NOVEL MECHANISMS OF MITOCHONDRIAL FREE RADIAL GENERATION

线粒体自由基产生的新机制

• 批准号: 6625924
• 负责人: GARY M FISKUM
• 金额: $ 18.56万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-11 至 2004-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6625924
• 关键词: Krebs' cycle; Parkinson's disease; alpha ketoglutarate; cell death; central neural pathway /tract; electron transport; enzyme activity; enzyme mechanism; free radical oxygen; gene environment interaction; genetic susceptibility; laboratory rat; methylphenyltetrahydropyridine; mitochondrial disease /disorder; nerve injury; neural degeneration; neurotoxicology; neurotoxins; oxidative stress; oxoglutarate dehydrogenase; substantia nigra; superoxides; tissue /cell culture

DESCRIPTION (provided by applicant) Although evidence suggests that mitochondrial dysfunction stimulates the production of reactive oxygen species (ROS) that trigger dopaminergic cell death in Parkinson's disease (PD) the molecular mechanisms responsible for mitochondrial ROS production are unknown. We have recently discovered that the multi-subunit enzyme alpha-ketoglutarate dehydrogenase (alpha-KGDC) is a substantial source of ROS production in brain mitochondria. The activity and immunoreactivity of this protein has been shown to be altered in neurons and in the brains of animals treated with MPP' and in the substantia nigra of patients with PD. We hypothesize that dysregulation in the intramolecular electron transfer within the subunits of alpha-KGDC is a primary mediator of oxidative stress associated with PD and to ROS-mediated neuronal cell death. The specific aims of this exploratory project are: 1. Quantify the contribution of alpha-KGDC to ROS production in isolated brain mitochondria in the absence and presence of PD-associated neurotoxins. We will compare the ROS production by alpha-KGDC, other mitochondrial dehydrogenases and electron transport chain Complex I. We will also determine if the ROS generated by alpha-KGDC and Complex I interact to decrease normal enzyme activity while increasing production of ROS. 2. Explore possible chemical mechanisms of ROS production by different enzyme subunits of the alpha-KGDC. 3. Develop a cell culture model for assessing the contribution of alpha-KGDC to oxidative stress and the interactions of alpha-KGDC and Complex I in the absence and presence of neurotoxins. We will measure the effects of MPP' in the absence and presence of high extracellular alpha-ketoglutarate and alpha-KGDC inhibitors on markers of protein and DNA oxidation. The effects of different culture conditions on alpha-KGDC and Complex I enzyme activities and on H2O2 production will be measured using mitochondria isolated from these cells. This project will lay the foundation for the molecular etiology of cell death in PD which could be activated by genetic and (or) environmental determinants. Verification of the role of alpha-KGDC in mitochondrial ROS generation and in oxidative cell death could lead to the development of genetic animal models of susceptibility to PD disease leading to the development of targeted neuroprotective interventions that will minimize the incidence or slow the progression of Parkinson's disease.

描述（由申请人提供） 尽管有证据表明线粒体功能障碍刺激了 产生触发多巴胺能细胞的活性氧（ROS） 帕金森氏病（PD）死亡的分子机制 线粒体ROS的产生尚不清楚。我们最近发现 多生酶α-酮戊二酸脱氢酶（alpha-kGDC）是一个 大脑线粒体中ROS产生的大量来源。活动和 该蛋白的免疫反应性已显示在神经元和 用MPP治疗的动物和患者的黑质的动物大脑 与PD。我们假设分子内电子失调 在α-KGDC亚基内转移是氧化的主要介体 与PD和ROS介导的神经元细胞死亡相关的应力。具体 该探索性项目的目的是：1。量化 在不存在的情况下，α-kGDC在孤立的脑线粒体中产生ROS，并且 PD相关的神经毒素的存在。我们将通过比较ROS的生产 α-KGDC，其他线粒体脱氢酶和电子传输链 复合物I。我们还将确定Alpha-KGDC产生的ROS是否和 复合物I相互作用以减少正常酶活性，同时增加 ROS的生产。 2。通过 α-KGDC的不同酶亚基。 3。开发细胞培养模型 评估α-kGDC对氧化应激的贡献和 在不存在和存在的情况下，α-KGDC和复合物I的相互作用 神经毒素。我们将在不存在和存在的情况下测量MPP的影响 高细胞外α-酮戊二酸和α-kGDC抑制剂在标记上 蛋白质和DNA氧化。不同培养条件对 alpha-kGDC和复杂的I酶活性以及H2O2的生产将是 使用从这些细胞分离的线粒体测量。这个项目将奠定 PD中细胞死亡分子病因的基础，这可能是 被遗传和（或）环境决定因素激活。验证 α-KGDC在线粒体ROS产生和氧化细胞死亡中的作用 可能导致发展PD易感性的遗传动物模型 导致靶向神经保护干预措施的发展 这将使帕金森氏症的发病率最小 疾病。