RESUMEN
Neural precursor cells (NPCs) critically regulate brain morphogenesis and recent studies have revealed an unexpectedly high frequency of NPC chromosomal abnormalities and apoptosis in the developing brain. We have shown previously that the apoptotic response of NPCs to genotoxic agents is dependent on p53 and caspase-9, but not Bax or caspase-3 expression. In this study, we found that NPCs deficient in Apaf-1, or both the pro-apoptotic multidomain Bcl-2 family members Bax and Bak, were resistant to cytosine arabinoside and gamma-irradiation-induced apoptosis. Inhibitors of gene transcription, protein translation, and caspase activity also blocked genotoxin-induced NPC apoptosis. Although caspase-3 and caspase-6 were both cleaved in response to DNA damage, neither of these effector caspases was critical for apoptosis. Genotoxin-induced NPC death was accompanied by the generation of reactive oxygen species and could be inhibited by several known antioxidants. Conversely, DNA damage-induced reactive oxygen species generation was inhibited significantly by gene disruption of p53, Apaf-1, or caspase-9, and combined deficiency of Bax and Bak, but not by caspase-3 or caspase-6 deficiency. These studies suggest that caspase-9 activation is both necessary and sufficient for genotoxin-induced neural precursor cell reactive oxygen species generation and death.
Asunto(s)
Caspasas/metabolismo , Muerte Celular/efectos de los fármacos , Imidazolinas , Mutágenos/farmacología , Neuronas/metabolismo , Envejecimiento , Animales , Factor Apoptótico 1 Activador de Proteasas , Western Blotting , Caspasas/genética , Catecolaminas/farmacología , Muerte Celular/efectos de la radiación , Células Cultivadas , Citarabina/farmacología , Daño del ADN , Agonistas de Dopamina/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Embrión de Mamíferos , Femenino , Inmunohistoquímica , Inmunosupresores/farmacología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos , Ratones Noqueados , Neuronas/efectos de los fármacos , Neuronas/efectos de la radiación , Embarazo , Proteínas/genética , Proteínas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Telencéfalo/citología , Telencéfalo/efectos de los fármacos , Telencéfalo/metabolismo , Telencéfalo/efectos de la radiación , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Rayos X , Proteína Destructora del Antagonista Homólogo bcl-2RESUMEN
Targeted gene disruptions have revealed significant roles for caspase family members in the regulation of neuronal programmed cell death. Both caspase-3- and caspase-9-deficient mice exhibit a variably severe neurodevelopmental phenotype that may include marked ventricular zone expansion, exencephaly, and ectopic neuronal structures. Our previous studies of caspase-3- and caspase-9-deficient mice were performed using mice on mixed genetic backgrounds, raising the possibility that strain-specific generic factors influence the effects of caspase deficiency on nervous system development. To directly test this hypothesis. we backcrossed the caspase-3 mutation for 7-10 generations onto pure C57BL/6J and 129X1/SvJ genetic backgrounds. Caspase-3-deficient 129X1/SvJ mice were uniformly and severely affected. These mice died during the perinatal period and exhibited marked neural precursor cell expansion and exencephaly. In contrast, caspase-3-deficient C57BL/6J mice reached adulthood, were fertile and showed minimal brain pathology. Intercrosses of C57BL/6J and 129X1/SvJ mutants revealed that the vast majority of caspase-3-/- F1 mice displayed the severe 129X1/SvJ-"like" phenotype. These findings are consistent with an incompletely penetrant strain-dependent genetic modifier (or modifiers) that alters the neurodevelopmental consequences of caspase-3 deficiency. Since caspase-9- and Apaf-1-deficient mice also display variably severe developmental neuropathology, this strain-dependent modifier(s) may be involved in the activation of a caspase-independent death pathway; alternatively, strain-dependent compensatory caspase activation and/or its inhibition may influence the severity of the caspase-3-deficient neuronal phenotype.
Asunto(s)
Caspasas/deficiencia , Malformaciones del Sistema Nervioso/embriología , Malformaciones del Sistema Nervioso/genética , Animales , Caspasa 3 , Caspasas/genética , Desarrollo Embrionario y Fetal , Ratones , Ratones Endogámicos , Ratones Noqueados/genética , Sistema Nervioso/embriología , Especificidad de la EspecieRESUMEN
Recently several methods have been described for triggering extensive apoptotic neurodegeneration in the developing in vivo mammalian brain. These methods include treatment with drugs that block NMDA glutamate receptors, drugs that promote GABA(A) neurotransmission, or treatment with ethanol, which has both NMDA antagonist and GABAmimetic properties. A single intoxication episode induced by any of these agents is sufficient to cause widespread neurodegeneration throughout many brain regions. The cell death process transpires rapidly from early to late stages within several hours. As the neurons die, they become TUNEL positive and show, by both light and electron microscopy, all of the classical morphological characteristics of apoptosis. In the present study, using immunocytochemical methods, we document that ethanol intoxication of 7-day-old infant mice causes a widespread pattern of caspase-3 activation corresponding to the pattern of apoptotic neurodegeneration that is occurring simultaneously.