Saudi Cultural Missions Theses & Dissertations
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Item Restricted THE IMPACT OF O-GLCNAC SIGNALING ON DNA HYDROXYMETHYLATION IN TEMPORAL LOBE EPILEPSY(the University of Alabama at Birmingham, 2024) Bahabry, Rudhab; Lubin, FarahTemporal 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.6 0Item Restricted The Role of HBP1 and Wnt signaling in Genetic and Idiopathis Epilepsy(2023-04-06) Alrubiaan, Sumaiah; Yee, AmyEpilepsy is one of the most prevalent neurological diseases which results from abnormal neuronal firing, leading to the development of spontaneous recurrent seizures. Despite the development of multiple anti-epileptic medications in the last decade, about one third of the patients eventually develop resistant to anti-epileptic drugs. One of the most important reasons behind the drug resistance is the inability to identify a clear mechanistic pathway to be targeted for treatment. Existing pharmaceutical treatments function primarily to reduce hyper-excitability thus providing only symptomatic control. Identifying alternative mechanistic targets for anti-epileptic treatment to prevent the progression of chronic epilepsy has been the main research focus recently. The Yee lab has shown that the Wnt/β-catenin pathway is aberrantly activated in both acute and chronic epilepsy, driving a metabolic reprogramming of aerobic glycolysis (Warburg-like metabolism) resulting in elevated mTOR signaling. Key to these changes was phosphorylation-dependent inhibition of Pyruvate Dehydrogenase (PDH) by the Wnt target gene PDK4. One of the known inhibitors of Wnt signaling, HMG box protein 1 (HBP1), was shown to decline in the epileptogenic period, suggesting a mechanism for Wnt activation in epileptogenesis. As a test of this mechanism, HBP1 knockout mice had elevated Wnt signaling in the hippocampus and recapitulated the metabolic reprogramming to aerobic glycolysis (Warburg-like metabolism) and elevated mTOR signaling. In-depth analysis of gene and protein expression, bioinformatics and confocal microscopy suggested additional changes. Among these, we observed a reduction in multiple GABA receptors and increased glutamate receptor expression along with altered glutamate transport. These results were consistent with a remarkably altered susceptibility to seizure induction along with occasional spontaneous seizures in HBP1-/- mice. Accompanying the seizure phenotype we observed gliosis, which is consistent with seizure and the altered signaling and metabolic alterations. Collectively, these data provide evidence that HBP1 alterations/Wnt signaling may determine the threshold of acquired seizure susceptibility. Consistent with the mouse results, several genome-wide studies suggest a disruption of Wnt/β-catenin in epilepsy patients. Moreover, multiple case reports were published highlighting deletions within the genomic region of the HBP1 and its possible correlation with neurological diseases including autism, intellectual disability and epilepsy. Human genetic sequencing data were obtained from 1600 patients and family members in the Epi4K database yielded 8 HBP1 variants and a near-significant association with Lennox-Gastault epilepsy tied to a specific variant (R375P). Mutagenesis experiments to evaluate the functional impact of these variants on HBP1 protein function demonstrated that R375P was disruptive, resulting in HBP1R375P failing to repress Wnt activity. These data suggest that loss of HBP1 function through deletion or mutation results in susceptibility to epilepsy.27 0