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

项目摘要/摘要 Munc18-1(也称为STXBP1)突变导致毁灭性的婴儿癫痫脑病 严重的智力残疾，顽固性癫痫，共济失调和许多其他症状。Munc18-1控制 神经递质在突触释放，但最近的证据表明，非突触Munc18-1-1- 依赖过程，在一般的秘密交易中扮演的角色，也被Munc18-1的损失所困扰。然而， Munc18-1-/-神经元出现失误的分子机制尚不清楚。初步 数据显示，Munc18-1的突变导致高尔基体圈套蛋白合成素-5(Stx5)水平降低和 含有stx5的圈套复合体，以及异常的高尔基体形态。这一点的中心假设是 基于强大的初步数据，提出Munc18-1突变是否会导致Synaxin-1错误定位和 诱导stx5功能丧失，导致高尔基体运输缺陷及随后的非突触 神经元功能障碍。这种分泌运输的损害可能部分地引发了发育障碍 见于Munc18-1相关综合征。这项提案的目标是演示stx5的丢失如何导致 神经元，非突触功能障碍。这些研究的基本原理是揭示stx5在维持 神经元功能将在Munc18-1合理治疗的发展中具有翻译重要性 连锁综合症。在强劲的初步数据的指导下，这一假设将在两个具体目标中进行检验：目标1) 确定突变Munc18-1如何导致异常高尔基表型，并目的2)确定stx5如何 Munc18-1-/-和突变型Munc18-1神经元的减少影响细胞内蛋白运输和神经元 活动。在第一个目的中，将使用PRIMAL来确定这种异常高尔基形态的性质和原因 神经元。首先，Munc18-1-/-和Munc18-1-/-将表征驻留高尔基体、ER和细胞骨架蛋白的变化 突变的Munc18-1神经元。此外，还将确定stx5减少对高尔基体表型的影响， 以及Synaxin-1错位的影响。在第二个目标中，更改秘密货物抽奖错误 由于stx5的减少将被确定。此外，stx5的减少对神经元功能和 将检查神经突起和突触的形态。这项研究意义重大，因为它将决定 神经元分泌转运紊乱的机制和重要性，特别是stx5的作用，以及 将在新的治疗策略的开发中具有翻译重要性。这项研究具有创新性， 由于(1)其新的假设，即Munc18-1的非突触功能障碍与这些综合征有关， 以及(2)结合生化和细胞生物学方法的多学科方法，以深入了解 Munc18-1基因突变如何导致非突触功能障碍。

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.