FREE RADICALS AND MITOCHONDRIA IN NEURONAL APOPTOSIS

描述（改编自申请人摘要）： 胚胎发生过程中产生的所有神经元中有一半会发生凋亡 死亡之前或之后不久。那些神经元获得 足够数量的所需神经营养因子逃脱了这种死亡。 神经元死亡的特征与在 中风后和神经退行性疾病也会发生发育 例如阿尔茨海默病和帕金森病。许多相似之处 发育性死亡和由侮辱或疾病引起的死亡之间 表明在这两种情况下类似的过程都会杀死神经元，并且 获得有关发育神经元死亡机制的信息 可能有助于理解和治疗病理性死亡。目标是 这项研究计划是为了了解自由基氧的作用 在神经元的凋亡性死亡中。神经元的主要模型 申请人将研究的细胞凋亡是交感神经元的细胞凋亡 在文化中。当这些细胞在体内和体外发生凋亡时 缺乏神经生长因子（NGF）。已公布的数据，以及 申请人初步结果显示，增幅显着 自由基氧（活性氧；ROS）的产生 NGF 剥夺后不久，这些细胞中就会出现线粒体。 ROS爆发 是细胞凋亡死亡的必要组成部分。申请人将测试 ROS 通过铁催化生成促进细胞凋亡的假设 线粒体中的羟基自由基。他假设这些极其 活性自由基直接或间接损伤线粒体 并导致它们释放细胞色素c或其他促凋亡蛋白， 进入细胞质。他计划使用生化技术、共聚焦技术 显微镜和电子显微镜来研究这一假设。他 还计划检验抗凋亡蛋白 Bcl-2 的假设 促凋亡蛋白 Bax 促进神经元存活或死亡 通过调节 ROS 爆发或 ROS 对线粒体完整性的影响。 他将通过在神经元中过度表达 Bcl-2 来测试这些假设， Bcl-2表达载体的细胞内显微注射，以及通过使用 来自 Bcl-2 转基因小鼠或 Bax 缺陷小鼠的神经元。至 确定这些发现关于作用的一般重要性 ROS、线粒体和 Bcl-2 家族在交感神经细胞凋亡中的作用 神经元，申请人将研究它们在中枢神经系统模型中的作用 系统，培养中的小脑颗粒细胞。这些研究将提供 关于自由基和线粒体的作用的明确答案 神经元凋亡以及确定操纵这种死亡的方法 药理学上。

DESCRIPTION (Adapted from applicant abstract): Half of all neurons produced during embryogenesis undergo apoptotic death shortly before or soon thereafter. Those neurons obtaining a sufficient quantity of a required neurotrophic factor escape this death. Neuronal death with characteristics similar to those seen during development also occurs after stroke and in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Many similarities between developmental death and death caused by insult or disease suggest that comparable processes kill neurons in both situations, and that information gained about mechanisms of developmental neuronal death may aid in understanding and treating pathological death. The goal of this research proposal is to understand the role of free radical oxygen in the apoptotic death of neurons. The principal model of neuronal apoptosis that the applicant will study is that of sympathetic neurons in culture. These cells undergo apoptosis both in vivo and in vitro when deprived of nerve growth factor (NGF). Published data, and the applicant's preliminary results show that there is a dramatic increase in production of free radical oxygen (reactive oxygen species; ROS) by mitochondria in these cells soon after NGF deprivation. This ROS burst is a required component of apoptotic death. The applicant will test the hypothesis that ROS contribute to apoptosis by iron-catalyzed production of hydroxyl radicals in mitochondria. He postulates that these extremely reactive radical species directly, or indirectly, damage mitochondria and cause them to release cytochrome c, or other pro-apoptotic proteins, into the cytoplasm. He plans to use biochemical techniques, confocal microscopy, and electron microscopy to investigate this hypothesis. He also plans to test the hypothesis that the anti-apoptotic protein, Bcl-2 and the pro-apoptotic protein, Bax, promote neuronal survival or death by regulating the ROS burst or ROS effects on mitochondrial integrity. He will test these hypotheses by over-expressing Bcl-2 in neurons, by intracellular microinjection of a Bcl-2 expression vector, and by use of neurons from Bcl-2 transgenic mice or Bax-deficient mice. To determine the general importance of these findings about the role of ROS, mitochondria, and the Bcl-2 family in apoptosis of sympathetic neurons, the applicant will investigate their role in a CNS model system, cerebellar granule cells in culture. These studies will provide clear answers about the role of free radicals and mitochondria in neuronal apoptosis and of identifying ways of manipulating this death pharmacologically.