The Impact of Beta- and Gamma-synucleins on Alpha-synuclein's Synaptic Function

项目摘要/摘要 α突触核蛋白(αSyn)在突触中起重要作用，通过聚集维持神经递质的释放 突触小泡(SV)和陪伴陷阱复合体组装。α-SYN聚集是一种重要的病理机制 帕金森氏病(PD)和阿尔茨海默氏症等多种年龄驱动的神经退行性疾病的特征 疾病相关痴呆(ADRD)，如路易体痴呆。尽管βSyn和γSyn参与了 包括路易体痴呆、高谢病和帕金森病在内的联体核病，几乎一无所知 它们在大脑中的生理功能。了解它们的功能是找出它们是如何 功能障碍导致帕金森病和阿尔茨海默病相关的痴呆，包括路易体痴呆，以及如何 这些疾病是可以预防或延迟的。此处的目标是确定β同步和γ同步的效果 关于α系统的突触功能。中心假设是β同步蛋白和γ同步蛋白与α同步蛋白的相互作用导致 α同步蛋白活性降低，导致SV簇和圈套-复合体组装减少，并改变了Neu- 罗纳尔活动。这一假设将在3个具体目标中得到验证：1)评估β同步和γ同步对 SNARE-复杂装配；2)评估β同步和γ同步对SV聚类的影响；以及3)确定不同的SNARs-复杂装配。 突触核蛋白相互作用在SV循环中的应用。在目标1下，陷阱-复杂装配将在#中量化。 从缺乏β突触蛋白和/或γ突触蛋白的小鼠中选择脑区和神经元，在异源细胞中，并使用重组- NANT蛋白。在目标2下，αSYN和SV池的Synaptobrevin-2结合和多聚体将是量子的 在缺乏β同步蛋白和/或γ同步蛋白的小鼠中使用重组蛋白。在AIM 3下，SV胞吐和循环 将在海马脑片和缺乏βSYN和/或γSYN的小鼠神经元中进行定量- 以慢病毒方式提高β同步或γ同步级别。这项研究具有创新性，因为它(1)检验了新的假设-- 认为β突触和γ突触影响α突触功能，(2)采用多学科相结合的方法 生物物理、生化、电生理和全动物方法，以及(3)分析新小鼠 缺少βSYN和/或γSYN的型号是由αβγSYN三基因敲除小鼠生成的，不仅支持 对突触核蛋白的直接比较，但可以作为突触核病和阿尔茨海默病的模型 相关痴呆症，包括路易体痴呆症。我们的工作具有重要意义，因为它(1)将澄清这一问题。 β突触和γ突触对神经元功能的重要性，(2)将为分子机制提供新的见解 αSYN的潜在SV结合，(3)可能揭示βSYN和γSYN对突触核病的贡献 阿尔茨海默病相关痴呆，包括路易体痴呆，以及(4)具有翻译重要性 有针对性地为上述年龄驱动的痴呆制定新的治疗策略，旨在 瞄准所有的突触核蛋白，而不是只关注α突触蛋白。与突触核蛋白病理学和共病理学相结合- 周一到帕金森氏症和阿尔茨海默病相关的痴呆，包括路易体痴呆，我们的研究有潜力- TIAL有助于从机制上理解这三种共核蛋白在这些联核病中的作用。

PROJECT SUMMARY/ABSTRACT αSynuclein (αSyn) plays an important role at the synapse, to maintain neurotransmitter release via clustering synaptic vesicles (SV) and chaperoning SNARE-complex assembly. Aggregation of αSyn is a key pathological feature in multiple age-driven neurodegenerative diseases such as Parkinson’s disease (PD) and Alzheimer’s disease related dementias (ADRD) such as Lewy body dementia. Despite the involvement of βSyn and γSyn in synucleinopathies including Lewy body dementia, Gaucher’s disease, and PD, virtually nothing is known about their physiological functions in the brain. Understanding their function is the first step to finding out how their dysfunction causes PD and Alzheimer’s disease related dementias including Lewy body dementia, and how these diseases can be prevented or delayed. The objective here is to determine the effects of βSyn and γSyn on αSyn’s synaptic function. The central hypothesis is that interaction of βSyn and γSyn with αSyn causes a reduction in αSyn’s activity, leading to reduced SV clusters and SNARE-complex assembly, and altered neu- ronal activity. This hypothesis will be tested in 3 specific aims: 1) Assess the effect of βSyn and γSyn on SNARE-complex assembly; 2) Assess the effect of βSyn and γSyn on SV clustering; and 3) Determine the im- plications of synuclein interactions on SV cycling. Under aim 1, SNARE-complex assembly will be quantified in select brain areas and neurons from mice lacking βSyn and/or γSyn, in heterologous cells and using recombi- nant proteins. Under aim 2, synaptobrevin-2 binding and multimerization of αSyn, and SV pools will be quanti- fied in mice lacking βSyn and/or γSyn and using recombinant proteins. Under aim 3, SV exocytosis and cycling will be quantified in hippocampal brain slices and in neurons from mice lacking βSyn and/or γSyn with or with- out lentivirally increasing βSyn or γSyn levels. This research is innovative because it (1) tests the novel hypoth- esis that βSyn and γSyn affect the synaptic function of αSyn, (2) uses a multidisciplinary approach combining biophysical, biochemical, electrophysiological and whole animal approaches, and (3) analyzes new mouse models lacking βSyn and/or γSyn that were generated from αβγSyn triple knockout mice, that not only enable a direct comparison of the synucleins but may serve as models for synucleinopathies and Alzheimer’s disease related dementias including Lewy body dementia. Our work is significant because it (1) will clarify the im- portance of βSyn and γSyn for neuronal function, (2) will provide new insights into the molecular mechanism underlying SV binding of αSyn, (3) may uncover the contributions of βSyn and γSyn to synucleinopathies and Alzheimer’s disease related dementias including Lewy body dementia, and (4) has translational importance for the targeted development of new treatment strategies for the above-mentioned age-driven dementias aimed at targeting all synucleins instead of focusing solely on αSyn. With synuclein pathology and co-pathologies com- mon to PD and Alzheimer’s disease related dementias including Lewy body dementia, our study has the poten- tial to contribute a mechanistic understanding of the role of the three synucleins in these synucleinopathies.