Abstract
Pathogenic variants of the eukaryotic elongation factor 1 alpha 2 (EEF1A2) gene have previously been identified in paediatric patients with epilepsy, leading to a diagnosis of developmental and epileptic encephalopathy 33 (DEE33). These patients present with developmental delays or regression, autistic behaviours and a range of different seizure types with the most common being myoclonic seizures. These DEE33 patients also show a range of resistance to anti-epileptic drugs, with no consistently effective treatment identified. Previous studies have identified that the expression of DEE33-associated EEF1A2 gene variants in mouse cortical neurons results in decreased branching and reduced length of dendritic spines Studies using mice models have found that mice carrying pathogenic gene variants of Eef1a2 have a muscle wastage (wst) phenotype and neuron loss in the spinal cord. However, these studies lack investigation into the epileptic phenotype that is seen in DEE33 patients. The aim of this thesis was to generate an EEF1A2 loss-of-function model of DEE33 using Xenopus laevis (X. laevis) to investigate EEF1A2 and its association with the epileptic phenotype present in DEE33 patients. Single stranded guide RNAs (sgRNA) that were designed to target the Eef1a2 gene successfully generated consistent, out of frame shifts in the X. laevis Eef1a2 gene which resulted in a premature stop codon, resulting in the translation of a truncated protein. Predicted consequences to the Eef1a2 protein revealed that these truncated proteins were missing two crucial domains, domain II which is important for binding and delivering tRNAs to the ribosome during protein synthesis, and domain III which is important for actin bundling and plays a role in dendritic spine outgrowth. Automated behavioural analysis revealed that Eef1a2 CRISPant tadpoles displayed increased levels of darting behaviour and higher swimming velocities compared to unedited controls, which is consistent with previously identified seizure-like behaviours displayed by X. laevis tadpoles. Additionally, manual behavioural analysis revealed that Eef1a2 CRISPant tadpoles also displayed higher numbers of C-Shaped contractions (CSC) compared to unedited controls. CSC are observed as large bilateral body contractions and have been associated with the display of more severe seizures. Neuronal calcium signal analysis of Eef1a2 CRISPant showed that CRISPants displayed more notable signal peaks compared to unedited control tadpoles, with CRISPant signal peaks reaching higher amplitudes. Neuronal calcium signal traces of Eef1a2 CRISPant also had higher root mean squared (RMS) values compared to unedited control tadpoles. Together, this evidence indicated that Eef1a2 CRISPant tadpoles had widespread neuronal hyper-activity associated with the manifestation of electrical seizures. Blood brain barrier (BBB) permeability assays revealed that Eef1a2 CRISPant tadpoles had increased leakage of sodium fluorescein (NaF) dye from the BBB into the extracellular space outside of the brain compared to unedited control tadpoles, indicating that Eef1a2 CRISPant tadpoles had compromised integrity to the BBB associated with seizures. The findings of this thesis provide evidence that an EEF1A2 loss-of-function model of DEE33 was successfully and consistently generated using X. laevis, and that Eef1a2 loss-of-function leads to the onset of seizures.