Alpha-Synuclein and the Synaptic Vesicle Cycle

描述（由申请人提供）：大量证据表明该蛋白质？-突触核蛋白在帕金森病（PD）发病机制中的作用。点突变？-突触核蛋白可以引起常染色体显性遗传的PD，而且？突触核蛋白在散发性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疾病中起作用。重要的是，野生型基因的剂量增加也会导致PD，表明正常蛋白质的致病作用及其正常功能。然而，角色？突触核蛋白在PD中及其正常功能仍然知之甚少。 在酵母和哺乳动物系统中的研究表明？突触核蛋白膜运输。该蛋白在体外与膜相互作用，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然存在争议，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，在神经末梢。既然PD似乎涉及到增加？-对于突触核蛋白，策略是在原代神经元培养物中过表达该蛋白，并通过活细胞的光学成像评估其对突触囊泡周期的影响。 在初步实验中，使用的光学成像，电生理学，生物化学和电子显微镜相结合，我们发现，过度表达？突触核蛋白通过减小突触囊泡再循环池的大小而损害神经递质释放，这为其在神经元中的作用提供了一些第一批明确的证据。我们现建议：1）通过研究三重突触核蛋白敲除小鼠，确定突触核蛋白对递质释放的抑制是否涉及其正常功能的获得; 2）表征？突触核蛋白对胞吐后突触囊泡蛋白和膜的分散和再聚簇的影响; 3）评估突触核蛋白、突触蛋白和突触亲蛋白在突触囊泡动员中的关系;和4）确定突触核蛋白对神经递质释放的影响是否有助于变性。 该结果将扩展之前的工作？-突触核蛋白在神经元中的适当生物学背景，并提供了一个重要的生理学框架，以了解？共核蛋白在这个过程中，他们将解决一种可能导致帕金森病中观察到的退化的机制，并提出治疗策略，以逆转由于存活神经元释放递质受损而导致的功能障碍。公共卫生相关性：目前帕金森病的治疗方法可以改善症状，而不会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？突触核蛋白是一种功能未知的蛋白质，在帕金森病的发病机制中起着重要作用。我们最近发现，突触核蛋白抑制神经递质的释放，损害突触囊泡动员，现在将表征的机制负责。除了探索防止帕金森氏症中观察到的退化的机制外，这些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.