Abstract
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.
Highlights
Epigenetic mechanisms are known to maintain long-lasting gene expression programs
kainic acid (KA) treatment of mature organotypic cultured hippocampus slices is a well-established in vitro model for inducing epileptiform activity [25]. Using this model we examined the state of DNA methylation of the proximal promoter of the gria2 gene (Figure 1A) as well as a second region upstream to the proximal promoter in hippocampal slices after 2 hours of treatment with KA compared to drug-free cultured slices using pyrosequencing (Figure 1B)
The analysis identified several transcription factor binding sites, including CCAAT/enhancer-binding protein beta (C/EBP beta) and the Glucocorticoid Receptor (GR), which was previously shown to localize in Gria2 positive cells in the hippocampus and this colocalization was affected by epilepsy [27,28]
Summary
Epigenetic mechanisms are known to maintain long-lasting gene expression programs These mechanisms involve several levels of regulation, including chemical modification of the DNA molecule by adding methyl groups at specific positions, often involving the dinucleotide sequence CpG [1]. Recent data supports the hypothesis that differential DNA methylation patterns can form in response to experiences after birth [5], can be long lasting, and can affect brain-related phenotypes in both rodents and humans [6]. It has been previously shown, that inhibition of DNA methyl transferases (DNMTs) could affect excitatory neurotransmission in the hippocampus [7,8]. These mechanisms may explain the persistence of acquired epilepsy long after the original trigger has receded and account for interindividual variations in development of epilepsy, in addition to or in the absence of genetic heterogeneity
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
