RESUMEN
Apoptotic protease-activating factor-1 (Apaf-1), dATP, and procaspase-9 form a multimeric complex that triggers programmed cell death through the activation of caspases upon release of cytochrome c from the mitochondria into the cytosol. Although cell death pathways exist that can bypass the requirement for cytochrome c release and caspase activation, several gene knockout studies have shown that the cytochrome c-mediated apoptotic pathway is critical for neural development. Specifically, the number of neuronal progenitor cells is abnormally increased in Apaf-1-, caspase-9-, caspase-3-deficient mice. However, the role of the cytochrome c cell death pathway for apoptosis of postmitotic, differentiated neurons in the developing brain has not been investigated in vivo. In this study we investigated embryonic neuronal cell death caused by trophic factor deprivation or lack of neurotransmitter release by analyzing Apaf-1/tyrosine kinase receptor A (TrkA) and Apaf-1/Munc-18 double mutant mice. Histological analysis of the double mutants' brains (including cell counting and terminal (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) staining) reveals that neuronal cell death caused by these stimuli can proceed independent of Apaf-1. We propose that a switch between apoptotic programs (and their respective proteins) characterizes the transition of a neuronal precursor cell from the progenitor pool to the postmitotic population of differentiated neurons.
Asunto(s)
Apoptosis/genética , Factores de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso , Sistema Nervioso/embriología , Neuronas/metabolismo , Neurotransmisores/metabolismo , Proteínas/metabolismo , Células Madre/metabolismo , Proteínas de Transporte Vesicular , Animales , Factor Apoptótico 1 Activador de Proteasas , Caspasas/metabolismo , Ciclo Celular/genética , Diferenciación Celular/genética , Grupo Citocromo c/metabolismo , Ganglios Sensoriales/citología , Ganglios Sensoriales/embriología , Ganglios Sensoriales/metabolismo , Ratones , Ratones Noqueados , Proteínas Munc18 , Sistema Nervioso/citología , Sistema Nervioso/metabolismo , Neuronas/citología , Proteínas/genética , Receptor trkA/deficiencia , Receptor trkA/genética , Transducción de Señal/genética , Células Madre/citologíaRESUMEN
The forebrain overgrowth mutation (fog) was originally described as a spontaneous autosomal recessive mutation mapping to mouse chromosome 10 that produces forebrain defects, facial defects, and spina bifida. Although the fog mutant has been characterized and available to investigators for several years, the underlying mutation causing the pathology has not been known. Because of its phenotypic resemblance to apoptotic protease activating factor-1 (Apaf-1) knockout mice, we have investigated the possibility that the fog mutation is in the Apaf-1 gene. Allelic complementation, Western blot analysis, and caspase activation assays indicate that fog mutant mice lack Apaf-1 activity. Northern blot and reverse transcription-PCR analysis show that Apaf-1 mRNA is aberrantly processed, resulting in greatly reduced expression levels of normal Apaf-1 mRNA. These findings are strongly suggestive of the fog mutation being a hypomorphic Apaf-1 defect and implicate neural progenitor cell death in the pathogenesis of spina bifida-a common human congenital malformation. Because a complete deficiency in Apaf-1 usually results in perinatal lethality and fog/fog mice more readily survive into adulthood, these mutants serve as a valuable model with which apoptotic cell death can be studied in vivo.
Asunto(s)
Defectos del Tubo Neural/genética , Proteínas/fisiología , Anomalías Múltiples/genética , Alelos , Animales , Apoptosis , Factor Apoptótico 1 Activador de Proteasas , Northern Blotting , Caspasa 3 , Caspasas/metabolismo , Mapeo Cromosómico , Cruzamientos Genéticos , Grupo Citocromo c/metabolismo , Activación Enzimática , Cara/anomalías , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes Neurológicos , Modelos Animales , Muridae , Prosencéfalo/anomalías , Proteínas/genética , Procesamiento Postranscripcional del ARN/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Disrafia Espinal/genética , Células Madre/patologíaRESUMEN
Cell death via apoptosis is a prominent feature in mammalian neural development. Recent studies into the basic mechanism of apoptosis have revealed biochemical pathways that control and execute apoptosis in mammalian cells. Protein factors in these pathways play important roles during development in regulating the balance between neuronal life and death. Additionally, mounting evidence indicates such pathways may also be activated during several neurodegenerative diseases, resulting in improper loss of neurons.
Asunto(s)
Enfermedad de Alzheimer/fisiopatología , Esclerosis Amiotrófica Lateral/fisiopatología , Apoptosis/fisiología , Enfermedad de Huntington/fisiopatología , Sistema Nervioso/crecimiento & desarrollo , Animales , Caspasas/metabolismo , Cromatina/fisiología , Grupo Citocromo c/metabolismo , Fragmentación del ADN/fisiología , Activación Enzimática , Humanos , Proteínas Proto-Oncogénicas c-bcl-2/fisiologíaRESUMEN
Release of cytochrome c from the mitochondria, and subsequent binding to apoptotic protease-activating factor-1 (Apaf-1), is a key trigger of apoptotic events. A complex composed of Apaf-1, dATP, and cytochrome c activates a series of cytoplasmic proteases called caspases, leading to apoptotic cell death. We have disrupted the Apaf-1 gene in the mouse. Like previous reports on this knockout model, we find that most Apaf-1 mutants die perinatally and frequently exhibit exencephaly and cranioschesis. We additionally find that the neural lesions that develop in the knockout are due to an excess of neural progenitor cells that manifests as early as embryonic day 9.5 in development. In contrast to previous reports on the Apaf-1 knockout mice, we find that 5% of the mutants successfully survive to adulthood. In these survivors, the brain develops normally, but in males, there is degeneration of spermatogonia resulting in the virtual absence of sperm. Thus, cytochrome c-mediated apoptosis is not absolutely required for normal neural development, but is essential for spermatogenesis. These findings strongly suggest that alternative apoptotic pathways work in conjunction with and parallel to Apaf-1 and can modify its effect on programmed cell death.