THE IMPACT OF O-GLCNAC SIGNALING ON DNA HYDROXYMETHYLATION IN TEMPORAL LOBE EPILEPSY

Abstract

Temporal Lobe Epilepsy (TLE) is the most prevalent form of focal epilepsy and the most treatment-resistant type. This condition is characterized by recurrent, unprovoked seizures typically originating in the hippocampus. Epigenetic modifications such as DNAme changes have been implicated in brain functions such as synaptic plasticity, learning and, memory, and cognition. The oxidation of DNA 5-methylcytosine (5-mC) and its oxidized form, 5-hydroxymethylcytosine (5-hmC) catalyzed by Ten- Eleven Translocation (TET) family of dioxygenases and linked to epilepsy's hyperexcitable state. Post‐translational modifications (PTMs) of proteins, such as O- GlcNAcylation, facilitate cells' immediate responses to intracellular or extracellular environmental stimuli by modifying the functions of targeted proteins. Recent evidence suggests a significant interaction between O-GlcNAc transferase (OGT) and TET enzymes, affecting TET activity and chromatin structure, thus influencing gene expression. This dissertation hypothesized that decreased TET1 O-GlcNAcylation in the epileptic hippocampus contributes to pathological hyperexcitability via decreased 5-hmC levels in a TLE rat model. We found a significant reduction in 5-hmC levels in the hippocampi of both human TLE patients and kainic acid-induced TLE rats, without affecting 5-mC levels. hydroxyMethylated DNA immunoprecipitation sequencing (hMeDIP-Seq) analysis indicated a notable loss of 5-hmC in intergenic regions of the epileptic hippocampus, with identified pathways related to GABA signaling and ion transport. In-vivo manipulating hippocampal Tet1/5-hmC levels showed direct implications on seizure susceptibility and resilience. Furthermore, a co-immunoprecipitation (co-IP) assay was utilized to investigate the interactions between TET1 and OGT within the hippocampus of epileptic tissues, revealing a decrease in their interaction compared to the controls. Additionally, we further identified the presence of a TET1-OGT complex by Western blot assays, emphasizing the presence of physical interaction between these proteins. Moreover, we aimed at detecting protein O-GlcNAcylation levels through the use of sWGA assay demonstrating a significant reduction in the O-GlcNAcylation of TET1 in epileptic animals, to the point where it was nearly undetectable when compared to control animals. Finally, using Thiamet-G treatment, we were able to increase O-GlcNAcylation and global 5-hydroxymethylcytosine in our epileptic animals, restoring the levels to levels similar to the control. These results elucidate the significant role of TET1 levels and O-GlcNAcylation in epilepsy, establishing a foundational link between PTMs and epigenetic regulation in the disorder and suggesting the potential of targeting O-GlcNAcylation pathways as a novel therapeutic strategy for epilepsy.