The role of VAMP2 in alpha-synuclein function and pathology

项目总结 α-突触核蛋白(α-Syn)是一种广泛分布于突触前终末的蛋白质。α-SYN病理与 并核病包括帕金森氏病(PD)和路易体痴呆，以及α-SYN错义突变- 运动导致早发性帕金森病。从生理学角度看，α-SYN通过突触簇参与突触传递 囊泡(SVS)和促进SNARE-Complex组装。此功能强烈依赖于与SVS的绑定。 病理上，囊泡相关膜蛋白2(VAMP2)和功能性单体α-Syn的水平 同时随着痴呆症持续时间的增加而减少，这意味着 VAMP2和正常单体α-Syn.初步数据显示，VAMP2与α-SYN相互作用，阻止α-SYN 聚合。该项目的长期目标是定义和表征稳定的关键蛋白质因子 α-SYN与SVS的结合，并检测其对神经元功能的影响及α-SYN的毒性。中央高铁- 推测VAMP2能稳定α-syn在SVs上的功能构象，从而介导囊泡聚集和 陷阱-复杂的组装，并防止病理性聚集。在强劲的初步数据指引下，这 假说将在三个具体目标中进行检验：1)确定VAMP2如何阻止α-Syn聚集和 毒性；2)测定α-syn在SNARE介导的体外融合中的功能；3)测定 VAMP2对α-Syn功能及体内毒性的影响在第一个目标下，α-syn与VAMP2的相互作用，折叠和 齐聚反应将在溶液和膜上进行分析，使用核磁共振、硫黄素T、CD、FRET和固体... 重组α-syn变体的国家纳米孔实验。在第二个目标下，机制和基奈- 分析SNARE介导的膜融合的ICS，采用单分子融合、囊泡聚集和 重组α-syn变体的核磁共振实验。在第三个目标下，本土化、功能化和集聚化 将在原代小鼠神经元上使用生化和细胞生物学分析来分析α-syn变体，在 除了野生型和杂合子VAMP2小鼠的行为和病理读数。这项研究是前- 预期随着SV结合量的增加，α-Syn的功能得到改善，毒性降低。这项研究是 具有重要意义，因为它将(1)阐明α-Syn的SV结合对于SNARE-Complex组装和 神经元功能，(2)为最近发现的α-PD突变的功能/功能障碍提供了新的见解- Syn，(3)通过改变VAMP2，对α-Syn SV结合的分子机制提供了新的见解 水平，并具有(4)翻译重要性，以有针对性地制定旨在保护 α-SYN的功能是通过提高VAMP2水平来稳定其SV绑定池。我们的研究具有创新性-- 原因它(1)使用结合生物物理、生化和细胞生物学的多学科方法-- 方法，(2)使用独特的单颗粒检测分析来研究α-syn功能和早期齐聚， (3)验证了VAMP2是α-Syn结合SV的重要稳定剂的新假说，并证明了VAMP2是Sv结合的重要稳定剂。 α-Syn的SV结合池的解离可防止其聚集和毒性。

PROJECT SUMMARY α-Synuclein (α-Syn) is a protein abundantly distributed in presynaptic terminals. α-Syn pathology is linked to synucleinopathies including Parkinson’s disease (PD) and Lewy body dementia, and α-Syn missense muta- tions cause early onset PD. Physiologically, α-Syn functions in synaptic transmission via clustering synaptic vesicles (SVs) and promoting SNARE-complex assembly. This function strongly depends on binding to SVs. Pathologically, levels of vesicle-associated membrane protein 2 (VAMP2) and functional monomeric α-Syn simultaneously decrease with increasing duration of dementia, which implies a potential functional link between VAMP2 and normal monomeric α-Syn. Preliminary data showed VAMP2 interacts with α-Syn to prevent α-Syn aggregation. The long-term goal of this project is to define and characterize critical protein factors that stabilize binding of α-Syn on SVs and test their effect on neuron function and α-Syn induced toxicity. The central hy- pothesis is that VAMP2 stabilizes α-Syn in its functional conformation on SVs to mediate vesicle clustering and SNARE-complex assembly, and to prevent pathological aggregation. Guided by strong preliminary data, this hypothesis will be tested in three specific aims: 1) Determine how VAMP2 prevents α-Syn aggregation and toxicity; 2) Determine α-Syn’s function in SNARE-mediated fusion in vitro; 3) Determine the influence of VAMP2 on α-Syn’s function and toxicity in vivo. Under the first aim, α-Syn interaction with VAMP2, folding and oligomerization will be analyzed in solution and on membranes, using NMR, ThioflavinT, CD, FRET and solid- state nanopore experiments on recombinant α-Syn variants. Under the second aim, the mechanism and kinet- ics of SNARE-mediated membrane fusion will be analyzed, using single-molecule fusion, vesicle clustering and NMR experiments on recombinant α-Syn variants. Under the third aim, localization, function, and aggregation of α-Syn variants will be analyzed, using biochemical and cell biological assays on primary mouse neurons, in addition to behavioral and pathology readouts in wild-type and heterozygous VAMP2 mice. The study is ex- pected to show improved function and reduced toxicity of α-Syn with increased SV binding. This research is significant because it will (1) clarify the importance of SV binding of α-Syn for SNARE-complex assembly and neuron function, (2) provide novel insights into the function/dysfunction of recently identified PD-mutations of α- Syn, (3) provide new insights into the molecular mechanism underlying SV-binding of α-Syn by altering VAMP2 levels, and has (4) translational importance for the targeted development of new strategies aimed at preserving α-Syn’s function via stabilizing its SV-bound pool through elevating VAMP2 levels. Our study is innovative be- cause it (1) uses a multidisciplinary approach combining biophysical, biochemical, and cell biological ap- proaches, (2) uses unique single-particle detection assays to study α-Syn function and early oligomerization, and (3) tests the novel hypothesis that VAMP2 is important stabilizers of SV-binding of α-Syn and that stabili- zation of the SV-bound pool of α-Syn prevents its aggregation and toxicity.