Regulation of neuronal stem cell death

描述（由申请人提供）：在妊娠期E12至18天，胚胎小鼠前脑中约有一半有丝分裂神经元细胞因凋亡而死亡。对caspase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，目前尚不清楚哪些因子选择性地诱导或阻止个体分化神经干细胞的凋亡。我们首次发现胚胎小鼠脑（E14.5）凋亡的高峰时间与内源性神经酰胺的升高和caspase 3的激活同时发生。我们还发现，神经酰胺的浓度高到足以杀死培养的神经祖细胞。根据这些观察，我们提出神经酰胺的升高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。我们对体外分化胚胎干（ES）细胞的研究表明，神经酰胺诱导细胞凋亡与PAR-4的上调同时发生。我们认为PAR-4可能是几种促凋亡蛋白之一，当它们的表达和神经酰胺的表达升高时，会诱导细胞凋亡。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）的同时上调导致促凋亡蛋白复合物（PAC）的形成，PAC通过抑制抗凋亡细胞存活信号触发有丝分裂神经干细胞的凋亡。我们将以小鼠胚胎干细胞为模型系统，在三个特定目标中验证这一假设。在Specific Aim 1中，我们将验证神经酰胺上调诱导有丝分裂神经干细胞特异性凋亡的假设。在Specific Aim 2中，我们将验证神经酰胺/ cap诱导的PAC形成导致半胱天蛋白酶激活的假设。在Specific Aim 3中，我们将验证神经酰胺的升高和细胞存活信号的下调是由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调节细胞凋亡的机制，这对正常大脑发育和/或成年期后续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.