Regulation of neuronal stem cell death

描述(申请人提供)：在胚胎12天至18天期间，胚胎小鼠前脑中约有一半的有丝分裂神经细胞因细胞凋亡而死亡。对Caspase基因敲除小鼠的研究表明，细胞凋亡对于防止神经干细胞的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素选择性地诱导或阻止个体分化神经干细胞的凋亡。我们首次证明了胚胎小鼠脑内神经酰胺升高和caspase3激活的同时，胚胎小鼠脑内细胞凋亡的高峰时间(E14.5)。我们还发现神经酰胺的浓度足以杀死培养中生长的神经前体细胞。从这些观察中，我们认为神经酰胺的升高可能在诱导分化神经干细胞的凋亡中起关键作用。最近，一些研究报道神经酰胺介导的PAR-4与非典型PKC之间形成抑制复合体，诱导细胞凋亡。我们自己对体外分化的胚胎干细胞的研究表明，神经酰胺诱导细胞凋亡的同时也上调了PAR-4的表达。我们认为，PAR-4可能是几种促凋亡蛋白之一，当它们和神经酰胺的表达升高时，它们可能会诱导细胞凋亡。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)的同时上调导致了促凋亡蛋白复合体(PAC)的形成，该复合体通过抑制抗凋亡的细胞生存信号而触发有丝分裂神经元干细胞的凋亡。我们将使用小鼠胚胎干细胞作为模型系统，在三个特定的目标上检验这一假设。在特定的目标1中，我们将检验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。在特定的目标2中，我们将检验神经酰胺/CAP诱导的PAC形成导致caspase激活的假设。在特定的目标3中，我们将检验神经酰胺的上调和细胞生存信号的下调与细胞周期和生长因子同步的假设。总而言之，这项研究将确定神经酰胺调节细胞凋亡的机制，这些机制对于正常的大脑发育和/或成年后病理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.