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

项目总结/摘要 α-突触核蛋白（αSyn）病理与突触核蛋白病（包括帕金森病和路易体）有关 痴呆症，但潜在的疾病机制仍然知之甚少。普遍的观点是， 发现αSyn的聚集通过毒性功能获得机制触发神经病理学， 消除αSyn的方法代表了治疗研究的一个活跃领域。然而，α-Syn聚集可能 还通过从突触囊泡中去除αSyn（其生理相关的细胞内 位置），从而导致功能丧失。通过突触囊泡结合状态，αSyn调节 突触囊泡运输和伴侣SNARE复合物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。本申请的目的是 为了确定αSyn的突触囊泡结合对αSyn功能和神经元存活的影响，使用 合理设计的αSyn变体，稳定突触囊泡结合。核心假设是， 稳定αSyn在突触囊泡上的结合降低了αSyn毒性和病理学。以强为导 初步数据显示，这一假设将在三个具体目标中进行检验：1）确定增加的影响 αSyn对SNARE复合物组装的突触囊泡结合; 2）评估增加突触囊泡结合的影响。 αSyn对突触囊泡循环的囊泡结合;和3）测试αSyn的突触囊泡结合是否增加 挽救体内神经毒性和病理学。在第一个目标下，SNARE复合物组装将在 体内和体外，使用细胞生物学和生物化学技术。在第二个目标下，αSyn 将使用细胞计数法定量多聚化、突触囊泡池和聚集以及突触囊泡循环。 生物学、生物化学和生物物理学技术。在第三个目标下，小鼠模型将通过 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 突变αSyn变体对α Syn诱导的毒性和病理学的影响，使用小鼠行为试验， 对注射的大脑进行生物化学、组织学和超微结构分析。这项研究预计将显示， 在稳定αSyn的突触囊泡结合后，改善αSyn功能并延迟病理学。这 这项研究之所以具有创新性，是因为它1）验证了一个新的假设，即稳定突触囊泡结合的α-Syn 减少αSyn病理学，2）创造新的工具来研究αSyn的功能和功能障碍，3）使用 多学科的方法来测试我们的假设，从单分子和细胞系统活小鼠。这 这项工作具有重要意义，因为它将1）阐明αSyn与突触囊泡结合对神经元的重要性， 功能，2）为αSyn突触囊泡结合的分子机制提供新的见解，3）揭示 αSyn功能丧失对疾病发病机制的贡献，以及4）对于 开发新的治疗策略，旨在稳定突触囊泡结合的αSyn。

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.