Department of Pharmacology and Therapeutics McGill University, McGill University, Montreal, Quebec, Canada.
PLoS One. 2013 Oct 2;8(10):e76299. doi: 10.1371/journal.pone.0076299. eCollection 2013.
Epilepsy is a chronic brain disorder involving recurring seizures often precipitated by an earlier neuronal insult. The mechanisms that link the transient neuronal insult to the lasting state of epilepsy are unknown. Here we tested the possible role of DNA methylation in mediating long-term induction of epileptiform activity by transient kainic acid exposure using in vitro and in vivo rodent models. We analyzed changes in the gria2 gene, which encodes for the GluA2 subunit of the ionotropic glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid receptor and is well documented to play a role in epilepsy. We show that kainic acid exposure for two hours to mouse hippocampal slices triggers methylation of a 5' regulatory region of the gria2 gene. Increase in methylation persists one week after removal of the drug, with concurrent suppression of gria2 mRNA expression levels. The degree of kainic acid-induced hypermethylation of gria2 5' region varies between individual slices and correlates with the changes in excitability induced by kainic acid. In a rat in vivo model of post kainic acid-induced epilepsy, we show similar hypermethylation of the 5' region of gria2. Inter-individual variations in gria2 methylation, correlate with the frequency and intensity of seizures among epileptic rats. Luciferase reporter assays support a regulatory role for methylation of gria2 5' region. Inhibition of DNA methylation by RG108 blocked kainic acid-induced hypermethylation of gria2 5' region in hippocampal slice cultures and bursting activity. Our results suggest that DNA methylation of such genes as gria2 mediates persistent epileptiform activity and inter-individual differences in the epileptic response to neuronal insult and that pharmacological agents that block DNA methylation inhibit epileptiform activity raising the prospect of DNA methylation inhibitors in epilepsy therapeutics.
癫痫是一种涉及反复发作的慢性脑疾病，通常由先前的神经元损伤引发。将短暂的神经元损伤与持续的癫痫状态联系起来的机制尚不清楚。在这里，我们使用体外和体内啮齿动物模型测试了 DNA 甲基化在介导短暂的海人酸暴露引起的癫痫样活动的长期诱导中的可能作用。我们分析了 gria2 基因的变化，该基因编码离子型谷氨酸、α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体的 GluA2 亚基，并且该基因在癫痫中起作用已有充分的记录。我们表明，海人酸暴露两小时会触发 gria2 基因 5'调控区的甲基化。药物去除后一周，甲基化增加持续存在，同时 gria2 mRNA 表达水平受到抑制。海人酸诱导的 gria2 5'区域的甲基化程度在个体切片之间有所不同，并与海人酸诱导的兴奋性变化相关。在海人酸诱导的癫痫大鼠体内模型中，我们显示 gria2 的 5'区域也存在类似的过度甲基化。gria2 甲基化的个体间差异与癫痫大鼠的癫痫发作频率和强度相关。荧光素酶报告基因检测支持 gria2 5'区域甲基化的调节作用。RG108 抑制 DNA 甲基化可阻断海马切片培养中海人酸诱导的 gria2 5'区域过度甲基化和爆发活性。我们的结果表明，gria2 等基因的 DNA 甲基化介导了持续的癫痫样活动和神经元损伤后癫痫反应的个体间差异，并且抑制 DNA 甲基化的药物可抑制癫痫样活动，这为癫痫治疗中的 DNA 甲基化抑制剂提供了前景。
