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，美国的发病率将增加一倍，发展中国家的发病率将增加一倍以上；然而，对于最常见的PD形式，迟发散发性特发性PD，尚未确定明确的病因或统一的分子事件序列。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子的靶标，这是基于家族性PD的原因，这是由泛素途径的两个组成部分Parkin和UCHL1的功能突变丧失引起的。本应用程序中的实验以以下工作假设为指导：三嗪类和硫代氨基甲酸酯类除草剂，二酰基二硫代氨基甲酸酯类和苯甲酰类杀菌剂共价修饰和抑制UBA1激活酶，导致泛素类蛋白质加工和细胞信号传导受损，从而促进导致PD的神经退行性变化。该假设将通过以下具体目的进行验证：1)在大鼠体内确定所提出的农药或所选代谢物是否抑制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，通过霰弹枪LC/MS/MS表征UBA1共价修饰，绘制神经退行性变化，量化tau和-synuclein的表达和磷酸化。2)确定体外抑制UBA1是否有助于多巴胺能神经元的细胞毒性、Cdk5的激活以及与散发性PD相关的基因产物加工的改变。为了实现这一目标，MN9D细胞将被用来调节UBA1的表达，并确定UBA1功能的组成性受损和增加对暴露于农药、其代谢物或特定药理学UBA1抑制剂的细胞的活力、-synuclein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的表达和定位的影响。从拟议的研究中获得的数据将有助于制定更明智的风险管理和发展筛选方法，以确定和预测通过类似或附加机制起作用的其他药剂。识别直接或通过基因-环境相互作用促进PD发展的环境因素将有助于制定干预策略，以降低PD的风险并减缓PD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.