甲基化和非编码RNA调控Shanks表达在癫痫发生中的机制研究

Synaptic remodeling is an important cause of epilepsy. Substantial studies have suggested that synaptic protein Shanks play a critical role in the synaptic function maintaining and synaptic remodeling. However, its role in the epileptogenesis remains unknown. Our previous studies have showed the shanks gene expression was decreased in epilepsy animal models and the methylation degree of multiple CpG sites in shanks gene was increased in epilepsy patients. The following biological information analysis explored a group of miRNAs and lncRNAs which regulate shanks expression. In this study, we aim to figure out the effect of shank in epileptogenesis and the mechanism of how miRNAs, lncRNAs and methylation regulating shanks in the synaptic remodeling in epilepsy. The expression of shanks gene would be intervened by retrovirus transfection, and then immunoprecipitation, laser confocal and patch clamp, DNA methylation sequencing and chromatin immunoprecipitation (chip) would be applied. This project will pave a new way for developing therapeutic strategies for the treatment of epilepsy.

突触重塑是癫痫发生的重要原因之一。研究表明突触蛋白Shanks在维持突触正常功能和突触重塑中发挥关键作用，但其在癫痫发生中的作用还尚未明了。我们前期研究显示shanks基因在癫痫动物模型中表达下调，且shanks基因的多个CpG位点在癫痫患者中甲基化程度增高，结合生物信息学方法筛选到一批调控shanks表达的miRNAs和lncRNAs。本项目拟通过逆转录病毒干预shanks基因的表达，采用蛋白质共沉淀、激光共聚焦、膜片钳等技术探讨shanks调控突触重塑在癫痫发生中的作用。然后进一步应用DNA甲基化测序、染色质免疫沉淀（ChIP）等技术探讨miRNAs、lncRNAs和甲基化调控shanks表达方式，及其通过shanks参与突触重塑的机制，有望为癫痫的治疗提供新的突破口。

突触蛋白SHANKs(包括Shank1/2/3)在突触可塑性及功能方面发挥着重要作用，与多种神经精神疾病如自闭症、阿尔茨海默症等密切相关，而不同SHANKs在癫痫中扮演的角色尚未明确。本课题通过前期研究基础发现SHANK2和SHANK3在癫痫动物模型中差异显著。本课题首先分别在细胞及动物模型层面调控SHANK2表达，观察突触可塑性以及相关分子的表达变化，探索其在癫痫动物模型中的生物学作用功能；同时探究SHANK3在正常人脑组织中的表达和定位，在动物模型不同时间检测SHANK3表达及甲基化改变，同时生物信息预测调控SHANK3基因的转录因子。结果显示，SHANK2表达在癫痫大鼠海马中有显著差异，调控树突小棘可塑性及GKAP、PSD95、NMDAR的表达，并最终影响痫性放电波幅。SHANK3在大脑皮层、海马结构和杏仁核复合体等区域表达丰富；动物模型中SHANK3 CpG位点甲基化参与SHANK3 mRNA的表达调控，干预SHANK3表达可影响癫痫持续状态下的发作程度；转录组测序初步筛选出调控SHANK3的转录因子Mafb和JUN。SHANKs在突触重塑中的作用及其调控机制研究加深对癫痫疾病机制的理解，有望为癫痫的治疗提供新的突破口.