RESUMO
Mitochondrial dysfunction is a significant factor in the development of Alzheimer's disease (AD). Previous studies have demonstrated that the expression of tau cleaved at Asp421 by caspase-3 leads to mitochondrial abnormalities and bioenergetic impairment. However, the underlying mechanism behind these alterations and their impact on neuronal function remains unknown. To investigate the mechanism behind mitochondrial dysfunction caused by this tau form, we used transient transfection and pharmacological approaches in immortalized cortical neurons and mouse primary hippocampal neurons. We assessed mitochondrial morphology and bioenergetics function after expression of full-length tau and caspase-3-cleaved tau. We also evaluated the mitochondrial permeability transition pore (mPTP) opening and its conformation as a possible mechanism to explain mitochondrial impairment induced by caspase-3 cleaved tau. Our studies showed that pharmacological inhibition of mPTP by cyclosporine A (CsA) prevented all mitochondrial length and bioenergetics abnormalities in neuronal cells expressing caspase-3 cleaved tau. Neuronal cells expressing caspase-3-cleaved tau showed sustained mPTP opening which is mostly dependent on cyclophilin D (CypD) protein expression. Moreover, the impairment of mitochondrial length and bioenergetics induced by caspase-3-cleaved tau were prevented in hippocampal neurons obtained from CypD knock-out mice. Interestingly, previous studies using these mice showed a prevention of mPTP opening and a reduction of mitochondrial failure and neurodegeneration induced by AD. Therefore, our findings showed that caspase-3-cleaved tau negatively impacts mitochondrial bioenergetics through mPTP activation, highlighting the importance of this channel and its regulatory protein, CypD, in the neuronal damage induced by tau pathology in AD.
Assuntos
Doença de Alzheimer , Poro de Transição de Permeabilidade Mitocondrial , Animais , Camundongos , Doença de Alzheimer/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Peptidil-Prolil Isomerase F/metabolismo , Mitocôndrias/metabolismo , Poro de Transição de Permeabilidade Mitocondrial/metabolismoRESUMO
The interaction between supraphysiological cytosolic Ca2+ levels and mitochondrial redox imbalance mediates the mitochondrial permeability transition (MPT). The MPT is involved in cell death, diseases and aging. This study compared the liver mitochondrial Ca2+ retention capacity and oxygen consumption in the long-lived red-footed tortoise (Chelonoidis carbonaria) with those in the rat as a reference standard. Mitochondrial Ca2+ retention capacity, a quantitative measure of MPT sensitivity, was remarkably higher in tortoises than in rats. This difference was minimized in the presence of the MPT inhibitors ADP and cyclosporine A. However, the Ca2+ retention capacities of tortoise and rat liver mitochondria were similar when both MPT inhibitors were present simultaneously. NADH-linked phosphorylating respiration rates of tortoise liver mitochondria represented only 30% of the maximal electron transport system capacity, indicating a limitation imposed by the phosphorylation system. These results suggested underlying differences in putative MPT structural components [e.g. ATP synthase, adenine nucleotide translocase (ANT) and cyclophilin D] between tortoises and rats. Indeed, in tortoise mitochondria, titrations of inhibitors of the oxidative phosphorylation components revealed a higher limitation of ANT. Furthermore, cyclophilin D activity was approximately 70% lower in tortoises than in rats. Investigation of critical properties of mitochondrial redox control that affect MPT demonstrated that tortoise and rat liver mitochondria exhibited similar rates of H2O2 release and glutathione redox status. Overall, our findings suggest that constraints imposed by ANT and cyclophilin D, putative components or regulators of the MPT pore, are associated with the enhanced resistance to Ca2+-induced MPT in tortoises.
