颞叶癫痫早期抑制Gadd45b表达对海马齿状回病理结构形成的影响及表观遗传机制

Emerging evidences have linked early stage of temporal lobe epilepsy (TLE) to a dramatically increased neurogenesis in dentate gyrus. However, large numbers of newly generated granule cells exhibit heterogeneous integration, including ectopic location in dentate hilus, developing aberrant basal dendrites and mossy fiber sprouting, which all lead to the formation of recurrent excitatory circuits among granule cells and contribute to epileptogenesis. Therefore, inhibiting adult hippocampal neurogenesis at early stage of TLE should be potentially protective. To achieve this purpose, the choice of proper target in anti-neurogenic strategy is of great importance. Gadd45b (Growth arrest and DNAdamage-inducible protein 45 beta) is a gene related to synaptic plasticity and may be required for DNA demethylation of specific promoters and expression of corresponding genes critical for adult neurogenesis, including brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF) and Epidermal Growth Factor (EGF). Supporting by the results of a series of preliminary experiments, a great up-regulation of Gadd45b in acute epileptic hippocampus may link seizures to expression of neurotrophic factors in surrounding milieu for extrinsic modulation of hippocampal neurogenesis. We hence hypothesize that reducing the expression of Gadd45b at early stage of TLE can block the development of hyper-excitatory circuits in DG and alleviate spontaneous recurrent seizures and memory deficits during the chronic stage of disease. Labeling the newly generated cells in subgranular zone-granule cell layer (SGZ-GCL) of dentate gyrus using BrdU or RV-GFP and reducing the expression of endogenous Gadd45b in dentate gyrus by stereotactic injection of adenovirus-shRNA at early stage of pilocarpine induced TLE model in mice, the project will: 1) examine proliferation of progenitor cells in SGZ, survival, migration, neuronal fate-choice decision and recruitment of newly-generated cells by morphologic technologies to explore the effect of reducing Gadd45b at early stage of TLE on hippocampal neurogenesis; 2) explore the effects of reducing Gadd45b at early stage of TLE on development of pathologies in dentate gyrus, including ectopic location, basal dendrites and mossy fiber sprouting of newly generated neurons; 3) examine the effects of reducing Gadd45b at early stage of TLE on neural architecture of hippocampus (including cell loss and mossy fiber sprouting) and excitability of granule cells at the chronic stage of TLE by morphologic technology and whole cell patch clamp recording in brain slices respectively; 4) clarify the effects of reducing Gadd45b at early stage of TLE on spontaneous recurrent seizures and memory deficits by EEG+Video monitoring and Morris Water Maze respectively; 5) examine whether reducing Gadd45b at early stage of TLE may confer epigenetic modulation on the expression of neurogenic niche signals, i.e. BDNF, FGF and EGF by region-specific DNA methylation analysis using Bisulfite Genomic Sequence. The project will provide novel strategy and target for the future studies of treatment of epilepsy.

颞叶癫痫（TLE）早期，齿状回上调的Gadd45b可能通过甲基化调节作用促进海马神经再生，而部分新生细胞异常整合形成折返性高兴奋性神经环路。我们假设在TLE早期抑制齿状回Gadd45b的表达能有效阻遏齿状回病理环路形成并减轻慢性期癫痫发作。项目应用匹鲁卡品制作小鼠TLE模型，BrdU或携带GFP的逆转录病毒标记海马新生细胞。在TLE早期应用腺病毒-shRNA下调齿状回Gadd45b的表达，采用形态学方法研究对海马新生细胞病理性整合，包括异位迁移、形成底树突和轴突芽发的影响；脑片膜片钳全细胞记录分析对齿状回颗粒细胞兴奋性的影响；EEG+视频监测和Morris水迷宫分别研究对TLE慢性期癫痫发作和认知障碍的影响。在此基础上，采用BSP甲基化测序确证Gadd45b调节的相关基因及CpG位点，并证实上述调节与海马神经再生改变的因果关系。项目的完成将建立以干扰海马病理变化形成为目标的癫痫治疗新策略。

研究背景和科学假设：颞叶癫痫早期，齿状回颗粒细胞下层细胞增殖活动增强，而部分新生细胞形成轴突芽发等异常整合，促进了癫痫的发生和发展，成为颞叶癫痫慢性发作的重要病理因素。本项目拟证实：抑制癫痫持续状态发生后Gadd45b表达可以通过提高齿状回神经发生相关因子BDNF和FGF-2基因启动子区特定CpG位点的甲基化水平而有效压制颗粒细胞下区细胞增殖，并因此进一步遏制颞叶癫痫小鼠慢性期齿状回苔藓纤维芽发的形成、减轻癫痫的反复自发发作。.研究方案和研究内容：项目应用匹鲁卡品诱导的小鼠颞叶癫痫模型，BrdU或携带GFP的逆转录病毒标记海马齿状回新生细胞。应用腺病毒-shRNA下调癫痫持续状态后齿状回Gadd45b的表达，采用形态学方法研究对齿状回细胞增殖和新生细胞病理性整合，包括异位迁移和轴突芽发等的影响；EEG+视频监测和Morris水迷宫分别研究对慢性期癫痫发作和认知障碍的影响。在此基础上，采用甲基化定量分析方法确证Gadd45b调节的相关基因及CpG位点，并证实上述调节与海马神经发生改变的因果关系。.研究结果和结论：本项目主要证实了①小鼠癫痫持续状态可以诱导齿状回高表达Gadd45b；②抑制齿状回Gadd45b表达可以明显压制小鼠癫痫持续状态后齿状回颗粒细胞下区的细胞增殖活动。③抑制Gadd45b的表达可以明显提高癫痫持续状态后后齿状回BDNF和FGF-2基因启动子区特定CpG位点的甲基化水平并沉默基因表达；④抑制Gadd45b的表达可以明显抑制颞叶癫痫慢性期齿状回苔藓纤维芽发的形成。这些研究结果表明抑制Gadd45b的表达可以提高齿状回神经发生相关因子BDNF和FGF-2基因启动子特定CpG位点的甲基化水平并下调这些基因的表达，抑制齿状回细胞增殖，并进一步遏制颞叶癫痫小鼠齿状回苔藓纤维芽发的形成。.科学意义：研究结果将建立干阻断海马病理变化形成、改善疾病转归的新靶点和新策略。