Single-molecule study of alpha-synuclein

α-突触核蛋白的单分子研究

• 批准号: 10019462
• 负责人: Jiajie Diao
• 金额: $ 24.08万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-30 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10019462
• 关键词: Acetylation; Address; Alzheimer disease prevention; Alzheimer' s Disease; Amino Acids; Binding; Biochemical; Biological Assay; Biophysics; Brain; Communication; Complex; Dementia; Detection; Disease; Environment; Goals; Kinetics; Label; Lewy Bodies; Lewy Body Dementia; Lewy Body Disease; Link; Lysophospholipids; Mediating; Membrane; Membrane Fusion; Methods; Missense Mutation; Molecular; Molecular Conformation; Mutation; N-terminal; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Parkinson Disease; Pathologic; Pathology; Phospholipids; Physiological; Play; Presynaptic Terminals; Property; Proteins; Public Health; Research; Role; SNAP receptor; Synaptic Transmission; Synaptic Vesicles; Testing; Therapeutic Intervention; Toxic effect; Vesicle; alpha synuclein; alpha synuclein gene; base; combat; early onset; fluorescence imaging; innovation; mutant; nanopore; nervous system disorder; neuropathology; neurotoxic; new therapeutic target; novel; novel therapeutic intervention; particle; preservation; prevent; receptor; receptor function; single molecule; solid state; therapeutic target; tool

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.

α-突触核蛋白（α-Syn）是一种大量分布在突触前末梢的蛋白质。在阿尔茨海默病（AD）、帕金森病、痴呆和其他神经退行性疾病中观察到的路易体含有中至高百分比的α-Syn。越来越多的证据表明，α-Syn蓄积是AD的特征，并提出了神经毒性α-Syn物质在AD神经病理学中的作用。因此，保持α-Syn的正确构象有助于AD的预防。同时，神经元可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）是神经元间通讯所必需的。α-Syn通过聚集突触囊泡促进SNARE复合物的组装。这种生理功能高度依赖于与突触囊泡膜的结合。一些研究表明，维持SNARE功能对于预防包括AD在内的神经退行性疾病也很重要。 我们假设突触前末端含有溶血磷脂（lysoPL）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功能。在此，我们提出了两个具体的目的来解决我们的假设：（1）研究突触前末端与lysoPL和蛋白质相互作用对α-Syn早期瞬时寡聚化的影响;（2）确定lysoPL对α-Syn在SNARE介导的膜融合中的作用。为了实现这些目标，我们将使用多个单分子测定，包括单颗粒纳米孔检测，单分子荧光成像和单囊泡融合。我们的单囊泡融合试验是揭示生理功能和评估各种α-Syn种类对突触传递的毒性的理想工具。此外，我们对α-Syn寡聚化或聚集的研究中与突触传递相关的磷脂和蛋白质的参与将得到进一步阐明。该项目的结果将促进我们对α-Syn在大脑中的正常和病理功能的理解，并揭示治疗以α-Syn异常积累为特征的神经退行性疾病（包括AD）的新治疗靶点。