Role of Rab27b in synucleinopathies

该项目的目标是研究Rab27在共核病中的作用，包括帕金森病和 路易体痴呆。αSYN的错误折叠和细胞间传播被认为是主要的 在帕金森氏病和路易体痴呆发病机制中的作用。α-SYN中涉及的关键步骤 扩散包括主动释放、摄取和错误折叠，但调节α-syn扩散的关键机制 人们对此了解甚少。Rab蛋白是一种小的GTPase蛋白，它与Rab效应蛋白相互作用来控制 蛋白质的运输和降解与αSYN的发病机制有关。我们最近发现了一种 Rab27b在联体核病中的潜在作用。Rab27b在神经元中高度丰富，在 蛋白质分泌在多种细胞类型中，包括神经元，但也与自噬和内吞有关。 Rab27b基因的改变与阿尔茨海默病、路易体痴呆和X连锁相关 肌张力障碍帕金森综合征。我们最近发现人死后帕金森病的Rab27b基因急剧增加 脑和可诱导的αSYN细胞系(ISYN)。ISYN细胞中RAB 27b基因敲除(KD)可减少αSYN的释放和 损害自噬清除，从而促进α的同步毒性。相比之下，Rab27a/b双基因敲除 (Dko)导致暴露于细胞外α同源原纤维的神经元α同源蛋白聚集性显著降低 (Pff)在体外和体内，很可能是由于摄取减少。基于这些数据，我们假设Rab27b 影响αSYN处理的两个独立角色：1)它通过内吞作用调节细胞外αSYN的进入； 2)通过分泌和自噬促进细胞内α同源蛋白的清除。在疾病中，纤维状αSYN增选 通过Rab27b进入细胞的途径，从而间接干扰Rab27b对细胞内αSYN的清除。我们认为，Rab27b对自噬、分泌和摄取的不同影响是通过不同的Rab27效应通路介导的。在目标1中，我们将测试Rab27b介导αSYN进入神经元的能力以及介导摄取的相关Rab27效应器。在目标2中，我们将重点研究Rab27b通过主动分泌和自噬对细胞内α-syn清除的影响，确定介导这些作用的相关Rab27效应分子。 自噬和分泌，并测试纤维状αSYN是否导致Rab27b重新分布，远离内溶酶体结构，从而干扰清除活动。在目标3中，我们将在活体α-SYN PFF模型中测试Rab27b和相关效应物对认知功能、α突触包涵体形成和神经元丢失的影响。进一步了解Rab27b影响αSYN运输的机制对于了解其在疾病中的作用及其治疗潜力至关重要。

The goal of this project is to examine the role of Rab27 in synucleinopathies, including Parkinson's disease and Dementia with Lewy Bodies. Misfolding and cell-to-cell propagation of αsyn are hypothesized to play a major role in the pathogenesis of Parkinson's Disease and Dementia with Lewy Bodies. Key steps implicated in α-syn spread include active release, uptake, and misfolding, yet the key mechanisms that regulate the spread of α-syn are poorly understood. Rab proteins are small GTPase proteins that interact with Rab effector proteins to control protein trafficking and degradation and have been implicated in αsyn pathogenesis. We recently identified a potential role for Rab27b in synucleinopathies. Rab27b, which is highly enriched in neurons, plays a role in protein secretion in multiple cell types including neurons, but has also been linked to autophagy and endocytosis. Alterations in Rab27b are associated with Alzheimer's disease, Dementia with Lewy Bodies, and X-linked dystonia parkinsonism syndrome. We recently found a dramatic increase in Rab27b in human PD postmortem brains and in an inducible αsyn cell line (isyn). Rab 27b knockdown (KD) in isyn cells reduces αsyn release and impairs autophagic clearance, consequently promoting αsyn toxicity. In contrast, Rab27a/b double knockout (DKO) leads to a dramatic reduction in αsyn aggregation in neurons exposed to extracellular αsyn protofibrils (PFFs) in vitro and in vivo, most likely due to reduced uptake. Based on these data, we hypothesize that Rab27b plays two independent roles that affect αsyn handling: 1) it regulates entry of extracellular αsyn via endocytosis; and 2) it promotes clearance of intracellular αsyn via secretion and autophagy. In disease, fibrillar αsyn co-opts cell entry pathways via Rab27b and thus indirectly disrupts Rab27b's clearance of intracellular αsyn. We propose that differential effects of Rab27b on autophagy, secretion, and uptake are mediated by different Rab27 effector pathways. In Aim 1, we will test the ability of Rab27b to mediate αsyn entry into neurons and the relevant Rab27 effectors that mediate uptake. In Aim 2, we will focus on the impact of Rab27b on clearance of intracellular α-syn by active secretion and autophagy, determine the relevant Rab27 effectors that mediate these effects on autophagy and secretion, and test whether fibrillar αsyn induces a redistribution of Rab27b away from endolysosomal structures to interfere with clearance activities. In Aim 3, we will test the consequences of Rab27b and relevant effectors on cognitive function, αsyn inclusion formation, and neuronal loss in the in vivo α-syn PFF model. Further understanding of the mechanisms by which Rab27b affects αsyn trafficking is critical to understand its role in disease and its therapeutic potential.