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

描述(申请人提供)：帕金森氏病(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。一些潜在的危险因素基因和环境毒素(特别是杀虫剂)已被牵连，但这些因素是否真的导致帕金森病仍不清楚。目前用动物模型来检验这些因素之间的因果关系以及两者之间的相互作用是不够的。我们建议使用斑马鱼来测试帕金森病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，体型小，生命周期短，相对容易植入转基因，幼虫是透明的，能够成像活动物的分子和细胞过程。行为也可以很容易地衡量出来。我们建议利用这些独特的特征来确定帕金森病危险因子基因和环境毒素在帕金森病中的潜在病因学作用，以及基因和环境之间的相互作用。我们假设一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病理，类似于帕金森病。也有可能的是，某些基因和毒素的毒性只有在组合中才会显现(基因-环境相互作用)。具体地说，我们将表征由双光子激光制作并使用斑马鱼酪氨酸羟化酶启动子驱动绿色荧光蛋白(Zth-GFP)鉴定的特定多巴胺能细胞团中的损伤所造成的行为影响。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。待检测的帕金森病相关基因包括α-突触核蛋白和LRRK2。将测试的毒素将包括泛素-蛋白酶体系统(UPS)抑制剂环氧米星，以及使用在神经母细胞瘤细胞系中进行的高通量筛选确定为UPS抑制剂的杀虫剂。ZTH-GFP转基因的表达还将有助于对毒素暴露的斑马鱼和表达PD基因的鱼类进行病理学评估。这些研究不仅将提供PD基因和环境毒素在PD发病机制中所起作用的有价值的信息，而且还将创建一个强大的模型来测试潜在的疾病修改治疗方法。 公共卫生相关性：帕金森氏症(PD)影响了大约100万美国人，但病因仍然难以捉摸。我们建议使用一种新的斑马鱼模型来研究遗传和环境因素在帕金森病发生中的作用。这些实验的结果将为帕金森病的病因提供有价值的信息，并使我们更接近有意义的疾病修改疗法的开发。

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.