Pathogenic mechanisms of gene-environment interactions in Parkinson's disease

描述(申请人提供)：这项工作的长期目标是通过阐明致病机制来开发对常见的散发性帕金森病(PD)的有效治疗方法，从而促进识别神经保护干预措施。帕金森病的特征是中枢神经系统、自主神经系统和肠神经系统的神经元群变性，存活神经元中形成嗜酸性的细胞内包涵体路易小体；帕金森病的显著运动症状可归因于黑质多巴胺能神经元的严重丧失。一致的遗传、生化和病理证据表明，(I)突触前蛋白和路易小体的主要成分α-突触核蛋白，以及(Ii)线粒体功能异常，氧化应激和由此导致的氧化损伤，在发病机制中起作用。在初步研究中，我们表明这些因素在机制上是相互依存的。大鼠长期暴露于鱼藤酮(一种在流行病学上与帕金森病有关的杀虫剂)会导致全身线粒体复合体I抑制，导致与帕金森病非常相似的特定神经病理(包括α-突触核蛋白聚集体形成)。取消大鼠黑质α-突触核蛋白基因的表达可防止神经变性，表明这种与病因相关的环境触发导致的帕金森样神经病理依赖于内源性α-突触核蛋白。α-突触核蛋白和线粒体之间的相互作用已经被提出，但在体内α-突触核蛋白是多巴胺神经元对病因性相关线粒体毒素易感性所必需的机制尚不清楚。为了解决这个问题，我们将采用一系列创新的模型和工具，包括：体内消除SNCA表达的病毒载体；由线粒体毒素引发的神经退化依赖于人的α-突触核蛋白的转基因斑马鱼模型；以及表达荧光报告基因的新型转基因斑马鱼系，从而能够检测体内活体多巴胺神经元中的活性氧物种、谷胱甘肽氧化、ATP水平以及线粒体的裂变、融合和运输的动态变化。使用这些独特的工具，我们将确定在哺乳动物和斑马鱼多巴胺神经元暴露于与帕金森病发病有关的线粒体毒素后，α-突触核蛋白如何影响体内ROS的产生和氧化应激(AIM 1)、细胞呼吸和生物能量学(AIM 2)和线粒体动力学(AIM 3)。在目标4中，我们将利用96孔板中斑马鱼幼虫的自动行为测量来发现多巴胺神经元中线粒体抑制物的α-突触核蛋白依赖毒性的小分子修饰物。这些数据将阐明依赖α-突触核蛋白的多巴胺神经元对线粒体毒素反应的机制，线粒体毒素与帕金森病的发病有关。独特的模型系统和研究团队将使我们能够了解散发性帕金森病中两种最突出的病理异常之间的机制联系，从而解决帕金森病治疗发展中的一个关键障碍。

DESCRIPTION (provided by applicant): The long-term objective of this work is to develop effective treatments for the common sporadic form of Parkinson's disease (PD) by elucidating pathogenic mechanisms, thereby facilitating identification of neuroprotective interventions. PD is characterized by degeneration of groups of neurons in the central, autonomic and enteric nervous systems with formation of eosinophilic intracellular inclusion, Lewy bodies, in surviving neurons; the prominent motor symptoms of PD are attributable to severe loss of substantia nigra dopaminergic neurons. Convergent genetic, biochemical and pathological evidence implicates (i) alpha-synuclein, a presynaptic protein and a major component of Lewy bodies, and (ii) abnormalities of mitochondrial function, oxidative stress and resulting oxidative damage, in pathogenesis. In preliminary studies, we showed that these factors are mechanistically interdependent. Chronic exposure of rats to rotenone, a pesticide that is epidemiologically linked to PD, causes systemic mitochondrial complex I inhibition, resulting in specific neuropathology closely resembling PD (including alpha-synuclein aggregate formation). Abrogation of alpha-synuclein gene expression in the substantia nigra of rats prevented neurodegeneration, demonstrating that the PD-like neuropathology resulting from this etiologically-relevant environmental trigger is dependent on endogenous alpha-synuclein. Interactions between alpha-synuclein and mitochondria have been suggested previously, but the mechanisms whereby alpha-synuclein is necessary for susceptibility of dopamine neurons to etiologically-relevant mitochondrial toxins in vivo are not known. In order to address this question, we will employ a range of innovative models and tools, including: viral vectors that abrogate SNCA expression in vivo; transgenic zebrafish models in which neurodegeneration triggered by mitochondrial toxins is dependent on human alpha-synuclein; and novel transgenic zebrafish lines that express fluorescent reporters, allowing detection of dynamic changes in reactive oxygen species, glutathione oxidation, ATP levels, and mitochondrial fission, fusion and transport, in live dopamine neurons in vivo. Using these unique tools, we will determine how alpha- synuclein affects ROS production and oxidative stress (aim 1), cellular respiration and bioenergetics (aim 2) and mitochondrial dynamics (aim 3) following exposure to mitochondrial toxins implicated in PD pathogenesis, in mammalian and zebrafish dopamine neurons in vivo. In aim 4, we will exploit automated behavioral measurements in zebrafish larvae housed in 96-well plates to discover small molecule modifiers of alpha-synuclein-dependent toxicity of mitochondrial inhibitors in dopamine neurons. These data will elucidate the mechanisms underlying alpha-synuclein-dependent degeneration of dopamine neurons in response to mitochondrial toxins implicated in PD pathogenesis. The unique array of model systems and the team of investigators will enable us to understand the mechanistic link between the two most prominent pathological abnormalities in sporadic PD and thereby address a critical roadblock in the development of PD therapeutics.