Alpha-Synuclein and the Synaptic Vesicle Cycle

描述（由申请人提供）：有相当多的证据表明该蛋白质？-synuclein在帕金森病发病机制中的作用。点突变？-synuclein可引起常染色体显性PD，而？-synuclein在散发性PD的路易体和营养不良的神经突中积累，表明该蛋白在大多数形式的PD中起作用。重要的是，增加野生型基因的剂量也可以引起帕金森病，这表明正常蛋白的致病作用及其正常功能。然而，的作用？-synuclein在PD中的作用及其正常功能尚不清楚。酵母和哺乳动物系统的研究表明？-突触核蛋白参与细胞膜运输。该蛋白在体外与细胞膜相互作用，主要定位于神经元的轴突末端，但其对突触囊泡胞吐和再循环的影响仍有争议且知之甚少。本提案的长期目标是阐明？-突触核蛋白在其生理相关的背景下，在神经末端。因为PD似乎会增加？-synuclein，策略是在原代神经元培养中过表达该蛋白，并通过活细胞的光学成像评估其对突触囊泡周期的影响。在使用光学成像、电生理学、生物化学和电子显微镜相结合的初步实验中，我们发现？-synuclein通过减少突触囊泡再循环池的大小而损害神经递质释放，为其在神经元中的作用提供了一些第一个明确的证据。我们现在提出：1)通过研究三重synuclein敲除小鼠，确定synuclein对递质释放的抑制是否涉及其正常功能的增加；描述……的影响？-突触核蛋白对胞吐后突触囊泡蛋白和膜的弥散和重聚的影响；3)探讨突触核蛋白、突触蛋白和突触亲和蛋白在突触囊泡动员中的关系；4)确定突触核蛋白对神经递质释放的影响是否与变性有关。研究结果将扩展之前的工作。-synuclein在神经元中的生物学背景，并为理解-synuclein的功能提供了一个重要的生理框架。在此过程中，他们将研究帕金森病退化的机制，并提出治疗策略，以逆转因存活神经元的递质释放受损而导致的功能残疾。公共卫生相关性：目前帕金森病的治疗改善了症状，而不影响最终导致严重残疾的进行性神经变性。为了开发更有效的治疗方法，我们正在研究？突触核蛋白，一种功能未知的蛋白质，在帕金森病的发病机制中起着核心作用。我们最近发现突触核蛋白通过损害突触囊泡动员来抑制神经递质释放，现在将描述其机制。除了探索防止帕金森病中观察到的退化的机制外，实验还将提出改善剩余神经元功能的治疗策略。

DESCRIPTION (provided by applicant): Considerable evidence has implicated the protein ?-synuclein in the pathogenesis of Parkinson's disease (PD). Point mutations in ?-synuclein can cause autosomal dominant PD, and ?-synuclein accumulates in the Lewy bodies and dystrophic neurites of sporadic PD, suggesting a role for the protein in most forms of the disorder. Importantly, an increased dose of the wild type gene can also cause PD, indicating a pathogenic role for the normal protein and perhaps its normal function. However, the role of ?-synuclein in PD and its normal function remain poorly understood. Studies in yeast and mammalian systems have suggested a role for ?-synuclein in membrane trafficking. The protein interacts with membranes in vitro and localizes primarily to the axon terminal in neurons, but its effects on synaptic vesicle exocytosis and recycling remain controversial and poorly understood. The long-term objective of this proposal is to elucidate the function of ?-synuclein in its physiologically relevant context, at the nerve terminal. Since PD seems to involve an increase in ?-synuclein, the strategy has been to over-express the protein in primary neuronal culture, and assess its effects on the synaptic vesicle cycle by optical imaging of live cells. In preliminary experiments using a combination of optical imaging, electrophysiology, biochemistry and electron microscopy, we have found that over-expression ?-synuclein impairs neurotransmitter release by reducing the size of the synaptic vesicle recycling pool, providing some of the first unambiguous evidence for its role in neurons. We now propose to: 1) determine whether the inhibition of transmitter release by synuclein involves a gain in its normal function, by studying triple synuclein knock-out mice; 2) characterize the effect of ?-synuclein on dispersion and reclustering of synaptic vesicle protein and membrane after exocytosis; 3) assess the relationship between synuclein, synapsins and synphilin in synaptic vesicle mobilization; and 4) determine whether the effects of synuclein on neurotransmitter release contribute to degeneration. The results will extend previous work on ?-synuclein to its appropriate biological context in neurons, and provide a crucial physiological framework to understand the function of ?-synuclein. In the process, they will address a mechanism that may give rise to the degeneration observed in Parkinson's disease, and suggest therapeutic strategies to reverse functional disability due to the impaired release of transmitter from neurons that survive. PUBLIC HEALTH RELEVANCE: Current therapy for Parkinson's disease ameliorates symptoms without affecting the progressive neural degeneration that eventually results in severe disability. To develop more effective treatment, we are studying ?-synuclein, a protein of unknown function that has a central role in the pathogenesis of Parkinson's. We have recently found that synuclein inhibits neurotransmitter release by impairing synaptic vesicle mobilization, and will now characterize the mechanism responsible. In addition to exploring a mechanism to prevent the degeneration observed in Parkinson's, the experiments will suggest therapeutic strategies to improve the function of neurons that remain.