Functional Validation of Parkinsons Disease Susceptibility Genes in Drosophila

描述（由申请人提供）：帕金森病（PD）是一种常见的、致残的、不可治愈的神经退行性疾病，有证据表明其具有显著的遗传性。基于全基因组关联研究（GWAS），已经确定了大约30个不同的PD风险位点。然而，大多数涉及的基因组区域包含多个基因，这些基因同样可能受到相关多态性的影响，并且大多数这些候选基因的潜在功能完全未知。因此，PD基因组学转化研究的关键下一步是（1）确定每个易感基因座的责任基因，以及（2）确定它们对疾病机制的影响。解决这些问题对于将人类遗传学的见解转化为PD治疗的临床突破至关重要。基于大量证据，α-突触核蛋白（alphaSyn）神经毒性是PD易感性和发病机制的核心机制，我们假设许多风险位点可能调节相关的生物学途径。我们已经开发了一种改进的果蝇（Drosophila melanogaster）突触核蛋白病模型，该模型动态且稳健地重现了成年神经系统中的alphaSyn毒性，包括突触传递的早期功能障碍和进行性神经退行性细胞丢失。重要的是，该系统适用于高通量遗传筛选。在目标1中，我们将利用果蝇遗传学的快速能力和果蝇基因组近饱和试剂的可用性，全面评估来自人类GWAS的约30个PD易感基因座的候选基因。对于约100个基因，我们将鉴定果蝇的直系同源基因，获得预测会破坏或激活基因功能的品系，并筛选与体内alphaSyn神经毒性的遗传相互作用。在果蝇视网膜中的多功能和高通量筛选将允许平行评估αSyn-mediated结构和功能性神经变性。在目标2中，将在成年果蝇脑中验证最有前途的修饰基因，检查年龄依赖性、α-Syn诱导的多巴胺能神经元损失和由此产生的运动障碍。为了定义关键机制，将在体内分析表现出一致和稳健相互作用的顶级修饰基因对alphaSyn动力学的影响，包括蛋白质水平、磷酸化、错误折叠/聚集和包涵体形成。所提出的策略将使我们能够有效地从相关多态性的列表转移到致病基因的体内确认和αSyn-mediated神经退行性变的作用的验证。PD易感基因的确定性鉴定和功能阐明为发现这种毁灭性疾病的新治疗策略提供了巨大的希望。

DESCRIPTION (provided by applicant): Parkinson's disease (PD) is a common, disabling, and incurable neurodegenerative disorder with evidence for substantial heritability. Approximately 30 distinct PD risk loci have been identified based on genome-wide association studies (GWAS). However, most of the implicated genomic regions contain multiple genes that are equally likely to be affected by associated polymorphisms, and potential functions for the majority of these gene candidates are completely unknown. Therefore, the critical next steps for translational research in PD genomics are (1) to determine the responsible gene(s) at each susceptibility locus, and (2) to define their impact on disease mechanisms. Answering these questions will be essential for turning insights from human genetics into clinical breakthroughs in PD therapeutics. Based on overwhelming evidence, alpha-synuclein (alphaSyn) neurotoxicity is a central mechanism in PD susceptibility and pathogenesis, and we hypothesize that many risk loci may modulate the relevant biological pathways. We have developed an improved model of synucleinopathy in the fruit fly, Drosophila melanogaster, that dynamically and robustly recapitulates alphaSyn toxicity in the adult nervous system, including early dysfunction in synaptic transmission and progressive neurodegenerative cell loss. Importantly, this system is amenable to high-throughput genetic screening. In Aim 1, we will leverage the rapid capabilities of fly genetics and the availability of near-saturation reagents for the Drosophila genome to comprehensively evaluate candidate genes at ~30 PD susceptibility loci from human GWAS. For ~100 genes, we will identify Drosophila orthologs, obtain lines predicted to disrupt or activate gene function, and screen for genetic interactions with alphaSyn neurotoxicity in vivo. A versatile and high-throughput screen in the fly retina will allow parallel assessments of αSyn-mediated structural and functional neurodegeneration. In Aim 2, the most promising modifier genes will be validated in the adult Drosophila brain examining age-dependent, alphaSyn-induced dopaminergic neuronal loss and resulting locomotor impairment. In order to define the key mechanisms, the top modifier genes demonstrating consistent and robust interactions will be profiled in vivo for their impact on alphaSyn dynamics, including protein levels, phosphorylation, misfolding/aggregation, and inclusion formation. The proposed strategy will enable us to move efficiently from lists of associated polymorphisms to in vivo confirmation of the causal genes and validation of roles in αSyn-mediated neurodegeneration. The definitive identification and functional elucidation of PD susceptibility genes holds great promise for the discovery of new treatment strategies for this devastating disorder.