely clarified. The aim of this study is to evaluate the role of mtROS in cell death and their involvement in the activation of Drp1 and mitochondrial fission in a model of cell death of cultured cerebellar granule neurons (CGN). Neuronal death of CGN induced by potassium deprivation (K5) and staurosporine (ST) triggers mitochondrial ROS production and mitochondrial fragmentation. K5 condition evoked an increase of Drp1 phosphorylation at Ser616, but ST treatment led to a decrease of Drp1 phosphorylation. Moreover, the death of CGN induced by both K5 and ST was markedly reduced in the presence of MitoTEMPO; however, mitochondrial morphology was not recovered. Here, we show that the mitochondria are the initial source of ROS involved in the neuronal death of CGN and that mitochondrial fragmentation is a common event in cell death; however, this process is not mediated by Drp1 phosphorylation at Ser616.
Assuntos
Dinaminas/genética , Mitocôndrias/metabolismo , Neurônios/metabolismo , Animais , Morte Celular , Humanos , Fosforilação , Ratos , Ratos Wistar , Espécies Reativas de OxigênioRESUMO
Synaptic aging has been associated with neuronal circuit dysfunction and cognitive decline. Reduced mitochondrial function may be an early event that compromises synaptic integrity and neurotransmission in vulnerable brain regions during physiological and pathological aging. Thus, we aimed to measure mitochondrial function in synapses from three brain regions at two different ages in the 3xTg-AD mouse model and in wild mice. We found that aging is the main factor associated with the decline in synaptic mitochondrial function, particularly in synapses isolated from the cerebellum. Accumulation of toxic compounds, such as tau and Aß, that occurred in the 3xTg-AD mouse model seemed to participate in the worsening of this decline in the hippocampus. The changes in synaptic bioenergetics were also associated with increased activation of the mitochondrial fission protein Drp1. These results suggest the presence of altered mechanisms of synaptic mitochondrial dynamics and their quality control during aging and in the 3xTg-AD mouse model; they also point to bioenergetic restoration as a useful therapeutic strategy to preserve synaptic function during aging and at the early stages of Alzheimer's disease (AD).
Assuntos
Envelhecimento/genética , Disfunção Cognitiva/genética , Dinaminas/genética , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/genética , Envelhecimento/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Cerebelo/metabolismo , Cerebelo/fisiopatologia , Córtex Cerebral/metabolismo , Córtex Cerebral/fisiopatologia , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/fisiopatologia , Modelos Animais de Doenças , Dinaminas/metabolismo , Feminino , Regulação da Expressão Gênica , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Humanos , Potencial da Membrana Mitocondrial/genética , Camundongos , Camundongos Transgênicos , Mitocôndrias/patologia , Neurônios/metabolismo , Neurônios/patologia , Especificidade de Órgãos , Sinapses/metabolismo , Sinapses/patologia , Sinaptossomos/metabolismo , Sinaptossomos/patologia , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
Melanoma is a malignant proliferative disease originated in melanocytes, characterized by high metastatic activity and by the activation of oncogenes, such as B-RAF (40-60% of cases). Recent studies have shown that vemurafenib (a MAPK inhibitor) promoted disturbance of mitochondrial bioenergetics, although underlying mechanisms are not fully comprehended. Here we showed that MAPK inhibition by vemurafenib in B-RAFV600E-mutated human melanoma culminated in the inhibition of DRP1 phosphorylation, associated to a large mitochondrial network remodeling to the hyperfused phenotype, and increased oxidative phosphorylation capacity. Such alterations may be associated to melanoma resistance to vemurafenib, since the impairment of oxidative phosphorylation increased the vemurafenib cytotoxicity. These results point to the potential of mitochondrial dynamics as a targetable pathway in melanoma.
Assuntos
Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica , Dinâmica Mitocondrial/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Vemurafenib/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Dinaminas/antagonistas & inibidores , Dinaminas/genética , Dinaminas/metabolismo , Humanos , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Melanócitos/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Terapia de Alvo Molecular , Mutação , Fosforilação Oxidativa/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas B-raf/metabolismo , Transdução de SinaisRESUMO
Mouse skin mesenchymal stem cells (msMSCs) are dermis CD105(+) CD90(+) CD73(+) CD29(+) CD34(-) mesodermal precursors which, after in vitro induction, undergo chondro, adipo, and osteogenesis. Extensive metabolic reconfiguration has been found to occur during differentiation, and the bioenergetic status of a cell is known to be dependent on the quality and abundance of the mitochondrial population, which may be regulated by fusion and fission. However, little is known regarding the impact of mitochondrial dynamics on the differentiation process. We addressed this knowledge gap by isolating MSCs from Swiss female mice, inducing these cells to differentiate into osteo, chondro, and adipocytes and measuring changes in mass, morphology, dynamics, and bioenergetics. Mitochondrial biogenesis was increased in adipogenesis, as evaluated through confocal microscopy, citrate synthase activity, and mtDNA content. The early steps of adipo and osteogenesis involved mitochondrial elongation, as well as increased expression of mitochondrial fusion proteins Mfn1 and 2. Chondrogenesis involved a fragmented mitochondrial phenotype, increased expression of fission proteins Drp1, Fis1, and 2, and enhanced mitophagy. These events were accompanied by profound bioenergetic alterations during the commitment period. Moreover, knockdown of Mfn2 in adipo and osteogenesis and the overexpression of a dominant negative form of Drp1 during chondrogenesis resulted in a loss of differentiation ability. Overall, we find that mitochondrial morphology and its regulating processes of fission/fusion are modulated early on during commitment, leading to alterations in the bioenergetic profile that are important for differentiation. We thus propose a central role for mitochondrial dynamics in the maintenance/commitment of mesenchymal stem cells.
Assuntos
Diferenciação Celular/genética , Dinaminas/biossíntese , GTP Fosfo-Hidrolases/biossíntese , Células-Tronco Mesenquimais , Mitocôndrias/metabolismo , Adipogenia/genética , Animais , Condrogênese/genética , DNA Mitocondrial/genética , Dinaminas/genética , Feminino , GTP Fosfo-Hidrolases/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , Mitocôndrias/genética , Dinâmica Mitocondrial/genética , Osteogênese/genética , Pele/citologia , Pele/metabolismoRESUMO
Iron overload contributes to the development of neurodegeneration and the exacerbation of normal apoptosis rates, largely due to its participation in the Fenton reaction and production of reactive oxygen species (ROS). Mitochondria constitute the major intracellular source of ROS and the main target of attack by free radicals. They are dynamic organelles that bind (fusion) and divide (fission) in response to environmental stimuli, developmental status, and energy needs of the cells. Sulforaphane (SFN) is a natural compound that displays antioxidant and anti-inflammatory activities. This study aims to investigate the effects of SFN on memory deficits and changes in markers of mitochondrial function, DNM1L and OPA1, and the synaptic marker, synaptophysin, induced by neonatal iron treatment. Male rats received vehicle or carbonyl iron (30mg/kg) from the 12th to the 14th postnatal day. In adulthood, they were treated with saline or SFN (0.5 or 5mg/kg) for 14days every other day. Memory deficits were assessed using the object recognition task. DNM1L, OPA1, and synaptophysin levels in the hippocampus were quantified by Western blotting. Results showed that SFN was able to reverse iron-induced decreases in mitochondrial fission protein, DNM1L, as well as synaptophysin levels in the hippocampus, leading to a recovery of recognition memory impairment induced by iron. These findings suggest that SFN may be further investigated as potential agent for the treatment of cognitive deficits associated with neurodegenerative disorders.