The Activation of LRRK2 by Alpha-Synuclein

描述（由申请人提供）：LRRK2常染色体显性突变是帕金森病（PD）最常见的遗传原因。值得注意的是，lrrk2相关性PD在临床上与特发性PD难以区分，并且通常伴有称为路易体的α-突触核蛋白的典型细胞内包涵体的存在，这是PD的病理标志。然而，确定α-synuclein聚集和LRRK2功能之间的生化联系已被证明是困难的。先前的研究工作可能因为假设涉及这两种疾病相关蛋白的共同途径必须发生在同一个细胞内而受阻。最近的证据表明，LRRK2在包括小胶质细胞在内的非神经元细胞中的表达和有效调控，特别是在神经炎症期间。另一方面，α-突触核蛋白仅由大脑神经元表达，但其分泌机制尚未明确。最近来自多个实验室的数据表明，细胞外α-突触核蛋白可以作为配体激活表达LRRK2的小胶质细胞。在本应用中，我们将筛选原代培养的小鼠小胶质细胞α-突触核蛋白的多种构象和组装的影响，识别那些激活小胶质细胞LRRK2的构象和组装，并在培养的人类小胶质细胞中证实这些作用。接下来，使用两种不同的敲入小鼠模型表达内源性突变LRRK2 (R1441C和G2019S)，我们将确定LRRK2的致病突变如何在体外和体内改变α-突触核蛋白依赖的小胶质细胞反应。这将涉及分析致病性LRRK2突变如何影响小胶质细胞对致病性α-突触核蛋白暴露的反应，探测LRRK2蛋白的重要生化特性以及对小胶质细胞行为和功能的影响。该探索性项目将验证LRRK2和α-突触核蛋白之间功能相互作用的新假设，并可能为整合两个最重要的pd相关基因产物的潜在途径提供有价值的见解。

DESCRIPTION (provided by applicant): Autosomal dominant mutations in LRRK2 are the most common genetic cause of Parkinson's disease (PD). Notably, LRRK2-associated PD is clinically indistinguishable from idiopathic PD, and generally is accompanied by the presence of the classic intracellular inclusions of α-synuclein called Lewy bodies, the pathological hallmark of PD. However, identifying biochemical links between α-synuclein aggregation and LRRK2 function has proven difficult. Prior work may have been thwarted by the assumption that the common pathways involving these two disease-linked proteins must occur within the same cell. Recent evidence demonstrates the expression and potent regulation of LRRK2 in non-neuronal cells including microglia, particularly during neuroinflammation. α-synuclein, on the other hand, is exclusively expressed by neurons in the brain but is secreted by a yet unidentified mechanism. Recent data from multiple labs suggest that extracellular α-synuclein can serve as a ligand to activate microglial cells, which express LRRK2. In this application we will screen primary cultured murine microglia for the effects of multiple conformations and assemblies of α-synuclein, identifying those that activate microglial LRRK2, and confirm these effects in cultured human microglia. Next, using two distinct knock-in mouse models expressing endogenous levels of mutant LRRK2 (R1441C and G2019S), we will determine how pathogenic mutations in LRRK2 alter the α-synuclein- dependent microglial responses, both in vitro and in vivo. This will involve an analysis of how pathogenic LRRK2 mutations influence microglial responses to pathogenic α-synuclein exposure probing important biochemical properties of the LRRK2 protein and the effects on microglial behavior and function. This exploratory project will test a novel hypothesis regarding the functional interactions between LRRK2 and α-synuclein, and may add valuable insight into potential pathways integrating what are arguably the two most important PD-linked gene products.