Abstracts

Rationale: Alterations in gene expression for NR2B, an NMDA receptor subunit, are associated with the development of epilepsy. However, the molecular mechanisms regulating expression of the NR2B gene in epilepsy have not been explored. Recent work has implicated epigenetics in the regulation of the NR2B gene in the adult CNS. Epigenetic mechanisms involve changes in gene activity without altering the DNA sequence. One such epigenetic mechanism involves the physical marking of DNA with a methyl group at the cytosine nucleotide within a CpG site, which is typically associated with gene silencing. Another common epigenetic mechanism involves posttranslational modification of histones including acetylation, phosphorylation, or methylation of the histone tail, which can either indicate gene silencing or activation. We hypothesized that epigenetic mechanisms contribute to abnormal regulation of the NR2B gene following seizures. Methods: In the present study, we first determined the gene expression pattern of the NR2B gene in the hippocampus of kainate-treated animals. Next, we evaluated the DNA methylation status of the NR2B promoter in animals experiencing kainate-induced status epilepticus (SE) using methylation-specific PCR and bisulfite sequencing. Finally, we assessed the effect of histone deacetylase (HDAC) inhibition on cognitive deficits associated with epilepsy using the contextual fear conditioning learning paradigm. Results: Using RT-PCR analysis, we found that NR2B mRNA levels are increased in area CA1 and dentate gyrus region of hippocampus 1 hour following kainate-induced SE (p < 0.05). No changes in NR2B mRNA levels were observed in area CA3 of hippocampus after 1 hour of SE. These results suggest altered NR2B gene expression in specific regions of the hippocampus during SE. In conjunction with NR2B gene expression changes, the DNA methylation status of the NR2B promoter was dynamically regulated in hippocampus of animals experiencing kainate-induced SE. These results suggest that in the hippocampus DNA methylation correlates to aberrant regulation of the NR2B gene during SE. Furthermore, in chronic epilepsy, we found that administration of a non-selective class I HDAC inhibitor (NaB; 1.2g/kg, IP) to kainate-treated animals for seven consecutive days prior to fear conditioning significantly improved long-term memory retention (p<0.001). These results suggest that enhancing the acetylated state of proteins through HDAC inhibition rescues at least one of the severe phenotypes associated with epilepsy; deficits in memory formation.