The Function of Synuclein

突触核蛋白的功能

• 批准号: 10335272
• 负责人: ROBERT H EDWARDS
• 金额: $ 50.35万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10335272
• 关键词: Actins; Behavior; Binding; Biological Assay; Catecholamines; Cell membrane; Chromaffin Cells; Chromaffin granule; Cytoplasmic Granules; Cytoskeleton; Data; Dense Core Vesicle; Disease; Dyes; Early Intervention; Electron Microscopy; Event; Exocytosis; Human Genetics; Idiopathic Parkinson Disease; Image; Impairment; In Vitro; Individual; Inherited; Knockout Mice; Membrane; Membrane Fusion; Membrane Potentials; Methods; Monitor; Mutation; N-terminal; Nerve Degeneration; Nervous system structure; Neuromodulator; Neurons; Neurotransmitters; Parkinson Disease; Pathogenesis; Pathogenicity; Patients; Peptides; Pharmacology; Phosphorylation; Phosphorylation Site; Process; Proteins; Regulation; Resolution; Role; Running; Secretory Vesicles; Signal Transduction; Structure; Synaptic Vesicles; System; Terminal Repeat Sequences; Testing; Time; Vesicle; Work; alpha synuclein; genetic manipulation; light microscopy; millisecond; postsynaptic; presynaptic; programs; response; synuclein; tool; vesicular release

Signaling in the nervous system depends on the regulated exocytosis of specialized secretory vesicles. Membrane fusion initiates the process of release, but behavior of the pore formed by fusion can control the rate, extent and identity of what is released. Indeed, the fusion pore can reseal before full vesicle collapse into the plasma membrane, potentially trapping unreleased cargo in a form of exocytosis known as `kiss-and-run', a regulatory mechanism well-established for large dense core vesicles (LDCVs), which release neuromodulators. However, the mechanisms that regulate behavior of the fusion pore have remained unclear and its role in the release of classical neurotransmitters from synaptic vesicles (SVs) has been controversial. The actin cytoskeleton and its associated proteins have been suggested to influence pore behavior but the role has remained unclear. The presynaptic protein α-synuclein has a central role in the pathogenesis of Parkinson's disease (PD). Human genetics shows that mutations in synuclein can cause the disease and the protein accumulates in all patients with the idiopathic disorder. Like other proteins important for neurodegeneration, however, its normal function has remained unknown. Using knockout mice and imaging by light and electron microscopy, we have found that endogenous synuclein normally regulates the fusion pore formed by both LDCVs and SVs, thus influencing the mode of release. The long-term objectives of this program are to understand how synuclein cooperates with other cellular factors to promote fusion pore dilation and how a disturbance in this activity contributes to disease. Since the available methods have limited analysis of the fusion pore and vesicle collapse, we have developed new methods to image the full scope of exocytosis by individual vesicles. Using these, we will study the role of synuclein in pore dilation and membrane collapse by both large dense core vesicles and synaptic vesicles. Specifically, we propose to 1) Characterize the role of synuclein in exocytosis by imaging at high resolution with several complementary methods, including false fluorescent neurotransmitters and Alexa dye entry. 2) Assess the interaction of synuclein with the actin cytoskeleton. Observations in multiple systems have implicated the actin cystoskeleton in exocytosis including pore dilation and we will determine whether synuclein acts through a common or independent mechanism. 3) Determine how the structure of α-synuclein contributes to its role in exocytosis. We will determine how the N- terminal repeats and C-terminus contribute to normal function. We will test the role of established phosphorylation sites since they may contribute to idiopathic disease by mimicking inherited mutations.

神经系统中的信号传导取决于专门分泌囊泡的受调节的胞吐作用。 膜融合启动释放过程，但融合形成的孔的行为可以控制释放速率， 事实上，融合孔可以在完全囊泡塌陷之前重新密封。 质膜，可能以一种称为“接吻并运行”的胞吐作用捕获未释放的货物， 大型致密核心囊泡（LDCV）的成熟调节机制，可释放神经调节剂。 然而，调节融合孔行为的机制及其在融合孔中的作用仍不清楚。 突触小泡（SV）释放经典神经递质一直存在争议。 细胞骨架及其相关蛋白被认为影响孔行为，但其作用 仍不清楚。 突触前蛋白 α-突触核蛋白在帕金森病 (PD) 的发病机制中发挥着核心作用。 人类遗传学表明，突触核蛋白的突变可导致这种疾病，并且该蛋白在所有细胞中积累 然而，与其他对神经退行性疾病很重要的蛋白质一样，它是正常的。 使用基因敲除小鼠并通过光学和电子显微镜成像，我们已经了解了其功能。 发现内源性突触核蛋白通常调节 LDCV 和 SV 形成的融合孔，因此 该计划的长期目标是了解突触核蛋白如何影响释放模式。 与其他细胞因子合作促进融合孔扩张以及如何干扰该活动 由于现有的方法对融合孔和囊泡塌陷的分析有限， 我们开发了新方法来对单个囊泡的胞吐作用进行全面成像。 将研究突触核蛋白在大致密核心囊泡和膜塌陷中的孔扩张和膜塌陷中的作用 具体来说，我们建议 1) 通过几个互补的高分辨率成像来表征突触核蛋白在胞吐作用中的作用 方法，包括假荧光神经递质和 Alexa 染料输入。 2) 评估突触核蛋白与肌动蛋白细胞骨架的相互作用。 暗示肌动蛋白胞骨架参与胞吐作用，包括孔扩张，我们将确定突触核蛋白是否 通过共同或独立的机制起作用。 3) 确定 α-突触核蛋白的结构如何促进其在胞吐作用中的作用 我们将确定 N-如何发挥作用。 我们将测试末端重复和C末端对正常功能的作用。 磷酸化位点，因为它们可能通过模仿遗传突变而导致特发性疾病。