Regulation of neuronal stem cell death

描述（由申请人提供）：在妊娠第E12天和第18天之间，胚胎小鼠前脑中约一半的有丝分裂神经元细胞因细胞凋亡而死亡。对半胱天冬酶基因敲除小鼠的研究表明，细胞凋亡对于防止神经干细胞过度增殖和随后的严重脑畸形是必要的。然而，尚不清楚哪些因子选择性地诱导或阻止个体分化的神经干细胞的凋亡。我们已经表明，第一次，在胚胎小鼠脑（E14.5）的细胞凋亡的峰值时间是同时与内源性神经酰胺和caspase 3的激活的升高。我们还表明，神经酰胺的浓度高到足以杀死培养中生长的神经元祖细胞。从这些观察，我们建议，神经酰胺的海拔可能是诱导分化的神经干细胞凋亡的关键。最近，一些研究报道了神经酰胺介导的PAR-4和非典型PKC之间的抑制复合物的形成诱导细胞凋亡。我们对体外分化的胚胎干（ES）细胞的研究表明，神经酰胺诱导的凋亡与PAR-4的上调同时发生。我们建议，PAR-4可能是几个促凋亡蛋白，诱导细胞凋亡时，他们的表达和神经酰胺升高。我们的主要假设是内源性神经酰胺和神经酰胺相关蛋白（CAPs）的同时上调导致形成促凋亡蛋白复合物（PAC），其通过抑制抗凋亡细胞存活信号而触发有丝分裂神经元干细胞的凋亡。我们将使用小鼠ES细胞作为模型系统在三个特定目的中检验这一假设。在具体目标1，我们将测试的假设，上调神经酰胺诱导细胞凋亡，特别是在有丝分裂的神经干细胞。在具体目标2中，我们将检验神经酰胺/CAP诱导的PAC形成导致半胱天冬酶激活的假设。在特定目标3中，我们将检验神经酰胺升高和细胞存活信号下调与细胞周期和生长因子同步的假设。总之，本研究将确定神经酰胺对细胞凋亡的调节机制，这些机制对正常的大脑发育和/或成年后病理性疾病的病因至关重要。

DESCRIPTION (provided by applicant): Between gestational day E12 and 18, about half of the mitotic neuronal cells die in embryonal mouse forebrain due to apoptosis. Studies with caspase knockout mice have demonstrated that apoptosis is necessary to prevent hyperproliferation of neuronal stem cells and subsequent, severe brain malformation. It is not known, however, which factors selectively induce or prevent apoptosis in individual, differentiating neuronal stem cells. We have shown for the first time, that the peak time of apoptosis in embryonal mouse brain (E14.5) is concurrent with elevation of endogenous ceramide and activation of caspase 3. We have also shown that the concentration of ceramide is high enough to kill neuronal progenitor cells grown in culture. From these observations, we propose that elevation of ceramide may be critical for induction of apoptosis in differentiating neuronal stem cells. Recently, several studies have reported that the ceramide-mediated formation of an inhibitory complex between PAR-4 and atypical PKC ,induces apoptosis. Our own studies with in vitro differentiated embryonic stem (ES) cells have shown that induction of apoptosis by ceramide is concurrent with up-regulation of PAR-4. We propose that PAR-4 is one of probably several pro-apoptotic proteins that induce apoptosis when their expression and that of ceramide is elevated. Our main hypothesis is that simultaneous upregulation of endogenous ceramide and ceramide-associated proteins (CAPs) results in formation of a pro-apoptotic protein complex (PAC) that triggers apoptosis in mitotic neuronal stem cells by suppression of anti-apoptotic, cell survival signaling. We will test this hypothesis in three Specific Aims using murine ES cells as model system. In Specific Aim 1, we will test the hypothesis that up-regulation of ceramide induces apoptosis specifically in mitotic neuronal stem cells. In Specific Aim 2, we will test the hypothesis that ceramide/CAP-induced PAC formation results in activation of caspases. In Specific Aim 3, we will test the hypothesis that elevation of ceramide and down-regulation of cell survival signaling is synchronized by the cell cycle and growth factors. In conclusion, this study will identify mechanisms for regulation of apoptosis by ceramide that are critical for normal brain development and/or the etiology of subsequent, pathological disorders in adulthood.