Free Radicals and Mitochondria in Neuronal Apoptosis

描述(申请人提供)：在胚胎发育过程中产生的所有神经元中，有一半在出生前不久或出生后不久经历了凋亡性死亡。中风后和阿尔茨海默病等神经退行性疾病中也会发生神经元死亡，其特征类似于发育过程中的神经元死亡。发育性死亡与侮辱或疾病引起的死亡之间的相似之处表明，类似的过程在两种情况下都会杀死神经元，获得的关于发育性死亡机制的信息可能有助于理解和治疗病理性死亡。我们使用两种细胞培养模型来研究神经元发育过程中的细胞凋亡机制：1)去神经生长因子的交感神经元和2)低钾条件下去血清的小脑颗粒神经元。在这笔赠款的初始资助期产生的数据显示，在这两种类型的细胞的凋亡性死亡期间，线粒体衍生的活性氧物种(ROS)急剧增加。这些ROS位于促凋亡蛋白Bax的下游，Bax通过使线粒体中的促凋亡因子释放到细胞质来诱导细胞凋亡。我们的证据表明，ROS对这次发布至关重要。这项研究计划的目的是了解这些RO在神经细胞凋亡中的作用。前四个具体目标将在交感神经元中完成。具体目标1将检验Bax通过增加线粒体产生超氧化物来诱导ROS升高的假设。具体目标2的目的是验证caspase蛋白酶通过攻击线粒体呼吸复合体增加ROS的假设。具体目标3将测试假设，即Bax诱导的ROS位于MLK/JNK激酶通路的下游。特定目标4的实验将验证这样的假设，即ROS通过打开线粒体膜外通道VDAC而导致线粒体释放促凋亡因子。特定目标5将测试小脑颗粒系统中前四个特定目标的共性。我们将使用遗传学、生物化学和共聚焦显微镜技术来研究这些假说。这些研究将为Bax在神经元凋亡过程中导致ROS增加的机制以及这些ROS如何导致细胞死亡提供明确的答案。它们将推动我们理解细胞凋亡机制和识别操纵这种死亡的方法的长期目标。

DESCRIPTION (provided by applicant): Half of all neurons produced during embryogenesis undergo apoptotic death shortly before birth or soon thereafter. Neuronal death with characteristics similar to those seen during development also occurs after stroke and in neurodegenerative diseases such as Alzheimer's disease. Similarities between developmental death and death caused by insult or disease suggests comparable processes kill neurons in both situations and that information gained about mechanisms of developmental death may aid in understanding and treating pathological death. We use two cell culture models to investigate mechanisms of apoptosis during neuronal development: 1) sympathetic neurons deprived of nerve growth factor and, 2) cerebellar granule neurons deprived of serum in low potassium medium. Data generated during the initial funding period of this grant shows that there is a dramatic increase in mitochondrial-derived reactive oxygen species (ROS) during the apoptotic death of both of these cell types. These ROS lie downstream of the pro-apoptotic protein, Bax, which induces apoptosis by causing release of apoptogenic factors from the mitochondria into the cytoplasm. Our evidence suggests that the ROS are critical for this release. The goal of this research proposal is to understand the role of these ROS in neuronal apoptosis. The first four specific aims will be done in sympathetic neurons. Specific aim 1 will test the hypotheses that Bax induces elevated ROS by increasing production of superoxide by mitochondria. The goal of specific aim 2 is to test the hypothesis that caspase proteases increase ROS by attacking mitochondrial respiratory complexes. Specific aim 3 will test the hypothesis that the Bax-induced ROS lie downstream from the MLK/JNK kinase pathway. Experiments in specific aim 4 will test the hypothesis that the ROS cause release of apoptogenic factors from mitochondria by opening the outer mitochondrial membrane channel, VDAC. Specific aim 5 will test the generality of the first four specific aims in the cerebellar granule system. We shall use genetic, biochemical, and confocal microscopic techniques to investigate these hypotheses. These studies will provide clear answers about the mechanism by which Bax causes increased ROS during neuronal apoptosis and how these ROS contribute to cell death. They will further our long-term goals of understanding mechanisms of apoptosis and of identifying ways of manipulating this death.