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

α突触核蛋白（αSyn）在突触中发挥重要作用，通过聚集突触囊泡（SV）和伴随snare复合物组装来维持神经递质释放。αSyn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（ADRD）如路易体痴呆的关键病理特征。尽管βSyn和γSyn参与了包括路易体痴呆、戈谢氏病和帕金森病在内的突触核蛋白病，但实际上对它们在大脑中的生理功能一无所知。了解它们的功能是找出它们的功能障碍如何导致PD和阿尔茨海默病相关的痴呆症，包括路易体痴呆，以及如何预防或延迟这些疾病的第一步。目的是确定βSyn和γSyn对αSyn突触功能的影响。核心假设是βSyn和γSyn与αSyn的相互作用导致αSyn活性降低，导致SV簇和snare复合物组装减少，并改变新区域活性。这一假设将在三个具体目标中得到验证：1)评估βSyn和γSyn对snare复合物组装的影响；2)评估βSyn和γSyn对SV聚类的影响；3)确定突触核蛋白相互作用对SV循环的影响。在目标1中，snare复合物的组装将在来自缺乏βSyn和/或γSyn的小鼠的特定脑区和神经元中，在异源细胞中使用重组蛋白进行量化。在目的2中，将在缺乏βSyn和/或γSyn的小鼠中使用重组蛋白量化synaptobrevin-2结合和αSyn和SV池的多聚。在目的3中，在缺乏βSyn和/或γSyn的小鼠海马脑切片和神经元中，SV的胞外分泌和循环将被量化，而βSyn或γSyn的水平则会慢病毒性地升高。本研究具有创新性，因为它(1)验证了βSyn和γSyn影响αSyn突触功能的新假设，(2)采用了生物物理、生化、电生理和全动物方法相结合的多学科方法，(3)分析了由αβγSyn三敲除小鼠产生的缺乏βSyn和/或γSyn的新小鼠模型。不仅可以直接比较突触核蛋白，而且可以作为突触核蛋白病和阿尔茨海默病相关痴呆（包括路易体痴呆）的模型。我们的工作意义重大，因为它(1)将阐明βSyn和γSyn对神经元功能的重要性，(2)将为αSyn结合SV的分子机制提供新的见解，(3)可能揭示βSyn和γSyn在突触核蛋白病和阿尔茨海默病相关痴呆（包括路易体痴呆）中的作用。(4)对于有针对性地开发针对上述年龄驱动型痴呆的新治疗策略具有重要的翻译意义，该策略旨在针对所有突触核蛋白，而不是仅仅关注αSyn。由于突触核蛋白病理以及PD和阿尔茨海默病相关痴呆（包括路易体痴呆）常见的共病理，我们的研究有可能有助于了解三种突触核蛋白在这些突触核蛋白病变中的作用机制。

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.