Alpha-Synuclein and the Synaptic Vesicle Cycle

摘要 相当多的证据表明，蛋白质<$-突触核蛋白参与了 帕金森病（PD）。突触核蛋白的点突变可导致常染色体显性PD， 和突触核蛋白积聚在路易体和营养不良的神经突散发性PD， 这表明这种蛋白质在大多数疾病中起作用。重要的是，增加剂量 野生型基因的突变也可导致PD，表明正常蛋白质的致病作用， 也许是它的正常功能。然而，<$-突触核蛋白在PD中的作用及其正常功能 仍然知之甚少。 对酵母和哺乳动物系统的研究表明，突触核蛋白在 膜运输该蛋白在体外与膜相互作用，主要定位于 轴突终末的神经元，但其对突触囊泡胞吐和回收的影响仍然存在 有争议的和不太了解的。本建议的长远目标是阐明 突触核蛋白在神经末梢的生理相关环境中的功能。自从PD 似乎涉及到突触核蛋白的增加，策略是过度表达蛋白质 在原代神经元培养，并评估其对突触囊泡周期的影响，通过光学 活细胞的成像。 在初步实验中，结合了光学成像，电生理学， 通过生物化学和电子显微镜，我们发现过表达的突触核蛋白 通过减小突触囊泡再循环池的大小来损害神经递质的释放， 为它在神经元中的作用提供了一些第一个明确的证据。我们现建议： 1)确定突触核蛋白对递质释放的抑制是否涉及其 正常功能，通过研究三重突触核蛋白敲除小鼠; 2）表征 突触核蛋白对突触囊泡蛋白和膜分散和再聚的影响 3）探讨突触核蛋白、突触蛋白和突触亲蛋白在突触后膜中的相互关系。 囊泡动员;以及4）确定突触核蛋白对神经递质的影响 释放导致退化。 这些结果将把以前关于突触核蛋白的工作扩展到其适当的生物学背景中， 神经元，并提供了一个重要的生理框架，以了解的功能， 共核蛋白在这个过程中，他们将解决一个可能导致退化的机制， 在帕金森病中观察到，并提出了治疗策略，以逆转功能性 由于存活的神经元释放的递质受损而导致的残疾。

Abstract 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 of ¿-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.