Abstracts

This abstract has been invited to present during the Basic Science Poster Highlights poster session

Rationale: Evidence has emerged to support the role of epigenetic mechanisms in the development and progression of temporal lobe epilepsy (TLE). DNA methylation (DNAme) is an epigenetic modification involved in the up and down-regulation of gene expression depending on the hypo- or hypermethylated state of cytosines within gene regions. Prior studies focused on investigating the role of DNAme in TLE have primarily focused on the 5-methylcytosine (5-mC) form of DNAme demonstrating cite specific changes at gene regions involved in epileptogenesis. However, very little is known about the 5-hydroxymethylcytosine (5-hmC) form of DNAme catalyzed by the ten-eleven translocation (TET) enzymes in TLE. Therefore, we sought to determine the role of TET/5-hmC in TLE._x000D_
Methods: Status epilepticus (SE) or prolonged seizure activity was induced in male Sprague-Dawley rats (125-150 g) via kainic acid (KA) injections (IP, 10 mg/kg). Rats were sacrificed at 8 weeks post-SE and the hippocampus from both brain hemispheres were microdissected out and processed for 5-hmC DNA immunoprecipitation followed by sequencing (hMeDIP-seq). For Tet1 knockdown in the dorsal hippocampus, animals were treated with either Tet1 siRNA or scrambled RNA treated controls for five days followed by K SE. In another cohort of animals, Tet1 was overexpressed for three weeks, SE was induced by KA, and behavioral seizures were observed using the Racine scale. All animals were perfused and the hippocampi were subdissected out. Tet1 mRNA levels were measured using q. 5-mC and 5-hmC levels were assessed using enzyme-linked immunosorbent assay (ELISA)._x000D_
Results: Mass spectrometry analysis revealed that 5-hmC, but not 5-mC, bulk levels were significantly decreased in the hippocampal tissue from drug-resistant human TLE patients compared to age-matched controls (average age, 39.6 yrs), which was recapitulated in the KA rat model of TLE (KA-TLE). hMeDIP-seq analysis revealed DNA 5-hmC changes at gene bodies (2531 genes hypomethylated and 2455 genes hypermethylated) and at gene promoters (298 hypomethylated and 290 hypermethylated) in the epileptic hippocampus compared to controls. Next, we found that Tet1 siRNA gene suppression in the hippocampus resulted in reduced 5-hmC levels. Tet1 knockdown was associated with decreased seizure threshold on the Racine scale. These results suggest that loss of Tet1 worsened the epileptic phenotype by accelerating the onset of SE. In contrast, Tet1 overexpression in the rat hippocampus increased 5-hmC levels and delayed SE onset and improved seizure resistance._x000D_
Conclusions: Here, we found deficits in 5-hmC levels in the epileptic hippocampus indicating that targeting Tet1 has possible acute antiseizure effects. Future studies will identify specific 5-hmC associated genes in epilepsy responsive cell types within the hippocampus. Targeting these epigenetic mechanisms hold promise for therapeutic intervention to ameliorate epileptic seizures and co-morbidities like memory deficits, which is one of the most difficult and challenging clinical problems for people with epilepsy._x000D_
Funding: NIH, NINDS