Single-molecule study of alpha-synuclein

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。在阿尔茨海默病(AD)、帕金森氏病、痴呆症和其他神经退行性疾病中观察到的路易小体含有中到高比例的α-Syn。越来越多的证据表明，α-Syn积聚是AD的特征，神经毒性α-Syn在AD神经病理中的作用已被提出。因此，保持α-Syn的正确构象有助于AD的预防。同时，神经元上的N-乙基马来酰亚胺敏感因子连接蛋白受体(SNARs)是神经元间通讯所必需的。α-SYN已被证明通过聚集突触小泡来促进SNARE复合体的组装。这一生理功能高度依赖于与突触囊泡膜的结合。一些研究表明，保持圈套功能对于预防包括阿尔茨海默病在内的神经退行性疾病也很重要。 我们假设，在突触前末端含有溶血磷脂的膜环境保留了α-Syn的正确构象，使其能够在膜融合中发挥作用。在这里，我们提出了两个具体的目标来解释我们的假设：(1)研究α-Syn的早期瞬时寡聚作用受与突触前末端的溶质PLs和蛋白质相互作用的影响；(2)确定溶质PLs在SNARE介导膜融合中对α-Syn功能的作用。为了实现这些目标，我们将使用多种单分子分析方法，包括单颗粒纳米孔检测、单分子荧光成像和单泡融合。我们的单囊泡融合实验是揭示生理功能和评估不同α-Syn物种对突触传递毒性的理想工具。此外，磷脂和与突触传递相关的蛋白质在我们的α-Syn寡聚或聚集研究中的参与将进一步阐明。本项目的研究结果将促进我们对α-Syn在脑内正常和病理功能的理解，并为包括AD在内的以α-Syn异常积聚为特征的神经退行性疾病的治疗提供新的治疗靶点。

α-Synuclein (α-Syn) is a protein abundantly distributed in presynaptic terminals. Lewy bodies observed in Alzheimer’s disease (AD), Parkinson’s disease, dementia, and other neurodegenerative conditions contain moderate to high percentages of α-Syn. Accumulating evidence suggest that α-Syn accumulation characterizes AD, and a role for neurotoxic α-Syn species in AD neuropathology has been proposed. Therefore, keeping the right conformation of α-Syn can contribute to AD prevention. Meanwhile, neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are essential for communication between neurons. α-Syn has been shown to function in promoting the assembly of SNARE complex by clustering synaptic vesicles. This physiological function is highly dependent on binding to the synaptic vesicle membrane. Several studies have suggested that maintaining SNARE function is also important for preventing neurodegenerative diseases including AD. We hypothesize that a membrane environment containing lysophospholipids (lysoPLs) at the presynaptic terminal preserves the right conformation of α-Syn, allowing it to perform its function in membrane fusion. Here, we propose two Specific Aims to address our hypothesis: (1) Study early transient oligomerization of α-Syn influenced by interaction with lysoPLs and proteins at the presynaptic terminal; (2) Determine the roles of lysoPLs on α-Syn’s function in SNARE-mediated membrane fusion. To accomplish these aims, we will use multiple single-molecule assays, including single-particle nanopore detection, single-molecule fluorescent imaging, and single-vesicle fusion. Our single-vesicle fusion assays are ideal tools to reveal the physiological function and to assess the toxicity of various α-Syn species on synaptic transmission. In addition, the involvement of phospholipids and proteins related to synaptic transmission in our studies of α-Syn oligomerization or aggregation will be further elucidated. The results of this project will advance our understanding of the normal and pathological function of α-Syn in the brain and reveal novel therapeutic targets for the treatment of neurodegenerative diseases characterized by abnormal accumulation of α-Syn, including AD.