齿状回颗粒细胞(GCs)形成轴突芽发等病理整合是颞叶癫痫(TLE)脑内高兴奋环路的重要病理基础，但其发生的细胞内信号机制远不清楚。神经元异常放电导致的基因甲基化修饰改变可能是探究该问题的一把钥匙。据此，我们择取甲基化结合蛋白MeCP2为切入点，依据预实验结果，拟求证MeCP2-miR682-PTEN-Akt-mTOR轴参与调控TLE齿状回GCs形成病理整合。项目采用小鼠TLE模型，首先敲低和过表达MeCP2、形态学方法确证MeCP2参与GCs病理整合；再应用ChIP-seq、Ago-RIP、甲基化PCR和荧光素酶报告系统等方法证实MeCP2/miR-682/PTEN分子间的靶向调控关系；继而通过调控各分子验证该轴对GCs病理整合的作用；最后应用脑片膜片钳和行为学方法阐明该轴对GCs兴奋性、癫痫发作和认知障碍的作用。项目的完成将发现MeCP2表观调控TLE齿状回GCs形成病理整合的信号机制。

Epilepsy is a prevalent neurological disorder affecting more than 50 million people worldwide. The aberrant integrations of hippocampal dentate granule cells, including the sprouting of their axons into the inner molecular layer of the dentate gyrus, their ectopic migration into the dentate hilus and development of aberrant basal dendrites, are the most consistent findings in the tissues from patients and animal models with mesial temporal lobe epilepsy (TLE). These aberrant integrations create recurrent excitatory connections in the hippocampus, thus resulting in the genesis of spontaneous recurrent seizures (SRSs). In the dentate granule cell, however, the intrinsic molecular mechanisms underlying the aberrant remodeling are yet to be determined. .One leading hypothesis for the development and progression of epilepsy is the large-scale changes in gene transcription and protein expression which contribute to the aberrant restructuring of networks. DNA methylation, one of the main mechanisms for epigenetic regulations, may exert important influences on these genetic networks. .The methyl CpG binding protein-2 (MeCP2) is a member of a family of proteins that specifically bind to methylated DNA sequences in the genome triggering the down-regulation of the target genes. To explore the epigenetic mechanisms by which the dentate granule cells develop aberrant integrations, we have identified the possible target genes of MeCP2 using CHIP-seq as well as analyzed related down-stream molecular pathways using bioinformatics databases and other molecular techniques: MeCP2-miR682-PTEN-Akt-mTOR axis may be essentially involved in the development of aberrant integrations. .To demonstrate the hypothesis, the pilocarpine-induced TLE mice models will be used and the project will: 1) examine the effect of MeCP2 on the aberrant integrations of granule cell by morphological observations; 2) verify the targeting regulation relationship between MeCP2 and miR-682 / miR-682 and PTEN by CHIP-seq, Ago-RNA binding protein immunoprecipitation (Ago-RIP), bisulfite sequencing PCR (BSP), DNA methylation analysis and double luciferase reporter assay and so on; 3) verify the role of MeCP2-miR682-PTEN-Akt-mTOR axis on the aberrant integrations of granule cells by morphological observations following up or down-regulation of expression of MeCP2, miR-682 and/or PTEN; 4) assess the potential of targeting MeCP2-miR682-PTEN-Akt-mTOR axis to reduce the excitability of granule cells and alleviate the SRSs and associated cognitive decline by using whole cell patch-clamp recording in brain slices, EEG+Video monitoring and Morris Water Maze respectively. The project will link the aberrant integrations of granule cells to intracellular epigenetic DNA modifications and will find out the underlying signaling pathway in the TLE mice.