The impact of beta- and gamma-synucleins on alpha-synuclein's synaptic function

α突触核蛋白(αSyn)在突触中起重要作用，通过聚集突触小泡(SV)和伴随SNARE-Complex组装来维持神经递质的释放。α突触神经元的聚集是多种年龄驱动的神经退行性疾病，如帕金森病(PD)和阿尔茨海默病相关痴呆(如路易体痴呆)的关键病理特征。尽管β-syn和γ-syn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，但对它们在大脑中的生理功能几乎一无所知。了解它们的功能是找出它们的功能障碍是如何导致帕金森氏症和阿尔茨海默病相关痴呆(包括路易体痴呆)的第一步，以及如何预防或推迟这些疾病。本研究的目的是确定β突触和γ突触对α突触功能的影响。中心假说是，β突触和γ突触与α突触的相互作用导致α突触活性降低，导致SV团簇和圈套复合体组装减少，并改变了神经细胞的活动。这一假说将在3个特定的目标中得到验证：1)评估β同步蛋白和γ同步蛋白对SNARE-复合体组装的影响；2)评估β同步蛋白和γ同步蛋白对SV聚集的影响；以及3)确定突触核蛋白相互作用对SV循环的影响。根据目标1，SNARE复合体的组装将在缺乏βSYN和/或γSYN的小鼠的选定脑区和神经元中、在异源细胞中和使用重组蛋白进行量化。在目标2下，将对缺乏α同步蛋白和/或β同步蛋白并使用重组蛋白的小鼠的突触短缩蛋白-2结合和γ同步蛋白和SV池进行量化。在目标3下，海马脑片和缺乏βSYN和/或γSYN的小鼠神经元中的SV胞吐和循环将被量化，无论有或没有透镜病毒增加βSYN或γSYN水平。这项研究之所以具有创新性，是因为它(1)检验了βSYN和γSYN影响αSYN突触功能的新假设，(2)使用了结合生物物理、生化、电生理学和全动物方法的多学科方法，(3)分析了缺乏βSYN和/或γSYN的新小鼠模型，这些模型来自αβγSYN三基因敲除小鼠，这些模型不仅能够直接比较突触核蛋白，而且可以作为联核病和阿尔茨海默病相关痴呆(包括路易体痴呆)的模型。我们的工作具有重要意义，因为它(1)将澄清β同步蛋白和γ同步蛋白对神经元功能的重要性，(2)将为α同步蛋白SV结合的分子机制提供新的见解，(3)可能揭示β同步蛋白和γ同步蛋白在突触核病和阿尔茨海默病相关痴呆(包括路易体痴呆)中的贡献，以及(4)对于有针对性地开发针对上述年龄驱动的痴呆的新治疗策略具有翻译上的重要性，旨在针对所有突触核蛋白，而不是仅关注α同步蛋白。由于突触核蛋白的病理学和与帕金森病和阿尔茨海默病相关的痴呆包括路易体痴呆的共同病理，我们的研究有可能有助于从机制上理解这三种突触核蛋白在这些突触核病中的作用。

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.