Abstracts

Rationale: It is established that abnormal regulation of epigenetic mechanisms like DNA methylation have critical implications in the development and progression of epilepsy. The ten-eleven translocation 1 (TET1) enzyme catalyzes the oxidation of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC). While 5-hmC DNA methylation is abundant in the hippocampus, prior studies on the role of DNA methylation in the pathogenesis of TLE have primarily focused on the 5-mC form of DNA methylation. Therefore, we sought to determine the potential therapeutic role of Tet1/5-hmC in Temporal Lobe Epilepsy (TLE).

Methods: In the present study, we used male Sprague Dawley rats (125-150g), status epilepticus (SE) was induced by intraperitoneal (IP) Kainic Acid (KA) injections (10 mg/kg). The onset of SE was defined as the time from KA injection to the occurrence of prolonged seizure activity (score 5 on the Racine scale). For selective modulation of Tet1 gene expression in the dorsal hippocampus, animals were divided into two experimental groups. The first group was treated with either Tet1 siRNA or scrambled RNA treated controls. After five days of Tet1 gene silencing, SE was induced, and behavior seizures were observed. The second experimental group was treated with either a lentiviral-mediated overexpression of the catalytic domain of Tet1 or catalytically inactive Tet1 treated controls. After three weeks of Tet1 overexpression, SE was induced, and behavior seizures were observed. All animals were then perfused, and the hippocampi subdissected out. Tet1 mRNA levels were measured using quantitative-PCR. 5-mC and 5-hmC levels were assessed using mass spectrometry or enzyme-linked immunosorbent assay (ELISA).

Results: Mass spectrometry analysis revealed a decrease in 5-hmC levels, but no changes in 5-mC levels, in resected hippocampal tissue from TLE patients compared to age-matched controls (Average age: 39.6yrs), which was recapitulated in the KA rat model of TLE. Next, we found that Tet1 siRNA gene suppression in the hippocampus resulted in reduced 5-hmC levels. Furthermore, 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 was sufficient to increase 5-hmC levels and delay SE onset, suggesting that elevated Tet1 expression delayed SE onset and improved seizure intensities.

Conclusions: Together these findings indicate that targeting Tet1 has possible acute antiseizure effects. Future studies will evaluate the impact of manipulating Tet1 on chronic epileptic seizures. Additionally, using next-generation sequencing, we will identify specific seizure-promoting genes modified by TET1 DNA hydroxymethylation mechanisms. Targeting these epigenetic mechanisms hold promise for therapeutic intervention to ameliorate epileptic seizures and improve the quality of life for TLE patients.

Funding: Please list any funding that was received in support of this abstract.: NIH, NINDS.