Free Radicals and Mitochondria in Neuronal Apoptosis

神经元凋亡中的自由基和线粒体

• 批准号: 7002170
• 负责人: JAMES Lee FRANKLIN
• 金额: $ 26.59万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-12-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7002170
• 关键词: Bax gene /protein; apoptosis; cerebellum; confocal scanning microscopy; cysteine endopeptidases; cytochrome c; developmental neurobiology; enzyme activity; free radical oxygen; genotype; laboratory mouse; microinjections; mitochondria; mitochondrial membrane; neurogenesis; neurons; polymerase chain reaction; protein purification; protein structure function; superoxide dismutase; sympathetic nervous system; tissue /cell culture; western blottings

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.

描述（由申请人提供）：胚胎发生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细胞凋亡。与发育过程中所见特征相似的神经元死亡也发生在中风后以及阿尔茨海默病等神经退行性疾病中。发育性死亡和由损伤或疾病引起的死亡之间的相似性表明，在这两种情况下，类似的过程都会杀死神经元，并且获得的有关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）缺乏神经生长因子的交感神经元，2）在低钾培养基中缺乏血清的小脑颗粒神经元。该资助的初始资助期间生成的数据表明，在这两种细胞类型的凋亡过程中，线粒体衍生的活性氧（ROS）急剧增加。这些 ROS 位于促凋亡蛋白 Bax 的下游，Bax 通过导致凋亡因子从线粒体释放到细胞质中来诱导细胞凋亡。我们的证据表明 ROS 对此版本至关重要。本研究计划的目标是了解这些 ROS 在神经元凋亡中的作用。前四个具体目标将在交感神经元中完成。具体目标 1 将测试 Bax 通过增加线粒体超氧化物的产生来诱导 ROS 升高的假设。具体目标 2 的目标是检验 Caspase 蛋白酶通过攻击线粒体呼吸复合物来增加 ROS 的假设。具体目标 3 将检验 Bax 诱导的 ROS 位于 MLK/JNK 激酶途径下游的假设。具体目标 4 中的实验将检验以下假设：ROS 通过打开线粒体外膜通道 VDAC 导致线粒体释放凋亡因子。具体目标 5 将测试小脑颗粒系统中前四个具体目标的通用性。我们将使用遗传、生化和共焦显微技术来研究这些假设。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。他们将进一步推进我们了解细胞凋亡机制和确定操纵这种死亡的方法的长期目标。