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取决于专门秘密蔬菜的调节胞吐作用。 膜融合启动释放过程，但是融合形成的孔的行为可以控制速率， 释放内容的范围和身份。实际上，融合孔可以在全囊泡倒入中之前重新密封 质膜，有可能捕获未发行的货物，形式为胞吞作用，称为“亲吻和跑”，一个 释放神经调节剂的大型核心蔬菜（LDCV）已建立的调节机制。 但是，调节融合孔行为的机制尚不清楚，其在 从突触蔬菜（SVS）中释放经典神经递质是有争议的。肌动蛋白 已经建议细胞骨架及其相关蛋白质影响孔的行为 仍然不清楚。 突触前蛋白α-核蛋白在帕金森氏病（PD）的发病机理中具有核心作用。 人遗传学表明，突触核蛋白的突变会引起疾病，蛋白质在所有人中积累 特发性疾病的患者。与其他对神经变性重要的蛋白质一样，它的正常 功能仍然未知。使用敲除小鼠和光和电子显微镜成像，我们有 发现内源突触蛋白通常调节由LDCV和SVS形成的融合孔，因此 影响释放方式。该计划的长期目标是了解合成蛋白 与其他细胞因素合作，以促进融合孔字典以及该活动中的灾难如何 有助于疾病。由于可用的方法对融合孔和囊泡塌陷的分析有限 我们开发了新的方法来图像单个蔬菜的全部胞吐作用范围。使用这些，我们 将研究突触核蛋白在大型密集的核心蔬菜和膜塌陷中的作用 突触蔬菜。具体来说，我们建议 1）表征突触核素在高分辨率的高分辨率成像中的作用 方法，包括假荧光神经递质和Alexa染料进入。 2）评估突触蛋白与肌动蛋白细胞骨架的相互作用。多个系统中的观察 在包括孔隙扩张在内的胞吞作用中实施了肌动蛋白囊肿骨骼，我们将确定突触核蛋白是否是否 通过共同或独立的机制起作用。 3）确定α-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- 末端重复序列和C末端有助于正常功能。我们将测试已建立的作用 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