NOVEL MECHANISMS OF MITOCHONDRIAL FREE RADIAL GENERATION

描述（由申请人提供） 尽管有证据表明线粒体功能障碍会刺激 引发多巴胺能细胞的活性氧（ROS）的产生 帕金森病（PD）死亡的分子机制 线粒体ROS的产生是未知的。我们最近发现， 多亚基酶α-酮戊二酸脱氢酶（α-KGDC）是一种 脑线粒体中ROS产生的重要来源。的活性和 这种蛋白质的免疫反应性在神经元和神经元中已被证明是改变的。 用MPP治疗的动物的脑和患者的黑质中 PD的。我们假设分子内电子的失调 在α-KGDC亚单位内的转移是氧化的主要介质， 应激与PD和ROS介导的神经元细胞死亡相关。具体 该探索性项目的目标是：1.量化的贡献 α-KGDC对分离的脑线粒体中ROS产生的影响， PD相关神经毒素的存在。我们将比较ROS的产生， α-KGDC、其他线粒体脱氢酶和电子传递链 综合体岛我们还将确定是否由α-KGDC产生的ROS和 复合物I相互作用以降低正常酶活性，同时增加 ROS的产生。2.探索ROS产生的可能化学机制， 不同的酶亚基的α-KGDC。3.建立细胞培养模型 用于评估α-KGDC对氧化应激的贡献， 在不存在和存在以下物质的情况下，α-KGDC和复合物I的相互作用 神经毒素我们将测量MPP在不存在和存在下的影响， 高细胞外α-酮戊二酸和α-KGDC抑制剂对 蛋白质和DNA氧化。不同培养条件对 α-KGDC和复合物I酶活性以及对H2 O2产生的影响将被 使用从这些细胞中分离的线粒体测量。该项目将奠定 PD细胞死亡的分子病因学基础， 由遗传和（或）环境决定因素激活。验证 α-KGDC在线粒体ROS生成和氧化性细胞死亡中的作用 可能导致开发对PD易感的遗传动物模型 疾病导致有针对性的神经保护干预措施的发展 这将最大限度地减少帕金森病的发病率或减缓帕金森病的进展， 疾病

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.