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

Munc 18 -1突变与三种婴儿癫痫性脑病相关，但突变与这些疾病之间的机制关系尚不清楚。长期目标是澄清机制， 哪些特定的突触功能障碍会引发神经系统疾病本申请的目的是确定Munc 18 -1突变如何引起神经元缺陷，并确定逆转这种缺陷的拯救策略。 赤字Munc 18 -1（也称为STXBP 1，酵母中的SEC 1，苍蝇中的Rop和蠕虫中的unc 18）通过与多种效应蛋白结合来控制突触处的神经递质释放。超过30个杂合子新生mu- 已经在Munc 18 -1基因中鉴定出导致婴儿癫痫性脑病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。基于强有力的初步数据，中心假设是Munc 18 -1突变导致 在其折叠、稳定性和定位方面的缺陷，并在野生型Munc 18 -1中引起相同的缺陷。这种功能性Munc 18 -1的丧失随后损害了其效应子的功能并触发突触功能障碍，这可以 通过稳定Munc 18 -1来恢复。这些研究的基本原理是，了解突变是如何在 munc 18 -1触发婴儿癫痫性脑病的突触功能障碍将为癫痫性脑病的治疗创造机会。 开发新的治疗方法，超越目前有限的基于胰岛素的治疗方法。在强有力的初步数据的指导下，这一假设将在三个具体目标中得到检验：1）确定疾病相关的影响。 Munc 18 -1突变对蛋白质稳定性的影响; 2）确定Munc 18 -1突变体如何影响其效应子和突触功能的稳定性;和3）确定稳定Munc 18 -1并恢复其功能的拯救策略。在第一个目标下，将定量Munc 18 -1野生型的稳定性、折叠、聚集和细胞内靶向。 型和突变体，结合纯化的重组蛋白和原代神经元与生物化学和细胞生物学技术。在第二个目标下，Munc 18 -1的效应子突触融合蛋白的稳定性、相互作用和靶向作用是研究的重点。 1，Doc 2，Mint 1，Mint 2和rab 3，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，以及体内小鼠和蠕虫模型进行分析。在第三个目标下，化学和分子 分子伴侣将用于恢复Munc 18 -1和突触结构和功能的缺陷， 与目标1和目标2的模式相同。这项研究意义重大，因为它将阐明Munc 18 -1连锁癫痫的分子机制，并将在开发新的治疗方法方面具有重要意义。 合理治疗。这项研究是创新的，因为它1）测试了Munc 18 -1突变通过显性负性机制导致突触功能障碍的新假设，2）使用了以前未在该研究领域使用过的多学科和系统方法，3）技术上是创新的，因为 新生成的C. elegans菌株，以及4）将焦点从以微生物为中心的观点转移到一种方法 重点是了解以Munc 18 -1为中心的会聚性潜在疾病机制。

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.