Assuntos
Tartarugas , Animais , Cálcio/metabolismo , Peptidil-Prolil Isomerase F , Peróxido de Hidrogênio , Mitocôndrias Hepáticas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Necrose Dirigida por Permeabilidade Transmembrânica da Mitocôndria , Permeabilidade , Ratos , Tartarugas/metabolismoRESUMO
Cytotoxic effects of cannabidiol (CBD) and tamoxifen (TAM) have been observed in several cancer types. We have recently shown that CBD primarily targets mitochondria, inducing a stable mitochondrial permeability transition pore (mPTP) and, consequently, the death of acute lymphoblastic leukemia (T-ALL) cells. Mitochondria have also been documented among cellular targets for the TAM action. In the present study we have demonstrated a synergistic cytotoxic effect of TAM and CBD against T-ALL cells. By measuring the mitochondrial membrane potential (ΔΨm), mitochondrial calcium ([Ca2+]m) and protein-ligand docking analysis we determined that TAM targets cyclophilin D (CypD) to inhibit mPTP formation. This results in a sustained [Ca2+]m overload upon the consequent CBD administration. Thus, TAM acting on CypD sensitizes T-ALL to mitocans such as CBD by altering the mitochondrial Ca2+ homeostasis.
Assuntos
Cálcio/metabolismo , Canabidiol/farmacologia , Peptidil-Prolil Isomerase F/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Tamoxifeno/farmacologia , Linhagem Celular Tumoral , Peptidil-Prolil Isomerase F/química , Sinergismo Farmacológico , Homeostase/efeitos dos fármacos , Humanos , Células Jurkat , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Conformação ProteicaRESUMO
Trypanosoma cruzi is the causative agent of Chagas disease, which is endemic in Latin America and around the world through mother to child transmission. The heart is the organ most frequently affected in the chronic stage of the human infection and depends on mitochondria for the required energy for its activity. Cyclophilins are involved in protein folding and the mitochondrial isoform, Cyclophilin D (CyPD), has a crucial role in the opening of the mitochondrial permeability transition pore. In the present study, we infected CyPD deficient mice, with ablation of the Ppif gene, with T. cruzi parasites and the course of the infection was analyzed. Parasite load, quantified by PCR, was significantly lower in skeletal and cardiac tissues of Ppif-/- mice compared to wild type mice. In vitro cultured cardiomyocytes and macrophages from mice lacking CyPD exhibited lower percentage of infected cells and number of intracellular parasites than those observed for wild type mice. Although histopathological analysis of heart and mRNA of heart cytokines showed differences between T. cruzi-infected mice compared to the uninfected animals, no significant differences were found mice due to the ablation of the Ppif gene. Our results suggest that cells deficient for mitochondrial CyPD, inhibited for the mitochondrial membrane potential collapse, reduces the severity of parasite aggression and spread of cellular infection.
Assuntos
Doença de Chagas/parasitologia , Peptidil-Prolil Isomerase F/deficiência , Trypanosoma cruzi/fisiologia , Animais , Citocinas/análise , Citocinas/genética , DNA de Protozoário/isolamento & purificação , Coração/parasitologia , Fígado/patologia , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/parasitologia , Músculo Esquelético/patologia , Miocárdio/patologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/parasitologia , Carga Parasitária , RNA Mensageiro/análise , RNA de Protozoário/análise , RNA de Protozoário/isolamento & purificação , Baço/patologia , Trypanosoma cruzi/genéticaRESUMO
BACKGROUND/AIMS: Cyclophilin D (CypD) mediates the mitochondrial permeability transition pore (mPTP) opening that contributes to mitochondrial dysfunction. CypD is regulated by its acetylation/deacetylation state that depends on Sirtuin-3 (SIRT3) mitochondrial deacetylase. Since obesity and metabolic syndrome decrease SIRT3 activity and expression, we tested the hypothesis that CypD hyperacetylation promotes mitochondrial dysfunction under this pathophysiological state, which is associated with ventricular dysfunction and heart failure. METHODS: Myocardial tissue samples from patients with left ventricular heart failure, with either obesity or normal weight, were processed for the expression of SIRT3 and acetylation profile by Western Blot (WB). In addition, a rat model of obesity and metabolic syndrome induced by 30% (w/v) of sucrose was conducted. The WB analysis was used to determine the levels of mitochondrial expression of SIRT3, Adenine Nucleotide