Investigating Neuroprotective Targets for Parkinson Disease Associated with Autop

描述(由申请人提供)：帕金森病(PD)的治疗进展取决于对影响神经元存活的细胞因素的识别和机制了解。我们利用线虫模型系统的属性，秀丽线虫，开发了有助于筛选和分离保守的遗传因素的方法，这些因素介导了帕金森病的临床特征，包括a-突触核蛋白(a-syn)和多巴胺(DA)神经元的细胞内聚集。这个R15应用扩展了之前的研究，在这些研究中，我们已经确定了翻译发育的神经保护靶点，并建立了遗传平台来评估年龄依赖性神经退行性变的功能修饰物。越来越多的证据表明，溶酶体功能是影响帕金森病的重要机制。在这种情况下，这一应用的焦点是ULK2蛋白--一种与蠕虫UNC-51基因产物同源的人类蛋白，一种与自噬有关的激酶，以及轴突的延长和引导。我们之前发现了ULK2在提高转基因线虫中DA神经元存活方面的神经保护活性。我们的数据在人类全基因组关联研究报告之后具有更大的意义，该研究也发现ULK2基因的一个多态性与PD患者相关。我们建议通过一系列结构-功能分析以及对ULK2修饰物的评估来加深我们对ULK2介导的神经保护的理解，以机械地确定ULK2激酶活性在减轻神经退行性变中的作用。在衰老机制方面，线虫也是被最好理解的动物模型之一，发现了与寿命有关的广泛因素。因此，由于衰老是帕金森病明确和明确的危险因素，我们将在线虫中使用RNA干扰(RNAi)进行功能基因组筛选，以敲除数百个以前与衰老相关的途径相关的基因，以评估它们对a-syn错误折叠和清除的不同贡献。我们实验室的初步研究表明，线虫daf-2/胰岛素样信号通路关键成分的特定突变会对DA神经元的存活产生实质性影响。我们已经产生了一系列转基因线虫菌株，以便于在a-syn修饰物对这一途径的潜在依赖性以及自噬的背景下进行功能分析。将产生转基因和突变的蠕虫，以检查RNAi筛选的基因靶标对DA神经保护的影响。这一系统的方法提供了一个前所未有的机会来辨别神经退行性变的年龄相关调节因子，这些调节因子可能是帕金森病发病或进展的遗传易感标记。总而言之，这些研究代表了一个综合的研究计划，旨在快速确定以前未确定的影响神经变性的因素的重要性。此外，我们的实验战略与R15区域项目的具体标准不谋而合，因为这一应用程序的更广泛影响涉及到在一个非常适合以学生为中心的研究的环境中提供广泛的本科生和研究生培训机会。 与公共卫生相关：超过100万美国人被诊断出患有帕金森氏症(PD)--这是一种最常见的运动障碍，几十年来医学一直没有治愈它。这项应用解决了一个尚未解决的挑战，即识别可能导致这种神经退行性疾病的遗传因素，同时加速我们对帕金森病潜在细胞机制的理解。在这项研究中，我们使用一个简单的动物模型系统来探索成熟但鲜为人知的衰老与帕金森病的关系，以及调节产生多巴胺的神经元存活的遗传因素。概述的实验策略在涉及遗传和基因组分析的系统方法的背景下整合了以学生为中心的研究，以揭示有助于识别具有对抗帕金森病潜力的新治疗靶点的机制。

DESCRIPTION (provided by applicant): Therapeutic advances to treat Parkinson disease (PD) are incumbent upon the identification and mechanistic understanding of cellular factors that influence neuronal survival. We have exploited attributes of the nematode model system, Caenorhabditis elegans, to develop assays that facilitate the screening and isolation of conserved genetic factors that mediate clinical hallmarks of PD, including intracellular aggregation of a-synuclein (a-syn) and dopamine (DA) neuron loss. This R15 application extends prior studies where we have identified neuroprotective targets for translational development and established genetic platforms to evaluate functional modifiers of age-dependent neurodegeneration. Increasing evidence is mounting for a significant role for lysosomal function as a mechanism influencing PD. In this context, a focal point of this application is the ULK2 protein - a human ortholog of the worm unc-51 gene product, a kinase implicated in autophagy, as well as axon elongation and guidance. We previously uncovered a neuroprotective activity for ULK2 in enhancing DA neuron survival in transgenic nematodes. Our data took on greater significance following the report of a human genome-wide association study that also identified a polymorphism in ULK2 as being associated with PD patients. We propose to advance our understanding of ULK2-mediated neuroprotection through a series of structure-function analyses, as well as evaluation of modifiers of ULK2, to mechanistically define the role of ULK2 kinase activity in attenuating neurodegeneration. C. elegans is also among the best-understood animal models in terms of aging mechanisms, with extensive sets of factors implicated in lifespan identified. Thus, as aging represents an unequivocal and defined risk factor for PD, we will use functional genomic screening via RNA interference (RNAi) in C. elegans to knockdown hundreds of genes previously linked to aging-associated pathways to evaluate their distinct contribution to a-syn misfolding and clearance. Preliminary studies in our lab have shown that specific mutations in key components of the daf-2/insulin-like signaling pathway of C. elegans result in substantial effects on DA neuron survival. We have generated a series of transgenic nematode strains to facilitate functional analysis of a-syn modifiers in the context of their potential dependence on this pathway, as well as in autophagy. Transgenic and mutant worms will be generated to examine gene targets from RNAi screening for their impact on DA neuroprotection. This systematic approach provides an unprecedented opportunity to discern age-associated regulators of neurodegeneration that may represent genetic susceptibly markers for PD onset or progression. Collectively, these studies represent an integrated research plan designed to rapidly define the significance of previously uncharacterized factors influencing neurodegeneration. Moreover, our experimental strategy coincides with the specific criteria of the R15 AREA program, as the broader impacts of this application involve extensive undergraduate and graduate student training opportunities in an environment ideally suited to student-centered research. PUBLIC HEALTH RELEVANCE: Over 1 million Americans have been diagnosed with Parkinson's Disease (PD) - the most common movement disorder for which a cure has eluded medical science for decades. This application addresses an unmet challenge of discerning genetic factors that may contribute to this neurodegenerative disease, while accelerating our understanding of underlying cellular mechanisms of PD. In this research, we use a simple animal model system to explore the well-established but poorly understood relationship of aging to PD, in addition to genetic factors that mediate the survival of dopamine-producing neurons. The experimental strategy outlined integrates student-centered research in the context of a systematic approach involving genetic and genomic analysis to uncover mechanisms that facilitate identification of new therapeutic targets with the potential to combat PD.