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

描述（由申请人提供）：这项工作的长期目标是通过阐明致病机制，从而促进神经保护干预措施的识别，为帕金森病（PD）的常见散发形式开发有效的治疗方法。PD的特征在于中枢、自主神经系统和肠神经系统中的神经元群变性，在存活的神经元中形成嗜酸性细胞内包涵体、路易体; PD的突出运动症状可归因于黑质多巴胺能神经元的严重损失。聚合的遗传，生化和病理学证据涉及（i）α-突触核蛋白，突触前蛋白和路易体的主要成分，和（ii）线粒体功能异常，氧化应激和由此产生的氧化损伤，在发病机制。在初步研究中，我们发现这些因素在机械上相互依赖。大鼠长期暴露于鱼藤酮（一种在流行病学上与PD相关的杀虫剂），会引起全身性线粒体复合物I抑制，导致与PD非常相似的特定神经病理学（包括α-突触核蛋白聚集体形成）。在大鼠黑质中α-突触核蛋白基因表达的消除防止了神经变性，表明由这种病因相关的环境触发引起的PD样神经病理学依赖于内源性α-突触核蛋白。之前已经提出了α-突触核蛋白和线粒体之间的相互作用，但α-突触核蛋白对于体内多巴胺神经元对病因相关线粒体毒素的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：废除SNCA在体内表达的病毒载体;转基因斑马鱼模型，其中线粒体毒素引发的神经变性依赖于人α-突触核蛋白;以及表达荧光报告基因的新型转基因斑马鱼系，其允许检测活性氧物质、谷胱甘肽氧化、ATP水平以及线粒体分裂、融合和转运。使用这些独特的工具，我们将确定α-突触核蛋白如何影响ROS的产生和氧化应激（目的1），细胞呼吸和生物能量学（目的2）和线粒体动力学（目的3）后，涉及PD发病机制的线粒体毒素，在哺乳动物和斑马鱼多巴胺神经元体内。在目标4中，我们将利用96孔板中斑马鱼幼体的自动行为测量来发现多巴胺神经元中线粒体抑制剂的α-突触核蛋白依赖性毒性的小分子修饰剂。这些数据将阐明多巴胺神经元响应与PD发病机制有关的线粒体毒素而发生α-突触核蛋白依赖性变性的潜在机制。独特的模型系统阵列和研究人员团队将使我们能够了解散发性PD中两种最突出的病理异常之间的机制联系，从而解决PD治疗发展中的关键障碍。

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.