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

• 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
• 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

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 Munc 18 -1（也称为STXBP 1）的突变导致毁灭性的婴儿癫痫性脑病， 严重的智力障碍，顽固性癫痫，共济失调和许多其他症状。Munc 18 -1对照品 神经递质在突触释放，但最近的证据表明，非突触Munc 18 -1- 依赖性过程，在一般分泌运输中的作用，也被Munc 18 -1丢失扰乱。然而 在Munc 18 -1-/-神经元中观察到的错误反应的致病分子机制仍不清楚。初步 数据显示，Munc 18 -1的突变导致高尔基体陷阱蛋白syntaxin-5（stx 5）水平降低， 含有stx 5的陷阱复合物，以及异常的高尔基体形态。这个问题的核心假设是 基于强有力的初步数据，Munc 18 -1突变导致syntaxin-1错误定位， 诱导stx 5功能丧失，导致高尔基体运输缺陷和随后的非突触 神经元功能障碍。这种分泌运输的障碍可能部分地触发了发育功能障碍 在Munc 18 -1相关综合征中可见。本提案的目的是证明stx 5的缺失如何导致 神经元非突触功能障碍这些研究的基本原理是揭示stx 5在维持 神经元功能在开发Munc 18 -1的合理治疗中具有重要意义 连锁综合征在强有力的初步数据的指导下，这一假设将在两个具体目标中得到检验： 确定突变体Munc 18 -1如何导致异常高尔基体表型，目的2）确定stx 5 Munc 18 -1-/-和突变Munc 18 -1神经元中观察到的减少影响细胞内蛋白质运输和神经元 活动在第一个目标，性质和原因，这种异常的高尔基体形态将确定使用初级 神经元首先，将在Munc 18 -1-/-和Munc 18 -1-/-中表征常驻高尔基体、ER和细胞骨架蛋白的变化。 突变的Munc 18 -1神经元。此外，将确定stx 5减少对高尔基体表型的影响， 以及syntaxin-1错误定位的影响。在第二个目标中，分泌性货物误转运的变化 由于STX 5的减少，将被确定。此外，STX 5减少对神经元功能和 检查神经突和突触形态。这项研究意义重大，因为它将决定 神经元分泌运输紊乱的机制和重要性，特别是stx 5的作用，以及 将在新治疗策略的开发中具有转化重要性。这项研究是创新的， 由于（1）其新的假设，即Munc 18 -1的非突触功能障碍促成这些综合征， 和（2）其多学科方法结合生物化学和细胞生物学方法，以深入了解 Munc 18 -1突变如何导致非突触功能障碍。