Investigating Genetic and Environmental Etiologies of Parkinson's Ds in Zebrafish

描述（由申请人提供）：帕金森病（PD）的病因仍然难以捉摸，但几乎可以肯定涉及遗传和环境因素。许多潜在的风险因素基因和环境毒素（尤其是农药）都与帕金森病有关，但目前尚不清楚这些因素是否真的导致帕金森病。目前用于测试这些因素的因果关系和两者之间相互作用的动物模型是不充分的。我们建议使用斑马鱼来测试PD的潜在遗传和环境原因，因为它们比目前的动物模型有几个优势。斑马鱼是脊椎动物，体积小，生命周期短，相对容易插入转基因，并且幼虫是透明的，可以成像活体动物的分子和细胞过程。行为也很容易被衡量。我们建议利用这些独特的特征来确定PD危险因素基因和PD相关的环境毒素的潜在病因作用以及基因与环境之间的相互作用。我们假设某些毒素会改变运动和嗅觉行为，并诱导选择性多巴胺能细胞死亡和类似PD的病理。也有可能某些基因和毒素的毒性只有在结合（基因-环境相互作用）时才会显现出来。具体来说，我们将描述由特定多巴胺能细胞团损伤引起的行为影响，这些损伤由双光子激光制成，并使用驱动绿色荧光蛋白（zTH-GFP）的斑马鱼酪氨酸羟化酶启动子识别。然后，我们将能够使用行为测量来筛选PD基因和毒素对多巴胺能系统的影响。PD相关基因检测包括α -突触核蛋白和LRRK2。待测试的毒素将包括泛素-蛋白酶体系统（UPS）抑制剂环氧霉素，以及在神经母细胞瘤细胞系中使用高通量筛选确定为UPS抑制剂的农药。zTH-GFP转基因的表达也将有助于毒素暴露的斑马鱼和表达PD基因的鱼的病理评估。这些研究不仅将为PD基因和环境毒素对PD发病机制的贡献提供有价值的信息，而且将为测试潜在的疾病修饰疗法创建一个强大的模型。

DESCRIPTION (provided by applicant): The cause of Parkinson's disease (PD) remains elusive but almost certainly involves genetic and environment factors. A number of potential risk factor genes and environmental toxins (especially pesticides) have been implicated but it is still not known if these factors actually cause PD. Current animal models to test causality of these factors and interactions between the two are inadequate. We propose to use zebrafish to test potential genetic and environmental causes of PD because they have several advantages over current animal models. Zebrafish are vertebrates, small, have a short life cycle, are relatively easy to insert transgenes, and the larvae are transparent enabling imaging of molecular and cellular processes in living animals. Behavior can also be readily measured. We propose to take advantage of these unique characteristics to determine the potential etiological roles of PD risk factor genes and environmental toxins implicated in PD and interactions between genes and the environment. We hypothesize that some toxins will alter locomotor and olfactory behavior and induce selective dopaminergic cell death and pathology similar to that seen in PD. It is also possible that the toxicity of some genes and toxins will only be apparent in combination (gene-environment interaction). Specifically, we will characterize the behavioral effects resulting from lesions in specific dopaminergic cell clusters made with a 2-photon laser and identified using a zebrafish tyrosine hydroxylase promoter driving green fluorescent protein (zTH-GFP). We will then be able to use behavioral measurements to screen for effects of PD genes and toxins on the dopaminergic system. PD- associated genes to be tested include alpha-synuclein and LRRK2. Toxins to be tested will include the ubiquitin-proteasome system (UPS) inhibitor epoxomicin, and pesticides identified to be inhibitors of the UPS using a high throughput screen performed in a neuroblastoma cell line. Expression of the zTH-GFP transgene will also facilitate pathological evaluation of toxin-exposed zebrafish and fish expressing PD genes. These studies will not only provide valuable information on the contributions PD genes and environmental toxins make to the pathogenesis of PD, but also will create a powerful model to test potential disease modifying therapies. PUBLIC HEALTH RELEVANCE: Parkinson's disease (PD) affects approximately 1 million Americans but the cause remains elusive. We propose to investigate genetic and environmental contributions to the development of PD using a novel zebrafish model. The results of these experiments will provide valuable information into the cause of PD and move us closer to the development of meaningful disease modifying therapies.