The Activation of LRRK2 by Alpha-Synuclein

描述(申请人提供)：LRRK2的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，与LRRK2相关的PD在临床上与特发性PD无法区分，通常伴随着经典的α-突触核蛋白细胞内包涵体的存在，称为路易小体，这是PD的病理标志。然而，确定α-突触核蛋白聚集和LRRK2功能之间的生化联系已经被证明是困难的。之前的工作可能因为假设涉及这两种与疾病相关的蛋白质的共同途径必须发生在同一细胞内而受挫。最近的证据表明，LRRK2在包括小胶质细胞在内的非神经细胞中的表达和有效的调节，特别是在神经炎症过程中。另一方面，α-突触核蛋白只由大脑中的神经元表达，但由一种尚未确定的机制分泌。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一种配体来激活表达LRRK2的小胶质细胞。在这项应用中，我们将筛选原代培养的小鼠小胶质细胞，以了解多种构象和组装的α-突触核蛋白的影响，鉴定那些激活小胶质细胞的LRRK2，并在培养的人小胶质细胞中证实这些影响。接下来，使用两种不同的表达内源性突变LRRK2(R1441C和G2019S)的敲入小鼠模型，我们将在体外和体内确定LRRK2的致病突变如何改变依赖α突触核蛋白的小胶质细胞反应。这将包括分析致病的LRRK2突变如何影响小胶质细胞对致病的α-突触核蛋白暴露的反应，探索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。这个探索性的项目将测试一个关于LRRK2和α-突触核蛋白之间功能相互作用的新假说，并可能增加对整合这两个最重要的PD相关基因产物的潜在途径的有价值的见解。

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.