Functional Analysis Of The Intersection of Mitochondrial Stress and Neurodegeneration

项目总结/摘要 随着我们的人口老龄化，神经退行性疾病如帕金森病（PD）构成了一个主要的疾病。 社会负担。虽然PD病因的机制仍在出现，但线粒体功能障碍的证据表明， 本病的发病机制十分丰富。PD病理学的另一个组成部分是α-突触核蛋白 （a-syn）;在路易体内含物中发现，但细胞毒性的原因仍不清楚。的策略 线粒体用于管理压力的是参与线粒体未折叠蛋白反应（UPRmt）， 其协调转移到线粒体的伴侣蛋白和蛋白酶的核表达， 处理受损和/或未折叠的蛋白质。当对急性应激源作出反应时，UPRmt被激活， 建立体内平衡并促进细胞存活。然而，当受到挑战时， 与长期遗传应激源如错误折叠的α-syn结合并变得具有细胞毒性。值得注意的是， 的α-syn可以与线粒体外膜蛋白TOMM 20相互作用，并启动对细胞的物理阻断。 线粒体蛋白输入。我们推测，在表达α-syn的小鼠中观察到的增加的UPRmt反应可能是由于在表达α-syn的小鼠中观察到的增加的UPRmt反应。 神经元是线粒体输入受阻的结果。虽然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越来越有见地，但存在一个关键的差距， 在证明UPRmt活性失调与细胞凋亡之间的直接功能相关性方面， 和神经退化重要的是，我们的研究说明了线粒体信号传导的一个潜在方面， 其中UPRmt途径加剧了体内多巴胺能神经元的破坏，导致神经元丢失， PD的特征。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，同基因人群可以对神经退行性疾病进行评分的严格性， 在单神经元水平上前所未有的精确度。我们将系统地研究 共表达α-syn结构变体和激活UPRmt的转录因子的转基因蠕虫， 识别UPRmt激活的功能要求，以神经退行性病变作为主要终点。的 目的1中的研究将调查α-syn-TOMM 20线粒体输入阻断触发线粒体转运的假设。 UPRmt通路，并将探索多巴胺在潜在加剧有害后果中的作用 这个过程。作为一项独特的战略，目标2将涉及鉴定与生物多样性有关的分子组分， 通过正向遗传筛选策略，利用我们拥有的菌株， 产生的，揭示了一个uncharacterized补偿机制UPRmt诱导。表型 利用C. elegans是我们实验室的本科生进行的常规实验， 将通过R15提案为学生提供一个极好的培训机会。这些研究代表了 一个及时和机械的战略，以确定核线粒体动力学，特别是在 多巴胺能神经变性，具有为治疗开发提供转化途径的潜力。

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.