RESUMO
The actin-modulating protein Wiskott-Aldrich syndrome protein verprolin homologous-1 (WAVE1) and a novel CNS-specific protein, pancortin, are highly enriched in adult cerebral cortex, but their functions are unknown. Here we show that WAVE1 and pancortin-2 interact in a novel cell death cascade in adult, but not embryonic, cerebral cortical neurons. Focal ischemic stroke induces the formation of a protein complex that includes pancortin-2, WAVE1, and the anti-apoptotic protein Bcl-xL. The three-protein complex is associated with mitochondria resulting in increased association of Bax with mitochondria, cytochrome c release, and neuronal apoptosis. In pancortin null mice generated using a Cre-loxP system, ischemia-induced WAVE1-Bcl-xL interaction is diminished, and cortical neurons in these mice are protected against ischemic injury. Thus, pancortin-2 is a mediator of ischemia-induced apoptosis of neurons in the adult cerebral cortex and functions in a novel mitochondrial/actin-associated protein complex that sequesters Bcl-xL.
Assuntos
Isquemia Encefálica/patologia , Proteínas da Matriz Extracelular/fisiologia , Glicoproteínas/fisiologia , Mitocôndrias/metabolismo , Neurônios , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Proteína bcl-X/metabolismo , Animais , Western Blotting/métodos , Isquemia Encefálica/genética , Morte Celular/fisiologia , Células Cultivadas , Córtex Cerebral/patologia , Citocromos c , Embrião de Mamíferos , Proteínas da Matriz Extracelular/deficiência , Lateralidade Funcional , Glicoproteínas/deficiência , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica/métodos , Imunoprecipitação/métodos , Masculino , Camundongos , Camundongos Knockout , Neurônios/patologia , Neurônios/fisiologia , Neurônios/ultraestrutura , Ratos , Ratos Wistar , Fatores de TempoRESUMO
The high-metabolic demand of neurons and their reliance on glucose as an energy source places them at risk for dysfunction and death under conditions of metabolic and oxidative stress. Uncoupling proteins (UCPs) are mitochondrial inner membrane proteins implicated in the regulation of mitochondrial membrane potential (Deltapsim) and cellular energy metabolism. The authors cloned UCP4 cDNA from mouse and rat brain, and demonstrate that UCP4 mRNA is expressed abundantly in brain and at particularly high levels in populations of neurons believed to have high-energy requirements. Neural cells with increased levels of UCP4 exhibit decreased Deltapsim, reduced reactive oxygen species (ROS) production and decreased mitochondrial calcium accumulation. UCP4 expressing cells also exhibited changes of oxygen-consumption rate, GDP sensitivity, and response of Deltapsim to oligomycin that were consistent with mitochondrial uncoupling. UCP4 modulates neuronal energy metabolism by increasing glucose uptake and shifting the mode of ATP production from mitochondrial respiration to glycolysis, thereby maintaining cellular ATP levels. The UCP4-mediated shift in energy metabolism reduces ROS production and increases the resistance of neurons to oxidative and mitochondrial stress. Knockdown of UCP4 expression by RNA interference in primary hippocampal neurons results in mitochondrial calcium overload and cell death. UCP4-mRNA expression is increased in neurons exposed to cold temperatures and in brain cells of rats maintained on caloric restriction, suggesting a role for UCP4 in the previously reported antiageing and neuroprotective effects of caloric restriction. By shifting energy metabolism to reduce ROS production and cellular reliance on mitochondrial respiration, UCP4 can protect neurons against oxidative stress and calcium overload.
Assuntos
Metabolismo Energético , Canais Iônicos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Neurônios/metabolismo , Estresse Oxidativo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/fisiologia , Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Cálcio/metabolismo , Restrição Calórica , Células Cultivadas , Temperatura Baixa , Glucose/metabolismo , Humanos , Hibridização In Situ , Canais Iônicos/química , Canais Iônicos/genética , Ácido Láctico/metabolismo , Masculino , Potenciais da Membrana/fisiologia , Camundongos , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Proteínas de Desacoplamento Mitocondrial , Dados de Sequência Molecular , Neurônios/citologia , Oxigênio/metabolismo , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Endogâmicos F344 , Espécies Reativas de Oxigênio/metabolismo , Alinhamento de SequênciaRESUMO
7,8-Dihydro-8-oxoguanine DNA glycosylase (OGG1) is a major DNA glycosylase involved in base-excision repair (BER) of oxidative DNA damage to nuclear and mitochondrial DNA (mtDNA). We used OGG1-deficient (OGG1(-/-)) mice to examine the possible roles of OGG1 in the vulnerability of neurons to ischemic and oxidative stress. After exposure of cultured neurons to oxidative and metabolic stress levels of OGG1 in the nucleus were elevated and mitochondria exhibited fragmentation and increased levels of the mitochondrial fission protein dynamin-related protein 1 (Drp1) and reduced membrane potential. Cortical neurons isolated from OGG1(-/-) mice were more vulnerable to oxidative insults than were OGG1(+/+) neurons, and OGG1(-/-) mice developed larger cortical infarcts and behavioral deficits after permanent middle cerebral artery occlusion compared with OGG1(+/+) mice. Accumulations of oxidative DNA base lesions (8-oxoG, FapyAde, and FapyGua) were elevated in response to ischemia in both the ipsilateral and contralateral hemispheres, and to a greater extent in the contralateral cortex of OGG1(-/-) mice compared with OGG1(+/+) mice. Ischemia-induced elevation of 8-oxoG incision activity involved increased levels of a nuclear isoform OGG1, suggesting an adaptive response to oxidative nuclear DNA damage. Thus, OGG1 has a pivotal role in repairing oxidative damage to nuclear DNA under ischemic conditions, thereby reducing brain damage and improving functional outcome.