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）病理学的标志。PD最常见的遗传风险因素是葡萄糖脑苷脂酶（GBA）突变，多达7%的PD患者携带这种突变。迄今为止的工作主要集中在GBA酶活性的丧失，这可能有助于突触核蛋白的积累和/或聚集。然而，有证据表明，不正确折叠的突变体GBA也可能导致不依赖于酶活性损失的突触核蛋白聚集。我们的中心假设是，突变GBA从溶酶体错误地定位到胞质溶胶，从而通过伴侣介导的自噬（CMA）干扰γ-突触核蛋白降解。我们将结合生物化学、细胞生物学、小鼠遗传学和神经病理学技术，在包括分离的溶酶体、小鼠模型、死后人脑和来自患者皮肤活检的成纤维细胞在内的几个系统中测试这一假设。我们的研究策略分为三个具体目标：目标1将研究突变GBA减弱CMA的机制，并通过使用分离的溶酶体，纯化的突变GBA和<$- synuclein蛋白在体外系统中检查CMA的每个步骤，导致<$-synuclein积累。目的2将通过在神经元培养中使用新型CMA报告基因来研究CMA改变是否发生在GBA突变小鼠模型中，并确定CMA机械组件的蛋白水平是否在GBA突变小鼠模型和具有GBA突变的死后PD脑中发生变化。目的3将使用报告基因在来自具有GBA突变的PD患者、不具有GBA突变的PD患者和年龄匹配的对照的皮肤成纤维细胞中确定CMA活性。这些数据将为突变型GBA是否导致CMA功能障碍，导致突触核蛋白聚集，以及CMA功能障碍是否是PD的典型特征提供证据。这项研究可能有助于未来识别基于机制的生物标志物和治疗靶点。本K 08建议书还概述了一个详细的5年培训计划，包括针对候选人Sheng-Han Kuo，M.D.的具体正式课程和结构化指导。拟议的工作将在哥伦比亚大学的神经病学系进行，这是一个培养医学科学家的极好环境。他将在PD领域世界知名的研究人员大卫·苏尔寿博士、安娜·玛丽亚·奎尔沃博士和凯伦·马尔德博士的指导下接受必要的培训，并获得必要的技能，成为一名独立的研究人员。候选人的长期目标是成为一名翻译医生-科学家，研究PD病理学的机制，并开发临床适用的PD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.