Translocator (ANT), CypD and the acetylation profile, as well as immunoprecipitation to establish the acetylation levels of CypD. Mitochondrial function was assessed by oxygen consumption analysis and maximum Ca2+ retention capacity. Oxidative stress was assessed by aconitase activity, protein carbonyl and thiol groups content. RESULTS: SIRT3 expression in the biopsies of the failing human hearts showed a 46% decrease in the expression levels of obese patients in comparison to the non-obese patients (p=0.0219). Remarkably, body mass index was associated with protein acetylation (0.627; p = 0.035), suggesting that the acetylation profiles of the failing hearts of obese patients are partly mediated by a reduction in SIRT3, which is also associated with higher BNP levels, indicating a more severe ventricular dysfunction (-0.636; p = 0.043). Accordingly, obese rats demonstrated a SIRT3 mitochondrial expression decrease of 22% concomitantly with a hyperacetylated mitochondrial profile, including CypD. Cardiac mitochondria from obese animals were 2.5-fold more prone to mPTP opening than the controls. CONCLUSION: Our results indicate that obesity reduces SIRT3 expression and that CypD hyperacetylation increases mPTP opening, suggesting that the activation of SIRT3 might be a potential target to decrease ventricular dysfunction and slow the progression of heart failure.
Assuntos
Mitocôndrias Cardíacas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Obesidade/metabolismo , Sirtuína 3/metabolismo , Acetilação , Adulto , Idoso , Animais , Índice de Massa Corporal , Cálcio/metabolismo , Peptidil-Prolil Isomerase F , Ciclofilinas/metabolismo , Feminino , Insuficiência Cardíaca/metabolismo , Humanos , Imunoprecipitação , Técnicas In Vitro , Masculino , Síndrome Metabólica/metabolismo , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Translocases Mitocondriais de ADP e ATP/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Consumo de Oxigênio/fisiologia , Ratos , Ratos WistarRESUMO
Tau is a key protein for microtubule stability; however, post-translationally modified tau contributes to neurodegenerative diseases by forming tau aggregates in the neurons. Previous reports from our group and others have shown that pathological forms of tau are toxic and impair mitochondrial function, whereas tau deletion is neuroprotective. However, the effects of tau ablation on brain structure and function in young mice have not been fully elucidated. Therefore, the aim of this study was to investigate the implications of tau ablation on the mitochondrial function and cognitive abilities of a litter of young mice (3 months old). Our results showed that tau deletion had positive effects on hippocampal cells by decreasing oxidative damage, favoring a mitochondrial pro-fusion state, and inhibiting mitochondrial permeability transition pore (mPTP) formation by reducing cyclophilin D (Cyp-D) protein. More importantly, tau deletion increased ATP production and improved the recognition memory and attentive capacity of juvenile mice. Therefore, the absence of tau enhanced brain function by improving mitochondrial health, which supplied more energy to the synapses. Thus, our work opens the possibility that preventing negative tau modifications could enhance brain function through the improvement of mitochondrial health.
Assuntos
Cognição , Deleção de Genes , Hipocampo/fisiologia , Mitocôndrias/metabolismo , Proteínas tau/genética , Animais , Peptidil-Prolil Isomerase F , Ciclofilinas/metabolismo , Hipocampo/metabolismo , Aprendizagem , Masculino , Aprendizagem em Labirinto , Memória , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Estresse Oxidativo , Proteínas tau/metabolismoRESUMO
Wallerian degeneration is an active program tightly associated with axonal degeneration, required for axonal regeneration and functional recovery after nerve damage. Here we provide a functional molecular foundation for our undertstanding of the complex non-cell autonomous role of glial cells in the regulation of axonal degeneration. To shed light on the complexity of the molecular machinery governing axonal degeneration we employ a multi-model, unbiased, in vivo approach combining morphological assesment and quantitative proteomics with in silico-based higher order functional clustering to genetically uncouple the intrinsic and extrinsic processes governing Wallerian degeneration. Highlighting a pivotal role for glial cells in the early stages fragmenting the axon by a cytokinesis-like process and a cell autonomous stage of axonal disintegration associated to mitochondrial dysfunction.
