Differential glia activation in early epileptogenesis - insights from cell-specific analysis of DNA methylation and gene expression in the contralateral hippocampus. Differential glia activation in early epileptogenesis - insights from cell-specific analysis of DNA methylation and gene expression in the contralateral hippocampus

Morphological changes in mesial Temporal Lobe Epilepsy with Hippocampal Sclerosis (mTLE-HS) are well characterized. Yet it remains elusive whether these are a consequence of seizures or originate from a hitherto unknown underlying pathology. We recently published data on changes in gene expression (GE) and DNA methylation (DNAm) in the ipsilateral hippocampus (ILH), using the intracortical kainate mouse model of mTLE-HS. In order to explore the effects of epileptic activity alone and also to further disentangle what triggers morphological alterations, we investigated glial and neuronal changes in GE and DNAm in the contralateral hippocampus (CLH). The intracortical kainic acid mouse model of mTLE-HS was used to elicit status epilepticus. Hippocampi contralateral to injection site from 8 kainate injected (KA) and 8 sham (SH) mice were extracted, and shock frozen at 24 hours post injection. Glial- and neuronal nuclei were sorted by flow cytometry. Alterations in GE and DNAm were assessed using reduced representation bisulfite sequencing (RRBS) and RNA sequencing (RNAseq). The R package edgeR was used for statistical analysis. The CLH featured substantial, mostly cell-specific changes in both GE and DNAm in glia and neurons. While changes in GE overlapped to a great degree between CLH and ILH, alterations in DNAm did not. In the CLH we found a significantly lower number of glial genes up- and downregulated compared to previous results from the ILH. Furthermore, several genes and pathways potentially involved in anti-epileptogenic effects were upregulated in the CLH. By comparing GE data from the CLH to previous results from the ILH (featuring hippocampal sclerosis), we derive potential upstream targets for epileptogenesis, including glial Cox2 and Cxcl10. Despite the absence of morphological changes, the CLH displays substantial changes in GE and DNAm. We find that GE changes related to potential anti-epileptogenic effects seem to dominate compared to pro-epileptogenic effects in the CLH and speculate whether this imbalance contributes to prevent morphological alterations like neuronal death and reactive gliosis. Overall design: DNA methylation (RRBS) and gene expression (mRNA-seq) changes in neurons and glia; Left hippocampi of mice exposed to a right sided suprahippocampal kainate injection (number of mice=8, number of samples=3) compared to left hippocampi of mice exposed to a right sided suprahippocampal NaCl injection (number of mice=8, number of samples=3) at 24 hrs past injection.

伴海马硬化的内侧颞叶癫痫（mesial Temporal Lobe Epilepsy with Hippocampal Sclerosis, mTLE-HS）的形态学改变已得到充分表征，但目前仍无法明确此类改变究竟是癫痫发作的继发结果，还是源自迄今尚未阐明的潜在病理机制。
本团队此前利用mTLE-HS的皮层内红藻氨酸小鼠模型，发表了关于同侧海马（ipsilateral hippocampus, ILH）内基因表达（gene expression, GE）与DNA甲基化（DNA methylation, DNAm）变化的相关数据。
为单独探究癫痫活动的独立影响，并进一步厘清究竟是何种因素触发了形态学改变，本研究对健侧海马（contralateral hippocampus, CLH）内胶质细胞与神经元的GE及DNAm变化进行了系统分析。本研究采用mTLE-HS的皮层内红藻氨酸小鼠模型诱导癫痫持续状态，于造模后24小时，分别提取8只红藻氨酸（kainic acid, KA）注射组小鼠与8只假手术（sham, SH）组小鼠的注射位点对侧海马组织，并进行速冻保存。通过流式细胞术分离胶质细胞核与神经元细胞核，分别采用简化亚硫酸氢盐测序（reduced representation bisulfite sequencing, RRBS）与RNA测序（RNA sequencing, RNAseq）对基因表达与DNA甲基化的改变进行检测，并使用R包edgeR完成统计学分析。
结果显示，健侧海马在胶质细胞与神经元的GE及DNAm层面均呈现出显著改变，且此类改变大多具有细胞类型特异性。尽管健侧海马与同侧海马的GE改变存在高度重叠，但二者的DNAm改变并无此共性特征。相较于此前同侧海马的研究结果，健侧海马中上调与下调的胶质细胞基因数量均显著更少。此外，健侧海马内若干潜在参与抗癫痫发生作用的基因与通路呈现上调表达。通过将健侧海马的GE数据与此前伴海马硬化的同侧海马研究结果进行比对，本研究确定了若干癫痫发生的潜在上游靶点，其中包括胶质细胞源性的Cox2与Cxcl10。
尽管健侧海马未出现形态学改变，但其GE与DNAm仍存在显著变化。本研究发现，相较于促癫痫发生的相关改变，健侧海马中与潜在抗癫痫发生作用相关的GE改变占据主导地位，并据此推测这种表达失衡可能有助于阻止神经元死亡、反应性胶质增生等形态学改变的发生。
实验设计：检测神经元与胶质细胞的DNA甲基化（RRBS）及基因表达（mRNA-seq）变化；以造模后24小时的右侧海马上区红藻氨酸注射小鼠的左侧海马（小鼠数=8，样本数=3），与同期右侧海马上区生理盐水注射小鼠的左侧海马（小鼠数=8，样本数=3）进行对比。