FREE RADICALS AND MITOCHONDRIA IN NEURONAL APOPTOSIS

神经元凋亡中的自由基和线粒体

• 批准号: 2750973
• 负责人: JAMES Lee FRANKLIN
• 金额: $ 9.36万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 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 2003-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2750973
• 关键词: BCL2 gene /protein; apoptosis; confocal scanning microscopy; cytochrome c; electron microscopy; free radical oxygen; genetically modified animals; granule cell; hydroxyl radical; iron; laboratory mouse; membrane potentials; microinjections; mitochondria; mitochondrial membrane; neurotrophic factors; sympathetic nervous system; tissue /cell culture; transfection /expression vector

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.

描述（改编自申请人摘要）： 在胚胎发育过程中产生的所有神经元中有一半经历凋亡 死亡前不久或之后不久。这些神经元获得了 足够量的所需神经营养因子逃脱了这种死亡。 神经元死亡，其特征与 发展也发生在中风后和神经退行性疾病中 如阿尔茨海默病和帕金森病。许多相似之处 发育性死亡和侮辱或疾病导致的死亡之间的关系 这表明，在两种情况下，类似的过程都会杀死神经元， 关于发育性神经元死亡机制的信息 有助于理解和治疗病理性死亡。的目标 这项研究计划是为了了解自由基氧的作用， 神经元的凋亡。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。这些细胞在体内和体外都经历凋亡， 缺乏神经生长因子（NGF）。公布的数据，以及 申请人的初步结果显示， 在通过以下方法产生自由基氧（活性氧; ROS）中， 这些细胞中的线粒体在NGF剥夺后不久。这个活性氧爆发 是凋亡性死亡的必要组成部分。申请人将测试 ROS通过铁催化产生促进细胞凋亡假说 线粒体中的羟基自由基他认为，这些极端 活性自由基直接或间接地损伤线粒体 并使它们释放细胞色素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 显微镜和电子显微镜来研究这一假设。他 同时也计划验证抗凋亡蛋白Bcl-2 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， 细胞内显微注射Bcl-2表达载体，并通过使用 Bcl-2转基因小鼠或Bcl-2缺陷小鼠的神经元。到 确定这些发现的一般重要性， 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，申请人将研究其在CNS模型中的作用 系统，小脑颗粒细胞培养。这些研究将提供 关于自由基和线粒体在 神经元凋亡和识别操纵这种死亡的方法 - 是的