Pesticide-mediated inhibition of UBA1 and cumulative risk for Parkinson's Disease

描述(由申请人提供)：帕金森病(PD)的患病率正在增加，预计在未来25年内在美国将翻一番，在发展中国家将翻一番以上；然而，最常见的PD形式-晚发性散发特发性PD的确切病因或统一的分子事件序列尚未确定。该项目的长期目标是描述导致帕金森病风险增加的环境因素的机制。泛素途径被认为是环境因素的靶点，基于泛素途径的两个组成部分Parkin和UCHL1的功能突变导致的家族性帕金森病。本申请中介绍的实验遵循以下工作假设：三嗪和硫代氨基甲酸酯除草剂，以及二烷基二硫代氨基甲酸酯和苯菌灵类杀菌剂共价修饰和抑制UBA1激活酶，导致基于泛素的蛋白质加工和细胞信号受损，从而促进导致帕金森病的神经退行性变化。这一假说将通过以下具体目标进行验证：1)在大鼠体内确定所建议的杀虫剂或选定的代谢物是否抑制UBA1激活酶并产生黑质纹状体毒性。为了实现这一目标，我们将确定脑UBA1酶抑制的剂量-反应，用鸟枪式LC/MS/MS表征UBA1共价修饰，绘制神经退行性变化图，并定量tau和-突触核蛋白的表达和磷酸化。2)确定体外抑制UBA1是否参与了多巴胺能神经元的细胞毒性、CDK5的激活以及与散发性帕金森病相关的基因产物的加工改变。为了实现这一目标，将利用MN9D细胞来调节UBA1的表达，并确定UBA1功能的结构性受损和增强对细胞活力、突触核蛋白和tau蛋白的表达和磷酸化状态、CDK5的激活以及多巴胺转运体在所建议的农药、其代谢物或特定的药物UBA1抑制剂中的表达和定位的影响。拟议研究得出的数据将有助于制定更知情的风险管理和制定筛选方法，以查明和预测通过类似或附加机制发挥作用的其他因素。识别直接或通过基因-环境相互作用导致帕金森病发生的环境因素，将有助于采取干预策略，降低帕金森病的风险，减缓帕金森病患者的疾病进展。

DESCRIPTION (provided by applicant): The prevalence of Parkinson's disease (PD) is increasing and predicted to double in the US and more than double in developing countries during the next 25 years; however a definite etiology or unifying sequence of molecular events for the most common form of PD, late onset sporadic idiopathic PD, has not been established. The long term goal of this project is to delineate mechanisms of environmental agents that contribute to increased risk for PD. The ubiquitin pathway has been implicated as a target for environmental agents based upon familial forms of PD resulting from loss of function mutations in Parkin and UCHL1 that are both components of the ubiquitin pathway. The experiments presented in this application are guided by the following working Hypothesis: The triazine and thiocarbamate herbicides, and the diaklydithiocarbamate and benomyl based fungicides covalently modify and inhibit UBA1 activating enzyme resulting in impaired ubiquitin based protein processing and cell signaling that promote neurodegenerative changes contributing to PD. This hypothesis will be tested through the following Specific Aims: 1) To determine in vivo in rats if the proposed pesticides or selected metabolites inhibit UBA1 activating enzyme and produce nigrostriatal toxicity. To achieve this aim we will determine the dose-response for brain UBA1 enzyme inhibition, characterize UBA1 covalent modifications by shotgun LC/MS/MS, map neurodegenerative changes and quantify the expression and phosphorylation of tau and -synuclein. 2) To determine if in vitro inhibition of UBA1 contributes to dopaminergic neuron cytotoxicity, activation of Cdk5 and altered processing of gene products associated with sporadic PD. To achieve this aim MN9D cells will be used to modulate UBA1 expression and determine the influence of constitutively compromised and increased UBA1 function on viability, the expression and phosphorylation state of -synuclein and tau proteins, activation of Cdk5 and expression and localization of the dopamine transporter in cells exposed to the proposed pesticides, their metabolites or specific pharmacologic UBA1 inhibitors. The data derived from the proposed studies will assist in the formulation of more informed risk management and the development of screening methods to identify and predict other agents that act through similar or additive mechanisms. Identification of environmental agents that contribute to the development of PD either directly or through gene-environment interactions will facilitate strategies of intervention to decrease the risk of PD and slow the progression of disease in PD patients.