Assuntos
Axônios/metabolismo , Neuroglia/metabolismo , Animais , Desdiferenciação Celular/efeitos dos fármacos , Células Cultivadas , Proteínas Contráteis/antagonistas & inibidores , Proteínas Contráteis/genética , Proteínas Contráteis/metabolismo , Peptidil-Prolil Isomerase F , Ciclofilinas/deficiência , Ciclofilinas/genética , Dactinomicina/farmacologia , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Bainha de Mielina/fisiologia , Neuroglia/citologia , Proteômica , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/lesões , Degeneração Walleriana/metabolismo , Degeneração Walleriana/patologia , Quinases Associadas a rho/metabolismoAssuntos
Trypanosoma cruzi/metabolismo , Morte Celular/efeitos dos fármacos , Peptidil-Prolil Isomerase F , Ciclofilinas/metabolismo , Ciclosporina/farmacologia , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Poro de Transição de Permeabilidade Mitocondrial , Trypanosoma cruzi/citologia , Trypanosoma cruzi/efeitos dos fármacosRESUMO
Growing number of studies provide strong evidence that the mitochondrial permeability transition pore (PTP), a non-selective channel in the inner mitochondrial membrane, is involved in the pathogenesis of cardiac ischemia-reperfusion and can be targeted to attenuate reperfusion-induced damage to the myocardium. The molecular identity of the PTP remains unknown and cyclophilin D is the only protein commonly accepted as a major regulator of the PTP opening. Therefore, cyclophilin D is an attractive target for pharmacological or genetic therapies to reduce ischemia-reperfusion injury in various animal models and humans. Most animal studies demonstrated cardioprotective effects of PTP inhibition; however, a recent large clinical trial conducted by international groups demonstrated that cyclosporine A, a cyclophilin D inhibitor, failed to protect the heart in patients with myocardial infarction. These studies, among others, raise the question of whether cyclophilin D, which plays an important physiological role in the regulation of cell metabolism and mitochondrial bioenergetics, is a viable target for cardioprotection. This review discusses previous studies to provide comprehensive information on the physiological role of cyclophilin D as well as PTP opening in the cell that can be taken into consideration for the development of new PTP inhibitors.
Assuntos
Cardiotônicos/farmacologia , Ciclofilinas/metabolismo , Descoberta de Drogas , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Isquemia Miocárdica/tratamento farmacológico , Isquemia Miocárdica/metabolismo , Animais , Cálcio/metabolismo , Peptidil-Prolil Isomerase F , Ciclofilinas/antagonistas & inibidores , Metabolismo Energético/efeitos dos fármacos , Coração/efeitos dos fármacos , Humanos , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Poro de Transição de Permeabilidade Mitocondrial , Terapia de Alvo Molecular , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Mapas de Interação de Proteínas/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacosRESUMO
Caloric restriction (CR) protects against many cerebral pathological conditions that are associated with excitotoxic damage and calcium overload, although the mechanisms are still poorly understood. Here we show that CR strongly protects against excitotoxic insults in vitro and in vivo in a manner associated with significant changes in mitochondrial function. CR increases electron transport chain activity, enhances antioxidant defenses, and favors mitochondrial calcium retention capacity in the brain. These changes are accompanied by a decrease in cyclophilin D activity and acetylation and an increase in Sirt3 expression. This suggests that Sirt3-mediated deacetylation and inhibition of cyclophilin D in CR promote the inhibition of mitochondrial permeability transition, resulting in enhanced mitochondrial calcium retention. Altogether, our results indicate that enhanced mitochondrial calcium retention capacity underlies the beneficial effects of CR against excitotoxic conditions. This protection may explain the many beneficial effects of CR in the aging brain.