The Activation of LRRK2 by Alpha-Synuclein

描述（由申请人提供）：LRRK 2中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，LRRK 2相关的PD在临床上与特发性PD无法区分，并且通常伴随着称为路易体的α-突触核蛋白的经典细胞内内含物的存在，这是PD的病理学标志。然而，鉴定α-突触核蛋白聚集和LRRK 2功能之间的生物化学联系已被证明是困难的。先前的工作可能已经被假设这两种疾病相关蛋白质的共同途径必须发生在同一个细胞内的假设所阻碍。最近的证据表明LRRK 2在非神经元细胞包括小胶质细胞中的表达和有效调节，特别是在神经炎症期间。另一方面，β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。来自多个实验室的最新数据表明，细胞外α-突触核蛋白可以作为激活表达LRRK 2的小胶质细胞的配体。在本申请中，我们将筛选原代培养的鼠小胶质细胞的α-突触核蛋白的多种构象和组装的影响，鉴定那些激活小胶质细胞LRRK 2，并确认这些影响在培养的人小胶质细胞。接下来，使用表达内源水平的突变体LRRK 2（R1441 C和G2019 S）的两种不同的敲入小鼠模型，我们将确定LRRK 2中的致病性突变如何在体外和体内改变α-突触核蛋白依赖性小胶质细胞反应。这将涉及致病性LRRK 2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的分析，探索LRRK 2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。该探索性项目将测试关于LRRK 2和α-突触核蛋白之间的功能相互作用的新假设，并可能增加对整合可以说是两种最重要的PD连锁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 a-synuclein called Lewy bodies, the pathological hallmark of PD. However, identifying biochemical links between a-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. �ynuclein, 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 a-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 a- 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 a-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 a-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 a- synuclein, and may add valuable insight into potential pathways integrating what are arguably the two most important PD-linked gene products.