Munc18-1蛋白通过表观修饰机制调节神经元功能的机制研究

Munc18-1 (also known as STXBP1) is an evolutionally conserved neuronal Sec1/Munc-18 (SM) protein, which is famous in exocytosis of neurotransmitters. Munc18-1 was initially found as an interacting partner of syntaxin-1. Now, it has been known that the process of synaptic vesicle exocytosis executed by SNARE proteins is regulated by Munc18-1. Recent evidence has revealed the mutations or haploinsufficiency of Munc18-1 causing progressive encephalopathy characterized by intellectual disability, epilepsy, movement disorders and autism. These neurological diseases are also known by the eponym of Ohtahara syndrome, West syndrome and Rett syndrome. Moreover, the abnormal expression and localization of munc18-1 were also found in many neurodegenerative diseases and neuropsychiatric disorder. The mechanism of how the mutations cause neurological disorders still remains elusive, because the inhibition of common system for synaptic vesicle release could not ideally explain the complexity of symptom in MUNC18-1 mutation caused diseases, and an interesting study found that a mutant MUNC18-1 lost the binding ability for SNAREs still could support normal synaptic transmission. Therefore, malfunction of other mechanism might be involved in mutation of Munc18-1..We and other have reported the nuclear localization of Munc18-1. In addition, we noted that the common mutant gene among Rett syndrome related genes is Methyl-CpG-binding protein 2 (MeCP2), which is important in epigenetic mechanism. Our preliminary experiments showed that Munc18-1 contains functional nuclear localization signal and nuclear export sequence, nuclear localization of Munc18-1 could be regulated by neural activity. It could bind with Mecp2, and regulate its phosphorylation, and regulate the expression of neuron-restrictive silencer factor (NRSF). Therefore, we suppose, in this study, that Munc18 might take a role in epigenetic mechanism, abnormality of Munc18 would cause a change expression of neuronal genes, which is related to the many neurological diseases. In this project, we will study the mechanism of Munc18-1 in nuclear translocation, explore the relationship between Munc18 phosphorylation and it nuclear location; study how Munc18-1 takes part in epigenetic mechanism via Mecp2 and NRSF; and resolve its abnormality linked to functional defects and clinical symptom. The results of this study would uncover the new function of Munc18-1 in nuclear, and the pathological mechanism of Munc18-1 malfunctional diseases, which should be helpful for developing the treatment of Munc18-1 related diseases.

Munc18-1病理突变可导致多种低年龄发病神经系统疾病，如Ohtahara、Rett综合症等；另，其它神经精神疾病中也有该蛋白表达、分布异常。已知Munc18参与突触囊泡释放，但该功能失调并不能完全解释相关疾病症状的复杂性。根据Munc18可在细胞核分布，及Rett综合症常见的突变基因是参与表观遗传修饰的Mecp2。预实验初步显示Munc18-1含核定位和出核序列，其核内分布受神经元兴奋调控，Munc18-1可与Mecp2结合并调控其磷酸化，并下游机制中涉及表观沉默因子NRSF。项目根据前期基础，提出研究Munc18-1出入核机制，探讨Munc18-1磷酸化与其核内分布的关系；研究Munc18-1如何通过Mecp2、NRSF参与表观修饰机制，调节基因表达；解析Munc18-1功能失调导致的表观修饰机制异常与疾病的关系。研究将展示Munc18-1的新功能，并对探索相关疾病的防治提供基础。

Munc18-1（也称STXBP1）病理突变可导致多种低年龄发病的神经系统疾病。本项目提出Munc18-1可能在细胞核内发挥作用，影响表观修饰系统；Munc18性脑病可能部分涉及基因表达调控异常。.研究确定Munc18-1细胞核分布，鉴定出其至少含两个核定位序列与两个出核序列。Munc18-1病理突变型可导致截断Munc18-1增加，或改变其在细胞核内分布。神经元兴奋性刺激和突触NMDA受体兴奋可诱导Munc18-1细胞核穿梭，伴随Mecp2与NRSF(REST)修饰或表达发生改变。Munc18-1敲减影响兴奋性刺激诱导的Mecp2与NRSF改变。Munc18-1敲减导致的基因表达改变，部分与突触功能抑制有关，一些为其特异。证明Munc18-1可与相关基因的调控序列结合，包括SCN7A基因，提示Munc18可能直接参与基因调控。证明了Munc18-1可与转录因子UXT结合, 参与UXT机制。.制备的Munc18-1+/-小鼠表现复杂多样的行为学异常，包括：焦虑、过度活跃、记忆受损、抑郁、社交偏好消失、攻击性增加等。高效液相色谱检测，发现5-羟色胺（5-HT）及其代谢产物5-羟吲哚乙胺（5-HIAA）显著下降；而多巴胺等有下降趋势。明确5-HT合成关键酶Tph2减少是5-HT水平下降的原因。研究确定Tph2表达下降，与DNA甲基化增加相关。证明Tph2调控序列DNA甲基化增加，而DNA甲基化酶DNMT3B增加，及Mecp2与Tph2调控序列DNA结合可能参与了Tph2调控序列DNA甲基化机制。5-HT系统异常及其涉及的表观修饰机制是本研究的重要发现。此外，研究确定Munc18-1+/-功能不足会对神经系统造成广泛的影响，包括GABA能神经元数目变化、及髓鞘水平降低等。本研究的结果对于发掘STXBP1功能提供了新资料，对STXBP1脑病发病机制拓展了新的研究思路与用药策略。