The Function of Synuclein

突触核蛋白的功能

• 批准号: 10569089
• 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/10569089
• 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; Methods; Monitor; Mutation; N-terminal; Nerve Degeneration; Nervous System; Neuromodulator; Neurons; Neurotransmitters; Parkinson Disease; Pathogenesis; Pathogenicity; Patients; Peptides; 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; pharmacologic; postsynaptic; presynaptic; programs; response; seal; 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依赖于特化分泌囊泡的受调节的胞吐作用。 膜融合启动了释放过程，但融合形成的孔的行为可以控制释放速率， 释放的范围和特性。事实上，融合孔可以在完整的囊泡塌陷到膜中之前重新密封。 质膜，潜在地以被称为“吻和跑”的胞吐作用的形式捕获未释放的货物， 对于释放神经调质的大致密核心囊泡（LDCV），已经建立了良好的调节机制。 然而，调节融合孔行为的机制仍然不清楚，其在细胞凋亡中的作用也不清楚。 从突触囊泡（SV）释放经典神经递质一直是有争议的。肌动蛋白 细胞骨架及其相关蛋白质被认为影响孔隙行为，但其作用 仍然不清楚。 突触前蛋白α-synuclein在帕金森病（PD）的发病机制中起着重要作用。 人类遗传学表明，突触核蛋白的突变可以引起疾病，并且蛋白质在所有细胞中积累。 患有特发性疾病的患者。然而，像其他对神经变性很重要的蛋白质一样， 功能仍然未知。使用基因敲除小鼠和光学和电子显微镜成像，我们有 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 影响释放方式。这个项目的长期目标是了解突触核蛋白 与其他细胞因子合作，促进融合孔扩张，以及如何干扰这种活动 有助于疾病。由于现有的方法对融合孔和囊泡塌陷的分析有限， 我们已经开发了新的方法来成像单个囊泡的胞吐作用的全部范围。利用这些，我们 将研究突触核蛋白在孔扩张和膜塌陷中的作用， 突触囊泡具体而言，我们建议 1)通过几种互补的高分辨率成像来表征突触核蛋白在胞吐中的作用 方法，包括假荧光神经递质和Alexa染料进入。 2)评估突触核蛋白与肌动蛋白细胞骨架的相互作用。多个系统中的观测结果 提示肌动蛋白囊骨架参与胞吐作用，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发挥作用。 3)确定α-突触核蛋白的结构如何影响其在胞吐作用中的作用。我们将确定N- 末端重复序列和C末端有助于正常功能。我们将测试既定的作用 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。

项目成果

High-speed imaging reveals the bimodal nature of dense core vesicle exocytosis.

• DOI: 10.1073/pnas.2214897120
• 发表时间: 2023-01-03
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Zhang P;Rumschitzki D;Edwards RH
• 通讯作者: Edwards RH