RESUMEN
Several different autosomal recessive genetic disorders characterized by ataxia with oculomotor apraxia (AOA) have been identified with the unifying feature of defective DNA damage recognition and/or repair. We describe here the characterization of a novel form of AOA showing increased sensitivity to agents that cause single-strand breaks (SSBs) in DNA but having no gross defect in the repair of these breaks. Evidence for the presence of residual SSBs in DNA was provided by dramatically increased levels of poly (ADP-ribose)polymerase (PARP-1) auto-poly (ADP-ribosyl)ation, the detection of increased levels of reactive oxygen/nitrogen species (ROS/RNS) and oxidative damage to DNA in the patient cells. There was also evidence for oxidative damage to proteins and lipids. Although these cells were hypersensitive to DNA damaging agents, the mode of death was not by apoptosis. These cells were also resistant to TRAIL-induced death. Consistent with these observations, failure to observe a decrease in mitochondrial membrane potential, reduced cytochrome c release and defective apoptosis-inducing factor translocation to the nucleus was observed. Apoptosis resistance and PARP-1 hyperactivation were overcome by incubating the patient's cells with antioxidants. These results provide evidence for a novel form of AOA characterized by sensitivity to DNA damaging agents, oxidative stress, PARP-1 hyperactivation but resistance to apoptosis.
Asunto(s)
Apoptosis/fisiología , Roturas del ADN de Cadena Simple , Estrés Oxidativo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Antineoplásicos Fitogénicos/farmacología , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Apraxias/metabolismo , Apraxias/patología , Apraxias/fisiopatología , Ataxia/metabolismo , Ataxia/patología , Ataxia/fisiopatología , Western Blotting , Camptotecina/farmacología , Células Cultivadas , Daño del ADN , Reparación del ADN , Etopósido/farmacología , Femenino , Citometría de Flujo , Humanos , Peróxido de Hidrógeno/farmacología , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potencial de la Membrana Mitocondrial/fisiología , Potencial de la Membrana Mitocondrial/efectos de la radiación , Metilnitronitrosoguanidina/farmacología , Mitomicina/farmacología , Poli(ADP-Ribosa) Polimerasa-1 , Radiación Ionizante , Especies de Nitrógeno Reactivo/metabolismoRESUMEN
Translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus can play a major role in neuronal death elicited by oxidant stress. The time course of nuclear translocation of AIF after experimental stroke may vary with the severity of injury and may be accelerated by oxidant stress associated with reperfusion and nitric oxide (NO) production. Western immunoblots of AIF on nuclear fractions of ischemic hemisphere of male mice showed no significant increase with 1 h of middle cerebral artery occlusion and no reperfusion, whereas increases were detectable after 6 and 24 h of permanent ischemia. However, as little as 20 min of reperfusion after 1 h of middle cerebral artery occlusion resulted in an increase in nuclear AIF coincident with an increase in poly(ADP-ribose) polymer (PAR) formation. Further nuclear AIF accumulation was seen at 6 and 24 h of reperfusion. In contrast, 20 min of reperfusion after 2 h of occlusion did not increase nuclear AIF. In this case, nuclear AIF became detectable at 6 and 24 h of reperfusion. With brief occlusion of 30 min duration, nuclear AIF remained undetectable at both 20 min and 6 h and became evident only after 24 h of reperfusion. Inhibition of neuronal NO synthase attenuated formation of PAR and nuclear AIF accumulation. Gene deletion of neuronal NO synthase also attenuated nuclear AIF accumulation. Therefore, reperfusion accelerates AIF translocation to the nucleus when focal ischemia is of moderate duration (1 h), but is markedly delayed after brief ischemia (30 min). Nuclear translocation of AIF eventually occurs with prolonged focal ischemia with or without reperfusion. Neuronally-derived NO is a major factor contributing to nuclear AIF accumulation after stroke.
Asunto(s)
Factor Inductor de la Apoptosis/metabolismo , Núcleo Celular/metabolismo , Ataque Isquémico Transitorio/patología , Neuronas/enzimología , Óxido Nítrico Sintasa de Tipo I/metabolismo , Animales , Conducta Animal/fisiología , Western Blotting , Inhibidores Enzimáticos/farmacología , Eliminación de Gen , Indazoles/farmacología , Infarto de la Arteria Cerebral Media/patología , Infarto de la Arteria Cerebral Media/psicología , Ataque Isquémico Transitorio/psicología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo I/genética , Poli Adenosina Difosfato Ribosa/metabolismo , Transporte de Proteínas , Daño por Reperfusión/patología , Daño por Reperfusión/psicología , Fracciones Subcelulares/metabolismo , Factores de TiempoRESUMEN
Poly(ADP-ribose) glycohydrolase (PARG) is a catabolic enzyme that cleaves ADP-ribose polymers formed by members of the PARP family of enzymes. Despite its discovery and subsequent partial purification in the 1970s and the cloning of its single gene in the late 1990s, little is known about the role of PARG in cell function. Because of its low abundance within cells and its extreme sensitivity to proteases, PARG has been difficult to study. The existence of several PARG isoforms with different subcellular localizations is still debated today after more than 30 years of intensive research. In this article, we want to summarize and discuss the current knowledge related to PARG, its different forms and subcellular distribution. We also examine the possible biological roles of PARG in modulating chromatin structure, transcription, DNA repair and apoptosis.