Role of UCH-L1 Oxidative Modification in Parkinson's Disease Pathogenesis

描述(申请人提供)：帕金森病(PD)是最常见的影响运动系统的神经退行性疾病，临床特征为运动迟缓和静止性震颤，病理特征为多巴胺能神经变性和细胞内路易体沉积。泛素羧基末端水解酶LI(UCH-L1)是一种丰富的神经元脱泛素酶，最近在一个常染色体显性遗传性帕金森病家系中发现了UCH-L1的I93M突变。UCH-L1参与了泛素蛋白酶体系统(UPS)和伴侣蛋白介导的自噬(CMA)蛋白降解途径。Pd连锁的I93M突变体Uch-L1表现出显著的去泛素化活性，此外，I93M突变体Uch-L1的过度表达与多巴胺能神经变性和CMA途径的抑制有关。缺乏功能性UCH-L1的小鼠表现为轴突变性，游离单体泛素水平降低，以及运动和学习障碍。散发性帕金森病患者大脑皮质中UCH-L1的表达降低，甲硫氨酸和半胱氨酸残基上的甲基化和氧化可引起广泛的修饰。综上所述，这些观察表明，UCH-L1功能障碍对神经元是有害的，并可能参与帕金森病的发病。暴露于环境毒物已被确定为帕金森病的危险因素，环境毒物与活性氧簇(ROS)的产生增加有关。环境毒物参与帕金森病发病的机制尚不清楚，但一种可能性是ROS增加导致特定靶蛋白的氧化修饰，如UCH-L1，导致其细胞功能的病理改变。这一提议将检验一种假设，即环境毒物诱导的氧化应激导致UCH-L1的氧化修饰，从而扰乱UCH-L1在UPS和CMA降解途径中的作用，从而导致有毒蛋白积累和多巴胺能细胞死亡。其具体目的是1)确定UCH-L1氧化修饰在帕金森病患者主要受影响的大脑区域黑质的严重程度，并评估UCH-L1作为环境毒物暴露引起的氧化修饰的靶点；2)表征UCH-L1氧化修饰对酶活性的影响以及与CMA机制的相互作用；以及3)确定UCH-L1氧化修饰对UPS和CMA蛋白降解途径和细胞活力的影响。了解UCH-L1氧化修饰及其功能的改变对蛋白质降解和细胞活性的影响将有助于理解PD的发病机制，并有助于开发新的治疗方法或治疗方法。

DESCRIPTION (provided by applicant): Parkinson's disease (PD) is the most common neurodegenerative disorder affecting the motor system, and is characterized clinically by bradykinesia and resting tremor, and pathologically by dopaminergic neurodegeneration and intracellular Lewy body deposition. Ubiquitin carboxyl-terminal-hydrolase LI (UCH- L1) is an abundant neuronal deubiquitinating enzyme, and recently the I93M mutation of UCH-L1 was described in a family with autosomal dominant PD. UCH-L1 is implicated in both the ubiquitin proteasome system (UPS) and chaperone mediated autophagy (CMA) protein degradation pathways. PD-linked I93M mutant UCH-L1 demonstrates significantly reduced deubiquitinating activity and additionally, I93M mutant UCH-L1 overexpression is associated with dopaminergic neurodegeneration and inhibition of the CMA pathway. Mice lacking functional UCH-L1 display axonal degeneration, reduced free monomeric ubiquitin level, and motor and learning deficits. Decreased expression of UCH-L1 is observed in cortex of sporadic PD patients, as well as extensive modification by carbonylation and oxidation at methionine and cysteine residues. Together these observations suggest that UCH-L1 dysfunction is detrimental to neurons, and may contribute to PD pathogenesis. Exposure to environmental toxicants which are linked to increased reactive oxygen species (ROS) generation has been identified as a PD risk factor. The mechanism by which environmental toxicants contribute to PD pathogenesis remains unclear, but one possibility is that increased ROS induce oxidative modification of particular target proteins, such as UCH-L1, leading to pathological alteration of their cellular function. This proposal will test the hypothesis that environmental-toxicant induced oxidative stress causes oxidative modification of UCH-L1, thereby disrupting the role of UCH-L1 in the UPS and CMA degradation pathways, which leads to toxic protein accumulation and dopaminergic cell death. The specific aims are to 1) determine the severity of UCH-L1 oxidative modification in the predominantly affected brain region in PD, the substantia nigra, and to evaluate UCH-L1 as a target of oxidative modification caused by environmental toxicant exposures; 2) characterize the effects of oxidative modification of UCH-L1 on enzymatic activity and interactions with CMA machinery; and 3) determine the effects of UCH-L1 oxidative modification on the UPS and CMA protein degradation pathways and cell viability. Understanding the role of UCH-L1 oxidative modification and subsequent alterations of its function on protein degradation and cell viability will contribute to the understanding of PD pathogenesis and to the development of novel therapeutics or treatments for this disease.