Mechanisms of LRRK2 Mediated Neurotoxicity

R 01-NS 064934“LRRK 2神经毒性机制”的更新请求帕金森病（PD）和相关路易体疾病是阿尔茨海默病相关痴呆（ADRD）的常见原因。根据国家阿尔茨海默病项目法案，路易体疾病 影响了超过一百万的美国人，这种疾病的发病通常比阿尔茨海默病引起的痴呆症更早。成千上万的美国人携带LRRK 2错义变异，可能导致PD。纵向研究表明，LRRK 2突变携带者随着时间的推移在很大程度上遵循典型的运动和认知障碍过程，反映了特发性疾病。尸检研究表明，LRRK 2突变是tau和α-突触核蛋白聚集的潜在遗传风险因素。LRRK 2变体与克罗恩病和分枝杆菌感染的易感性相关，突出了LRRK 2在免疫细胞中的功能。 在过去的项目期间，我们观察到一种双重激活现象，即内在LRRK 2修饰神经元中的α-突触核蛋白聚集，以及疾病模型中招募到大脑的髓样细胞中的高LRRK 2表达。在我们打算仔细检查的一种新的酶循环中，LRRK 2突变可能会增加 顺式LRRK 2激酶在Rab蛋白的一小部分的自磷酸化以及反式磷酸化中的活性。在我们继续努力了解LRRK 2在疾病中的功能，我们将注意力集中在LRRK 2- Rab底物相互作用。我们提出了一系列的定量邻近标记方法来探测 疾病相关细胞中LRRK 2-Rab酶循环的内部催化核心。这些实验将 通过冷冻电子显微镜分析确定的相关结构-功能关系锚定 蛋白质复合物我们预测这些实验将揭示不同的致病性突变是如何在 LRRK 2在Rab过度磷酸化中共享共同的最终输出。此外，我们将探讨LRRK 2 与疾病保护相关的变异可能会影响LRRK 2-Rab催化循环。LRRK 2及其Rab 基质组成表明免疫细胞中髓样细胞亚群中的一些最高表达。 系统作为对我们生物化学方法的补充，我们将剖析LRRK 2在促炎症反应中的作用。 在AAV-α-突触核蛋白小鼠模型中，巨噬细胞募集到脑中。我们预测突变的LRRK 2 在外周免疫细胞中的表达将加剧与免疫相关的有害神经炎症。 多巴胺能神经变性我们进一步预测，外周免疫细胞中的LRRK 2敲除将导致细胞凋亡。 减弱脑中的促炎反应和α-突触核蛋白诱导的多巴胺神经元损失。作为 几种靶向LRRK 2的探索性疗法最近通过临床安全性试验取得了进展， 项目的成功，我们预测我们的研究将进一步强调LRRK 2-Rab信号转导和外周免疫 LRRK 2相关疾病的反应。

Renewal Request for R01-NS064934 “Mechanisms of LRRK2 Neurotoxicity” Parkinson's disease (PD) and related Lewy body diseases represent common causes of Alzheimer's disease related dementias (ADRDs). According to the National Alzheimer's Project Act, Lewy body diseases affect more than one-million Americans, with onset of disease typically earlier in life than dementia caused by Alzheimer's disease. Tens of thousands of Americans harbor LRRK2 missense variants that can cause PD. Longitudinal studies show LRRK2 mutation carriers largely follow a typical course of motor and cognitive impairment over time, mirroring idiopathic disease. Post-mortem studies show that LRRK2 mutations are a potent genetic risk factor for the aggregation of tau and α-synuclein. LRRK2 variants associate with susceptibility to Crohn's disease and mycobacteria infection, highlighting LRRK2 function in immune cells. In the past project period, we observed a dual-activation phenomenon with intrinsic LRRK2 modifying α-synuclein aggregation in neurons, as well as high LRRK2 expression in myeloid cells recruited to the brain in disease models. In a novel enzymatic cycle that we intend to closely examine, LRRK2 mutations may increase cis LRRK2 kinase activity in autophosphorylation as well as trans phosphorylation of a small subset of Rab proteins. In our continued efforts to understand LRRK2 function in disease, we focus our attention to LRRK2- Rab substrate interactions. We propose a series of quantitative proximity labeling approaches to probe the inner catalytic core of LRRK2-Rab enzymatic cycling in disease-relevant cells. These experiments will be anchored through structure-function relationships identified by cryo-electron microscopy analysis of relevant protein complexes. We predict these experiments will shed light on how disparate pathogenic mutations in LRRK2 share a common final output in Rab hyper-phosphorylation. Further, we will explore how LRRK2 variants linked to protection from disease might affect the LRRK2-Rab catalytic cycle. Both LRRK2 and its Rab substrate constituency demonstrate some of the highest expression in subsets of myeloid cells in the immune system. In complement to our biochemical approach, we will dissect the role of LRRK2 in pro-inflammatory macrophages recruited to the brain in an AAV-α-synuclein mouse model. We anticipate mutant LRRK2 expression in peripheral immune cells will exacerbate deleterious neuroinflammation that correlates to dopaminergic neurodegeneration. We further predict that LRRK2 knockout in peripheral immune cells will attenuate pro-inflammatory responses in the brain and α-synuclein-induced loss of dopamine neurons. As several exploratory therapies that target LRRK2 have recently advanced through safety trials in the clinic, with project success, we predict our studies will further emphasize LRRK2-Rab signaling and peripheral immune responses in LRRK2-linked disease.