Changes in synaptic vesicle-binding of alpha-synuclein in the central and enteric nervous system

项目概要/摘要 α-突触核蛋白 (αSyn) 病理学与突触核蛋白病相关，包括帕金森病和路易体病 痴呆症，但其潜在的疾病机制仍知之甚少。普遍的观点有 发现 αSyn 的聚集通过毒性功能获得机制触发神经病理学，并且 消除 αSyn 的方法是治疗研究的一个活跃领域。然而，αSyn 聚集可能 还通过从突触小泡（其生理相关的细胞内 位置），从而导致功能丧失。通过其突触小泡结合状态，αSyn 调节 突触小泡运输和分子伴侣 SNARE 复合体组装以维持神经递质释放。 因此，从神经元中去除 αSyn 可能不是保护性的，而是有害的。该应用程序的目标是 确定突触小泡结合 αSyn 对 αSyn 功能和神经元存活的影响，使用 合理设计的 αSyn 变体可以稳定突触小泡的结合。中心假设是 稳定 αSyn 在突触小泡上的结合可降低 αSyn 毒性和病理学。强者引导 根据初步数据，该假设将在三个具体目标中进行检验：1）确定增加的效果 SNARE 复合体组装上 αSyn 的突触小泡结合； 2) 评估突触增加的效果 αSyn 的囊泡结合对突触囊泡循环的影响； 3) 测试 αSyn 的突触小泡结合是否增加 挽救体内神经毒性和病理学。在第一个目标下，SNARE 复合体的组装将被量化 体内和体外，使用细胞生物学和生化技术。在第二个目标下，αSyn 使用细胞对多聚化、突触小泡池和聚类以及突触小泡循环进行量化 生物、生物化学和生物物理技术。在第三个目标下，小鼠模型将通过以下方式生成 将慢病毒载体立体定向注射到 αSyn 敲除小鼠的黑质中，以评估 使用小鼠和小鼠的行为分析，研究αSyn突变体对αSyn诱导的毒性和病理学的影响 对注射大脑进行生化、组织学和超微结构分析。该研究预计将表明 在 αSyn 突触小泡结合稳定后，改善了 αSyn 功能并延迟了病理学。这个 该研究具有创新性，因为它 1) 测试了稳定突触小泡结合的 αSyn 的新假设 减少 αSyn 病理学，2) 创建新工具来研究 αSyn 的功能和功能障碍，3) 使用 多学科方法来检验我们的假设，从单分子和细胞系统到活体小鼠。这个 这项工作很重要，因为它将 1）阐明 αSyn 突触小泡结合对神经元的重要性 功能，2）提供对 αSyn 突触小泡结合的分子机制的新见解，3）揭示 αSyn 功能丧失对疾病发病机制的贡献，4) 对以下疾病具有转化重要性 旨在稳定突触小泡结合的 αSyn 的新治疗策略的开发。

PROJECT SUMMARY/ABSTRACT Alpha-synuclein (αSyn) pathology is linked to synucleinopathies including Parkinson's disease and Lewy body dementia, but the underlying disease mechanisms remain poorly understood. The prevalent viewpoint has emerged that aggregation of αSyn triggers neuropathology through a gain-of-toxic-function mechanism, and approaches to eliminate αSyn represent an active area of research for treatment. Yet, αSyn aggregation may also endanger neurons by removing αSyn from synaptic vesicles (its physiologically relevant intracellular location) and thereby causing loss-of-function. Through its synaptic vesicle-bound state, αSyn regulates synaptic vesicle trafficking, and chaperones SNARE-complex assembly to maintain neurotransmitter release. Thus, removing αSyn from neurons may not be protective, but detrimental. The objective in this application is to determine the impact of synaptic vesicle-binding of αSyn on αSyn function and neuron survival, using rationally designed variants of αSyn that stabilize synaptic vesicle-binding. The central hypothesis is that stabilizing binding of αSyn on synaptic vesicles reduces αSyn toxicity and pathology. Guided by strong preliminary data, this hypothesis will be tested in three specific aims: 1) Determine the effect of increased synaptic vesicle-binding of αSyn on SNARE-complex assembly; 2) Assess the effect of increased synaptic vesicle-binding of αSyn on synaptic vesicle cycling; and 3) Test if increased synaptic vesicle-binding of αSyn rescues neurotoxicity and pathology in vivo. Under the first aim, SNARE-complex assembly will be quantified in vivo and in vitro, using cell biological and biochemical techniques. Under the second aim, αSyn multimerization, synaptic vesicle pools and clustering, and synaptic vesicle cycling will be quantified, using cell biological, biochemical and biophysical techniques. Under the third aim, mouse models will be generated by stereotactic injections of lentiviral vectors into the substantia nigra of αSyn knockout mice to assess effects of mutant αSyn variants on αSyn-induced toxicity and pathology, using behavioral assays on mice and biochemical, histological and ultrastructural analyses on injected brains. The study is expected to show improved αSyn function and delayed pathology upon stabilization of synaptic vesicle-binding of αSyn. This research is innovative because it 1) tests the novel hypothesis that stabilizing synaptic vesicle-bound αSyn reduces αSyn pathology, 2) creates new tools to study function and dysfunction of αSyn, and 3) uses a multidisciplinary approach to test our hypothesis from single molecules and cellular systems to live mice. This work is significant, because it will 1) clarify the importance of synaptic vesicle-binding of αSyn for neuron function, 2) provide new insights into the molecular mechanism of synaptic vesicle-binding of αSyn, 3) uncover the contribution of loss-of-function of αSyn to disease pathogenesis, and 4) have translational importance for the development of new treatment strategies aimed at stabilizing synaptic vesicle-bound αSyn.