The role of glucocerebrosidase and chaperone_mediated autophagy in Parkinsons dis

描述(由申请人提供)：多巴胺能神经元中的突触核蛋白聚集是帕金森病(PD)病理的一个标志。帕金森病最常见的遗传风险因素是葡萄糖脑苷酶(GBA)突变，多达7%的帕金森病患者携带这种突变。到目前为止，工作主要集中在可能有助于�-突触核蛋白积累和/或聚集的GBa酶活性的丧失。然而，有证据表明，不正确折叠的突变体gba也可以促进�-突触核蛋白的聚集，而不是依赖于酶活性的丧失。我们的中心假设是，突变的gba被错误地从溶酶体定位到胞浆，这通过伴侣介导的自噬干扰了�-突触核蛋白的降解。我们将结合生化、细胞生物学、小鼠遗传学和神经路径学技术，在几个系统中验证这一假设，这些系统包括分离的溶酶体、小鼠模型、死后人脑和患者皮肤活检的成纤维细胞。我们的研究策略分为三个特定的目标：目的1通过使用分离的溶酶体、纯化的突变体gba和�-突触核蛋白在体外系统中检测cmA的每一步，来研究突变体gba减弱cmA并导致�-突触核蛋白积聚的机制。目的2通过在神经元培养中使用新的CMA报告基因，研究GBA突变小鼠模型中是否发生CMA改变，并确定GBA突变小鼠模型和死后GBA突变帕金森病脑中CMA机械部件的蛋白水平是否发生变化。目的3将利用该报告确定GBA突变的PD患者、无GBA突变的PD患者和年龄匹配的对照组的皮肤成纤维细胞中的CMA活性。这些数据将提供证据，证明突变的gba是否导致颈动脉功能障碍，导致�-突触核蛋白聚集，以及颈动脉功能障碍是否为帕金森病的典型特征。这项拟议的研究可能有助于未来识别基于机制的生物标记物和治疗靶点。这份K08提案还为候选人郭胜汉医学博士概述了详细的5年培训计划，包括具体的正式课程和结构化的指导。拟议的工作将在哥伦比亚大学神经病学系进行，这是培养内科科学家的绝佳环境。他将在PD领域的世界知名研究人员David Sulzer博士、Ana Maria Cuervo博士和Karen Marder博士的指导下接受必要的培训，并获得成为独立研究员所需的技能。候选人的长期目标是成为一名翻译内科医生-科学家，研究帕金森病的发病机制，并开发临床适用的帕金森病生物标志物。

DESCRIPTION (provided by applicant): Synuclein aggregation in dopaminergic neurons is a hallmark of Parkinson's disease (PD) pathology. The most common genetic risk factor for PD is glucocerebrosidase (GBA) mutation with as many as 7% of PD patients carrying this mutation. Work to date has primarily focused on the loss of GBA enzymatic activity that could contribute to �-synuclein accumulation and/or aggregation. However, evidence has indicated that improperly folded mutant GBA could also contribute to the �-synuclein aggregation independent of the loss of enzymatic activity. Our central hypothesis is that mutant GBA is mistargeted from lysosomes to the cytosol, which interferes with �-synuclein degradation by chaperone-mediated autophagy (CMA). We will test this hypothesis with a combination of biochemical, cell biological, mouse genetics, and neuropath logical techniques in several systems including isolated lysosomes, mouse models, post-mortem human brain, and fibroblasts from patients' skin biopsy. Our research strategies are divided into three specific aims: Aim 1 will investigate the mechanism by which mutant GBA attenuates CMA and leads to �-synuclein accumulation by examining each step of CMA in an in vitro system using isolated lysosomes, purified mutant GBA and �- synuclein proteins. Aim 2 will study whether CMA alteration occurs in GBA-mutant mouse models by using a novel CMA reporter in neuronal cultures and determine whether the protein levels of CMA machinery components change in GBA-mutant mouse models and post-mortem PD brain with GBA mutations. Aim 3 will determine CMA activity using the reporter in the skin fibroblasts from PD patients with GBA mutations, PD patients without GBA mutations, and age-matched controls. These data will provide evidence on whether mutant GBA causes CMA dysfunction, leading to �-synuclein aggregation, and whether CMA dysfunction is a typical feature of PD. The proposed study could contribute to the future identification of mechanism-based biomarker and therapeutic targets. This K08 proposal also outlines a detailed 5-year training program with specific formal coursework and structured mentoring for the candidate, Sheng-Han Kuo, M.D. The proposed work will be carried out in the Department of Neurology at Columbia University, an excellent environment for training physician-scientists. He will receive the necessary training under the mentorship of Drs. David Sulzer, Ana Maria Cuervo, and Karen Marder, world-renowned investigators in the PD field and acquire necessary skill set to become an independent researcher. The long-term goal of the candidate is to be a translational physician-scientist to investigate the mechanism underlying PD pathology and to develop clinical applicable biomarkers for PD.