Functional Analysis Of The Intersection of Mitochondrial Stress and Neurodegeneration

项目摘要/摘要 随着我们人口的老龄化，帕金森病(PD)等神经退行性疾病构成了主要的 社会负担。虽然帕金森病的发病机制仍在不断涌现，但有证据表明线粒体功能障碍在 该病的发病机制很多。帕金森病病理的另一个组成部分是蛋白a-突触核蛋白 (a-syn)；它存在于路易体包涵体中，但细胞毒性的原因尚不清楚。一种战略， 线粒体用于管理压力是参与线粒体未折叠蛋白反应(UPRmt)， 它协调转移到线粒体的伴侣和蛋白酶的核表达，以 处理受损和/或未折叠的蛋白质。当对急性应激源做出反应时，UPRmt反应 建立动态平衡，促进细胞存活。然而，当受到挑战时，它可能会变得不受监管 与长期的遗传应激源，如错误折叠的a-syn，并成为细胞毒。值得注意的是，分子变体 能与线粒体膜外膜蛋白TOMM20相互作用，并启动一种物理阻断 线粒体蛋白进口。我们推测，在a-syn表达中观察到的UPRmt反应增加 神经元是线粒体输入受阻的结果。尽管人们注意到线粒体的作用 神经退行性疾病的质量控制已被证明是越来越有洞察力的，有一个关键的差距是 在证明失调的UPRmt活动之间的直接功能相关性方面仍有待解决 和神经退化。重要的是，我们的研究说明了线粒体信号转导的一个潜在方面 其中UPRmt通路加剧了体内多巴胺能神经元的破坏，导致神经元丢失 帕金森病的特点。我们的方法利用了秀丽隐杆线虫基因操作的便利性 研究，以及大的、等基因的人群可以用来对神经变性进行评分的严谨程度 在单神经元水平上，前所未有的精确度。我们将系统地调查 共表达a-syn结构变体和激活UPRmt的转录因子的转基因蠕虫 识别以神经变性为主要终点的UPRmt激活的功能要求。这个 目标1中的研究将调查a-SYN-TOMM20线粒体输入阻断触发 并将探索多巴胺在潜在加剧有害后果中的作用 在这一过程中。作为一项独特的战略，目标2将涉及识别相关的分子成分 UPRmt通过一种前向基因筛选策略发出信号，这种策略利用了我们拥有的一种菌株 这揭示了UPRmT诱导的一种不具特征的补偿机制。表型 使用线虫的生物检测和遗传筛选是由我们实验室的本科生和 将成为学生通过此R15计划进行培训的绝佳机会。这些研究代表了 一种及时和机械的战略，以确定核-线粒体动力学，特别是关于 多巴胺能神经变性，有可能为治疗发展提供一条翻译路径。

Project Summary/Abstract As our population ages, neurodegenerative disorders such as Parkinson disease (PD) comprise a major societal burden. While mechanisms for PD etiology are still emerging, evidence of mitochondrial dysfunction in the pathogenesis of this disease is abundant. Another component of PD pathology is the protein a-synuclein (a-syn); it is found within Lewy Body inclusions, yet causes of cellular toxicity remain unclear. A strategy that mitochondria employ for managing stress is to engage the mitochondrial unfolded protein response (UPRmt), which coordinates nuclear expression of chaperones and proteases that translocate to the mitochondria to handle damaged and/or unfolded proteins. When activated in response to acute stressors, the UPRmt re- establishes homeostasis and promotes cell survival. However, it can become dysregulated when challenged with a long-term genetic stressor such as misfolded a-syn and becomes cytotoxic. Notably, molecular variants of a-syn can interact with TOMM20, an outer mitochondrial membrane protein, and initiate a physical block of mitochondrial protein import. We speculate that the increased UPRmt response observed in a-syn-expressing neurons is a consequence of blocked mitochondrial import. Although attention to a role for mitochondrial quality control in neurodegenerative disease has proven increasingly insightful, there is a pivotal gap that remains to be addressed in demonstrating a direct functional correlation between dysregulated UPRmt activity and neurodegeneration. Importantly, our research illustrates an insidious aspect of mitochondrial signaling in which the UPRmt pathway exacerbates disruption of dopaminergic neurons in vivo, resulting in the neuron loss characteristic of PD. Our approach exploits the expedience of genetic manipulation in Caenorhabditis elegans research, and the rigor with which large, isogenic populations can be scored for neurodegeneration with unprecedented accuracy, at the single-neuron level. We will systematically investigate combinations of transgenic worms co-expressing structural variants of a-syn and transcription factors that activate the UPRmt to discern functional requirements for UPRmt activation with neurodegeneration as the primary endpoint. The studies in Aim 1 will investigate the hypothesis that the a-syn-TOMM20 mitochondrial import block triggers the UPRmt pathway and will explore a role for dopamine in potentially exacerbating the deleterious consequences of this process. As a distinct strategy, Aim 2 will involve the identification of molecular components associated with UPRmt signaling through a forward genetic screening strategy that takes advantage of a strain we have generated that reveals an uncharacterized compensatory mechanism for UPRmt induction. Phenotypic bioassays and genetic screening using C. elegans are routinely conducted by undergraduates in our lab and will serve as an excellent training opportunity for students through this R15 proposal. These studies represent a timely and mechanistic strategy towards defining nuclear-mitochondrial dynamics, specifically with respect to dopaminergic neurodegeneration, with potential to inform a translational path for therapeutic development.