Molecular mechanisms of Munc18-1 linked infantile seizure disorders and rational rescue strategies

Munc18-1的突变与三种婴儿癫痫脑病有关，但突变与这些疾病之间的机制关系尚不清楚。长期目标是通过以下方式澄清机制 哪些特定的突触功能障碍会引发神经紊乱。本应用的目的是确定Munc18-1突变是如何导致神经元缺陷的，并确定逆转这种缺陷的补救策略 赤字。Munc18-1(在酵母中也称为STXBP1，在酵母中称为Sec1，在苍蝇中称为Rop，在蠕虫中称为unc18)通过与多种效应蛋白结合来控制突触处神经递质的释放。30多个新杂合子- 已经在Munc18-1基因中发现了导致婴儿癫痫脑病Ohtahara、West和Dravet综合征的突变，但尚不清楚这些突变如何影响神经元并引发三种不同的疾病。基于强大的初步数据，中心假设是Munc18-1的突变导致 在折叠、稳定性和定位方面的缺陷，并在野生型Munc18-1中引起相同的缺陷。这种功能性Munc18-1的缺失随后会损害其效应器的功能，并触发突触功能障碍，这可能 通过稳定Munc18-1来恢复。这些研究的基本原理是理解突变是如何在 Munc18-1在婴儿癫痫脑病中触发突触功能障碍将为 在目前有限的基于症状的治疗基础上开发新的治疗方法。在强大的初步数据的指导下，这一假设将在三个具体目标上进行检验：1)确定与疾病相关的影响 Munc18-1基因突变对蛋白质稳定性的影响；2)决定Munc18-1突变体如何影响其效应器和突触功能的稳定性；3)确定稳定Munc18-1并恢复其功能的救援策略。在第一个目标下，将量化Munc18-1野生型-1的稳定性、折叠、聚集和细胞内靶向。 类型和突变体，将纯化的重组蛋白和原代神经元与生化和细胞生物学技术相结合。在第二个目标下，Munc18-1‘S效应器的稳定性、相互作用和靶向性-- 1、Doc2、Mint1、Mint2和Rab3，以及突触的完整性和功能，将使用纯化的蛋白质、原代神经元以及活体小鼠和蠕虫模型进行分析。在第三个目标下，化学和分子 伴侣将被用来修复Munc18-1以及突触结构和功能的缺陷，使用 这项研究意义重大，因为它将阐明Munc18-1连锁癫痫的分子机制，并将在新的研究开发中具有翻译重要性。 理性的治疗。这项研究具有创新性，因为它1)验证了Munc18-1基因突变通过显性-负性机制导致突触功能障碍的新假说，2)使用了以前在该研究领域从未使用过的多学科和系统的方法，3)技术创新是因为 新产生的线虫菌株，以及4)将重点从以症状为中心的观点转移到一种方法 重点是了解以Munc18-1为中心的汇聚性潜在疾病机制。

Mutations in Munc18-1 are associated with three infantile epileptic encephalopathies, but the mechanistic relationship between mutations and these diseases is unknown. The long term goal is to clarify mechanisms by which specific synaptic dysfunctions trigger neurological disorders. The objective in this application is to determine how mutations in Munc18-1 cause neuronal defects, and to identify rescue strategies to reverse such deficits. Munc18-1 (also called STXBP1, SEC1 in yeast, Rop in flies, and unc18 in worms) controls neurotransmitter release at the synapse via binding to multiple effector proteins. Over 30 heterozygous de novo mu- tations have been identified in the Munc18-1 gene that cause the infantile epileptic encephalopathies Ohtahara, West, and Dravet syndrome, but it is unknown how these mutations affect neurons and trigger three different diseases. The central hypothesis, based on strong preliminary data, is that mutations in Munc18-1 result in defects in its folding, stability and localization, and elicit same defects in wild-type Munc18-1. This loss of functional Munc18-1 subsequently impairs the function of its effectors and triggers synaptic dysfunction, which can be restored by stabilizing Munc18-1. The rationale for these studies is that understanding of how mutations in Munc18-1 trigger synaptic dysfunction in infantile epileptic encephalopathies will create opportunities for the development of novel therapies beyond the current, limited symptom-based therapy. Guided by strong preliminary data, this hypothesis will be tested in three specific aims: 1) Determine the impact of disease-relevant mutations in Munc18-1 on protein stability; 2) Determine how Munc18-1 mutants affect the stability of its effectors and synapse function; and 3) Identify rescue strategies to stabilize Munc18-1 and restore its function. Under the first aim, stability, folding, aggregation and intracellular targeting will be quantified for Munc18-1 wild- type and mutants, combining purified recombinant proteins and primary neurons with biochemical and cell biological techniques. Under the second aim, stability, interaction, and targeting of Munc18-1's effectors syntaxin- 1, Doc2, Mint1, Mint2, and rab3, as well as synapse integrity and function will be analyzed, using purified proteins, primary neurons, and in vivo mouse and worm models. Under the third aim, chemical and molecular chaperones will be employed to restore deficits in Munc18-1 and in synapse structure and function, using same paradigms as for aims 1 and 2. This research is significant, because it will clarify the molecular mechanisms underlying Munc18-1-linked epilepsies, and will have translational importance in the development of new rational treatments. This research is innovative, because it 1) tests the novel hypothesis that Munc18-1 mutations cause synaptic dysfunction via a dominant-negative mechanism, 2) uses a multidisciplinary and systematic approach that has not previously been used in this research area, 3) is technically innovative because of newly generated C. elegans strains, and 4) shifts focus from a symptom-centered perspective to an approach that focusses on understanding convergent underlying disease mechanisms that pivot on Munc18-1.