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 触发多巴胺能细胞的活性氧的产生 帕金森病死亡的分子机制 线粒体ROS的产生是未知的。我们最近发现， 多亚单位酶α-酮戊二酸脱氢酶(α-KGDC)是一种 大脑线粒体中产生ROS的重要来源。活动和 这种蛋白的免疫反应性已被证明在神经元和脑内的 用MPP‘处理动物的脑和患者黑质 和警察在一起。我们假设分子内电子的失调 α-KGDC亚单位内的转移是氧化的主要介体 应激与帕金森病和ROS介导的神经细胞死亡有关。具体的 这一探索性项目的目标是：1.量化 α-KGDC对脑线粒体ROS产生的影响 存在帕金森病相关的神经毒素。我们将通过以下方式比较ROS产量 α-KGDC、其他线粒体脱氢酶与电子传递链 复合体I.我们还将确定由α-KGDC和 络合物I相互作用，降低正常酶活性，同时增加 ROS的生产。2.探索ROS产生的可能化学机制 α-KGDC的不同酶亚基。3.建立细胞培养模型 评估α-KGDC对氧化应激的贡献和 α-KGDC与络合物I在无或存在条件下的相互作用 神经毒素。我们将衡量MPP在没有和存在的情况下的影响 高胞外α-酮戊二酸和α-KGDC抑制剂对肿瘤标志物的影响 蛋白质和DNA氧化。不同培养条件对红曲霉生长的影响 α-KGDC和复合体I酶活性和对过氧化氢产生的影响 使用从这些细胞中分离的线粒体进行测量。这个项目将铺设 帕金森病细胞死亡的分子病因学基础可能是 由遗传和(或)环境决定因素激活的。核实 α-KGDC在线粒体ROS生成和细胞氧化死亡中的作用 可能导致帕金森病易感性的遗传动物模型的发展 导致制定有针对性的神经保护干预措施的疾病 这将使帕金森氏症的发病率降至最低或延缓病情发展 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.