The impact of synaptic vesicle-binding of alpha-synuclein on neuron function and neuropathology

α-突触核蛋白(ASyn)的病理与包括帕金森病和路易体在内的突触核病有关 痴呆症，但潜在的疾病机制仍然知之甚少。流行的观点有 研究发现，aSyn的聚集通过毒性功能获得机制触发神经病理，并且 消除aSyn的方法是治疗研究的一个活跃领域。然而，aSyn聚合可以 还通过从突触小泡(其生理上与细胞内相关的)中移除aSyn来危害神经元 位置)，从而导致功能丧失。通过其突触小泡结合状态，aSyn调节 突触小泡运输和伴侣诱捕复合体组装以维持神经递质的释放。 因此，从神经元中去除aSyn可能不是保护作用，而是有害的。此应用程序的目标是 为了确定突触囊泡结合aSyn对aSyn功能和神经元存活的影响，使用 合理设计的aSyn变体可以稳定突触小泡结合。中心假设是 稳定突触小泡上的aSyn结合可降低aSyn的毒性和病理学。以Strong为指导 初步数据显示，这一假设将在三个具体目标上进行检验：1)确定增加的效果 突触泡结合aSyn对SNARE复合体组装的影响；2)评估突触增加的影响 突触小泡结合对突触小泡循环的影响；3)检测α突触小泡结合是否增加 在体内挽救神经毒性和病理学。在第一个目标下，圈套-复杂组装将在 体内和体外，使用细胞生物学和生化技术。在第二个目标下，α同步 多聚体、突触小泡池和聚集，以及突触小泡循环将被量化，使用细胞 生物、生化和生物物理技术。在第三个目标下，鼠标模型将通过 慢病毒载体立体定向注射到aSyn基因敲除小鼠的黑质 突变的aSyn变异体对αSyn诱导的毒性和病理的影响 注射脑的生化、组织学和超微结构分析。这项研究预计将显示 α突触小泡结合稳定后可改善aSyn功能，延缓病理改变。这 研究是创新的，因为它1)测试了稳定突触囊泡结合的α同步的新假说 减少aSyn病理，2)创造新的工具来研究αsyn的功能和功能障碍，以及3)使用 从单分子和细胞系统到活老鼠的多学科方法来检验我们的假设。这 这项工作意义重大，因为它将1)阐明aSyn的突触小泡结合对神经元的重要性 功能，2)为α突触小泡结合的分子机制提供新的见解，3)揭示 ASyn功能丧失在疾病发病机制中的贡献，以及4)在翻译上对 旨在稳定突触囊泡结合的α突触的新治疗策略的开发。

Alpha-synuclein (aSyn) 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 aSyn triggers neuropathology through a gain-of-toxic-function mechanism, and approaches to eliminate aSyn represent an active area of research for treatment. Yet, aSyn aggregation may also endanger neurons by removing aSyn from synaptic vesicles (its physiologically relevant intracellular location) and thereby causing loss-of-function. Through its synaptic vesicle-bound state, aSyn regulates synaptic vesicle trafficking, and chaperones SNARE-complex assembly to maintain neurotransmitter release. Thus, removing aSyn from neurons may not be protective, but detrimental. The objective in this application is to determine the impact of synaptic vesicle-binding of aSyn on aSyn function and neuron survival, using rationally designed variants of aSyn that stabilize synaptic vesicle-binding. The central hypothesis is that stabilizing binding of aSyn on synaptic vesicles reduces aSyn 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 aSyn on SNARE-complex assembly; 2) Assess the effect of increased synaptic vesicle-binding of aSyn 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 aSyn knockout mice to assess effects of mutant aSyn 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 aSyn 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 aSyn 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 aSyn 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 aSyn to disease pathogenesis, and 4) have translational importance for the development of new treatment strategies aimed at stabilizing synaptic vesicle-bound αSyn.