The PARK10 gene USP24 affects Parkinson's Disease via regulation of autophagy

描述（由申请人提供）：最近的研究表明，自噬缺陷（溶酶体依赖的细胞内降解途径）与帕金森病（PD）之间存在因果关系。相反，自噬上调被认为是一种可能的PD干预策略。我们最近发现了一个位于PD相关的PARK10位点的功能未知的基因USP24，作为自噬的负调节因子。我们的数据表明，在PD患者的一个亚群中，USP24蛋白水平升高，这表明它也可能与特发性PD有关。另一方面，USP24失活可诱导自噬，在PD细胞模型中发挥保护作用。我们的目标是使用体外PD模型来验证USP24通过自噬依赖机制影响PD易感的假设，以及抑制USP24可以减轻PD病理。AIM 1将确定USP24过表达和pd相关编码区多态性如何改变自噬水平。我们假设USP24通过干扰III型PI3激酶的功能来抑制自噬通量。我们将通过在人类细胞系和原代大鼠神经元中表达野生型和突变型USP24，并使用免疫荧光、western blot和自噬通量分析来研究自噬水平、III型PI3激酶活性、溶酶体降解和线粒体功能的影响。AIM 2将确定usp24调节的自噬对基于细胞的PD模型结果的影响。我们假设通过抑制USP24诱导自噬将减轻PD病理。为了验证这一点，我们将在两种基于PD的细胞模型中评估USP24敲除、过表达和PD多态性对细胞活力、线粒体功能和蛋白质周转的影响：PD相关除草剂百草枯处理和突变α -突触核蛋白A53T的表达。我们的数据将是了解USP24如何调节自噬并促进PD的必要的第一步，并确定通过抑制USP24来上调自噬是否可以作为一种新的预防和治疗策略。

DESCRIPTION (provided by applicant): Recent studies indicate a causative link between defects in autophagy, a lysosome-dependent intracellular degradation pathway, and Parkinson's disease (PD). Conversely, up-regulation of autophagy has been proposed as a possible PD intervention strategy. We recently identified USP24, a gene of unknown function located in the PD associated PARK10 locus, as a negative regulator of autophagy. Our data indicate that USP24 protein levels are increased in the substantia nigra of a subpopulation of PD patients, suggesting possible involvement also in idiopathic PD. On the other hand, inactivation of USP24 induces autophagy and can play a protective function in cellular models of PD. The goal of our proposal is to use in vitro PD models to test the hypothesis that USP24 affects PD predisposition via autophagy-dependent mechanism and that inhibition of USP24 can attenuate PD pathology. AIM 1 will determine how USP24 overexpression and PD-associated coding region polymorphisms alter levels of autophagy. We hypothesize that USP24 inhibits autophagy flux by interfering with function of the type III PI3 kinase. We will test this by expressing wild-type and mutant USP24 in human cell lines and primary rat neurons and using immunofluorescence, western blot and autophagic flux analysis to investigate the effects on the levels of autophagy, type III PI3 kinase activity, lysosomal degradation and mitochondrial function. AIM 2 will determine the influence of USP24-regulated autophagy on outcomes in cell based PD models. We hypothesize that induction of autophagy by USP24 inhibition will attenuate PD pathology. To test this we will evaluate the effects of USP24 knock-down, overexpression and PD polymorphisms on cell viability, mitochondrial function and protein turnover in two cell based PD models: treatment with PD associated herbicide paraquat and expression of mutant alpha-synuclein A53T. Our data will be the necessary first step to understand how USP24 regulates autophagy and contributes to PD and to determine if up-regulation of autophagy by inhibition of USP24 can be used as a novel prevention and treatment strategy.