Munc18-1 mutations disrupt syntaxin-5 stability and general secretory trafficking

Munc18-1 突变破坏 Syntaxin-5 稳定性和一般分泌物运输

基本信息

批准号：
10451793
负责人：
Debra Abramov
金额：
$ 4.63万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-03 至 2023-05-18
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10451793
关键词：
Address Affect Ataxia Binding Biochemical Biological Biological Assay Cells Child Complex Cytoskeletal Proteins DNA Sequence Alteration Data Defect Development Disease Epilepsy Functional disorder Future Genetic Golgi Apparatus Impairment Induced Mutation Intellectual functioning disability Intractable Epilepsy Knockout Mice Lead Lewy body pathology Link Methods Missense Mutation Molecular Morphology Mutation Nature Neurites Neurologic Symptoms Neuronal Dysfunction Neurons Pathogenesis Patients Phenotype Physical condensation Process Protein Isoforms Proteins Research Role SNAP receptor Seizures Shiga-Like Toxin I Specificity Symptoms Synapses Syndrome Testing Therapeutic Vesicular stomatitis Indiana virus Work alpha synuclein autism spectrum disorder base cohort effective therapy epileptic encephalopathies experimental study glycosylation infancy innovation insight interdisciplinary approach knock-down loss of function mutant neurotransmitter release novel protein transport severe intellectual disability small hairpin RNA syntaxin 1 syntaxin 5 trafficking treatment strategy

项目摘要

PROJECT SUMMARY/ABSTRACT Mutations in Munc18-1 (also called STXBP1) lead to devastating infantile epileptic encephalopathies with profound intellectual disability, intractable seizures, ataxia, and numerous other symptoms. Munc18-1 controls neurotransmitter release at the synapse, but recent evidence demonstrates that a non-synaptic Munc18-1- dependent process, a role in general secretory trafficking, is also perturbed with Munc18-1 loss. Yet, the causative molecular mechanism for the mistrafficking seen in Munc18-1-/- neurons remains unclear. Preliminary data reveal that mutations in Munc18-1 cause reduced levels of the Golgi SNARE protein syntaxin-5 (stx5) and stx5-containing SNARE complexes, as well as an abnormal Golgi morphology. The central hypothesis of this proposal, based on strong preliminary data, is Munc18-1 mutations cause syntaxin-1 mislocalization and induce loss of function of stx5, leading to trafficking defects in the Golgi and subsequent non-synaptic neuronal dysfunction. This impairment in secretory trafficking may partly trigger the developmental dysfunction seen in Munc18-1 related syndromes. The objective of this proposal to demonstrate how loss of stx5 leads to neuronal, non-synaptic dysfunction. The rationale for these studies is that revealing the role of stx5 in maintaining neuronal function will have translational importance in the development of rational treatments for Munc18-1 linked syndromes. Guided by strong preliminary data, this hypothesis will be tested in two specific aims: Aim 1) Determine how mutant Munc18-1 causes an abnormal Golgi phenotype, and Aim 2) Determine how the stx5 reduction seen in Munc18-1-/- and mutant Munc18-1 neurons affects intracellular protein trafficking and neuron activity. In the first aim, the nature and cause of this abnormal Golgi morphology will be determined using primary neurons. First, changes to resident Golgi, ER, and cytoskeletal proteins will be characterized in Munc18-1-/- and mutant Munc18-1 neurons. Additionally, the effect of stx5 reduction on the Golgi phenotype will be determined, as well as the effect of syntaxin-1 mislocalization. In the second aim, changes to secretory cargo mistrafficking due to reduction in stx5 will be determined. Furthermore, the effect of stx5 reduction on neuronal function and neurite and synapse morphology will be examined. This research is significant, because it will determine the mechanisms and importance of disturbed secretory trafficking in neurons and the role of stx5 in particular, and will have translational importance in the development of new treatment strategies. This research is innovative, because of (1) its novel hypothesis that non-synaptic dysfunction of Munc18-1 contributes to these syndromes, and (2) its multidisciplinary approach combining biochemical and cell biological approaches to gain insight into how mutations in Munc18-1 lead to non-synaptic dysfunction.

项目摘要/摘要 Munc18-1的突变（也称为STXBP1）导致毁灭性的婴儿癫痫脑病与深刻的智力残疾，顽固性癫痫发作，共济素和许多其他症状。 MUNC18-1对照神经递质在突触中释放，但最近的证据表明，非突触Munc18-1-- 依赖过程是大分泌贩运中的角色，也与Munc18-1损失受到干扰。但是，在Munc18-1 - / - 神经元中看到的失去贩运的致病分子机制尚不清楚。初步的数据表明，Munc18-1的突变导致高尔基蛋白索引素5（STX5）和含STX5的圈圈复合物以及异常的高尔基形态。中心假设提案基于强大的初步数据，是Munc18-1突变导致语法素1错误定位和诱导STX5功能的丧失，导致高尔基体的运输缺陷和随后的非突触神经元功能障碍。分泌贩运的这种损害可能部分触发发育功能障碍在Munc18-1相关综合征中可见。该提案的目的是证明STX5的损失如何导致神经元，非突触功能障碍。这些研究的理由是揭示了STX5在维持神经元功能将在Munc18-1的合理处理中具有翻译重要性链接综合征。在强大的初步数据的指导下，该假设将以两个具体的目的进行检验：AIM 1）确定突变体Munc18-1如何引起异常高尔基表型，目标2）确定STX5如何在Munc18-1 - / - 和突变体Munc18-1神经元中看到的还原会影响细胞内蛋白运输和神经元活动。在第一个目的中，这种异常高尔基形态的性质和原因将使用原发性确定神经元。首先，在munc18-1 - / - 和突变Munc18-1神经元。另外，将确定STX5降低对高尔基表型的影响以及语法-1错误定位的效果。在第二个目标中，分泌货物的变化陷入困境由于STX5的减少，将确定。此外，STX5降低对神经元功能的影响和将检查神经突和突触形态。这项研究很重要，因为它将决定神经元中分泌型贩运的机制和重要性，尤其是STX5的作用，将在制定新的治疗策略方面具有翻译重要性。这项研究是创新的，由于（1）其新的假设，即Munc18-1的非突触功能障碍有助于这些综合症，所以（2）结合生化和细胞生物学方法以深入了解的多学科方法 MUNC18-1中的突变如何导致非突触功能障碍。