Mechanisms of Cell Death in the Nervous System

描述(申请人提供)：程序性细胞死亡(PCD)是一种严格的 调控过程及其破坏导致无数发育缺陷 以及病理性后遗症。PCD在哺乳动物的神经中尤其重要 其扰动会导致神经发育异常的系统 会导致许多神经紊乱。 关于半胱氨酸酶在细胞死亡中的作用，已经有很多研究。然而， 越来越多的证据表明caspase非依赖性细胞的重要性。 在哺乳动物的神经系统和其他组织中杀戮。很难做到 确定后一条途径的组成部分或确定其对 在体内消除细胞，因为它紧密地交织在一起，并被 无处不在的半胱氨酸天冬氨酸酶。我们已经开发出一种模式，可以 绕过这一限制。CED-4S是线虫的一种促凋亡蛋白 当在酿酒酵母中表达时，这是致命的。CED-4S致命性 酵母表现出生理特异性，因为它被其天然的 拮抗剂CED-9，不被其抗凋亡剪接变异体模仿， CED-4L。然而，CED-4S在酵母中的毒性不需要caspase。给定 细胞自杀性成分之间的高度结构保守 机械，我们建议使用酵母中的CED-4S致死性作为范例来分离 参与caspase非依赖性杀伤的分子。随后，我们将 识别这些分子的哺乳动物对应物，并研究它们的 在脊椎动物神经系统中的功能。使用CED-4S抑制器屏幕，我们 分离到2个具有较高同源物的酵母AAA-ATPase CDC48和YAPO-1 与神经元死亡有牵连的真核生物。CDC48与CED-4结合 而雅培-L则不这么认为。这表明CED-4S复合体 并改变其功能，从而导致死亡。雅培-L可能会有一个 CDC48的冗余功能，或者它可能位于 半胱氨酸天冬氨酸酶非依赖性死亡途径。基于这些发现，我们将使用酵母 和哺乳动物模型来表征caspase非依赖的死亡途径和 确定这些和其他CED-4抑制物在神经元中所起的作用 老鼠的死亡。在具体目标1中，我们将确定组成和 酵母中含CED-4复合体的功能结构域。在具体目标2中， 将使用CED-4S在酵母中鉴定CED-4的下游靶点 抑制器屏幕。在具体目标3中，我们将确定表达式和 CED-4抑制因子的哺乳动物同源物在发育和发育中的作用 并评估他们对正常和病理性细胞死亡的贡献 在神经系统中。

DESCRIPTION (provided by applicant): Programmed cell death (PCD) is a strictly regulated process and its disruption results in myriad developmental deficits and pathological sequelae. PCD is especially critical in the mammalian nervous system where its perturbation results in aberrant neural development and contributes to many neural disorders. There has been much research into the role of caspases in cell death. However, there is growing evidence for the importance of caspase-independent cell killing in the mammalian nervous system and other tissues. It is difficult to identify the components of the latter pathway or establish its contribution to cell elimination in vivo as it is intimately interwoven with, and masked by, the ubiquitous caspase cascades. We have developed a paradigm that can circumventthis limitation. CED-4S is a pro-apoptotic protein from C. elegans that is lethal when expressed in Saccharomyces cerevisiae. CED-4S lethality in yeast shows physiological specificity as it is blocked by its natural antagonist, CED-9 and is not mimicked by its anti-apoptotic splice variant, CED-4L. However, CED-4S toxicity in yeast does not require a caspase. Given the high degree of structural conservation amongst components of the cell suicide machinery, we propose to use CED-4S lethality in yeast as a paradigm to isolate molecules involved in caspase-independent killing. Subsequently, we will identify the mammalian counterparts of these molecules and investigate their function in the vertebrate nervous system. Using a CED-4S suppresser screen, we isolated 2 yeast AAA-ATPases, Cdc48 and yAPO-1 that have homologs in higher eukaryotes that have been implicated in neuronal death. Cdc48 binds to CED-4 whereas yAPO-l does not. This suggests a scenario in which CED-4S complexes with Cdc48 and alters its function, thereby leading to death. yAPO-l may have a redundant function with Cdc48 or it may lie downstream in the caspase-independent death pathway. Based upon these findings, we will use yeast and mammalian models to characterize the caspase-independent death pathway and determine the role that these and other CED-4 suppressers play in neuronal death in mice. In Specific Aim 1, we will determine the composition and functional domains of CED-4-containing complexes in yeast. In Specific Aim 2, downstream targets of CED-4 will be identified in yeast using a CED-4S suppresser screen. In Specific Aim 3, we will determine the expression and function of the mammalian homologs of the CED-4 suppressers in developing and adult brain and assess their contribution to normal and pathological cell death in the nervous system.