Eukaryotic Elongation Factor 1A (eEF1A) exists in two forms in vertebrates. The first form, eEF1A1, is expressed ubiquitously throughout development but is downregulated postdevelopmentally and replaced with eEF1A2, an isoform sharing 92% amino acid identity, in neurons and muscle. The primary function of eEF1A is to recruit amino-acylated tRNAs in a GTP-dependent manner to the A site of the ribosome during protein translation, but it also has non-canonical roles in the cell, some of which are isoform dependent. The reasons for the cell-type dependent switch from eEF1A1 to eEF1A2 are poorly understood. The first aim of this project was to examine the role played by eEF1A isoforms in neuritogenesis. To do this I used RNAi to significantly reduce expression of one or other isoform in neuronal cells and measure the effects this had on neurite outgrowth. Neurite outgrowth was significantly reduced in cells depleted of eEF1A1, but not eEF1A2. The complete loss of eEF1A2 is fatal, as has been demonstrated in the wasted mouse, an eEF1A2-null model characterised by muscle wastage, neurodegeneration and death at 4 weeks of age. Mice heterozygous for the wasted mutation have normal motor function. Recent work has found that heterozygous missense mutations in eEF1A2 can cause epilepsy and intellectual disability. It is not yet known whether the seven different de novo mutations identified to date confer loss or gain of function – a crucial piece of information required before possible treatments can be sought. The second aim of this project therefore was to investigate the role of eEF1A2 in epilepsy and intellectual disability. I achieved this by using CRISPR in two ways; firstly to model one of the mutations, D252H, in vitro in a neuronal cell line, and secondly to model another of the mutations, G70S, in vivo. No mice that recapitulated the human disease condition of EEF1A2G70S/+ were obtained however, due to the error-prone nature of the non-homologous end joining repair pathway activated by CRISPR-mediated DNA cleavage, 17 of the 35 mice born were found to be homozygous nulls at the Eef1a2 locus. Nine of these had fatal audiogenic seizures caused by sudden loud noises within the animal unit. Three mice were Eef1a2G70S/- and one Eef1a2G70S/G70S but these nonetheless showed a wasted phenotype, indicating that this mutant form of eEF1A2 has compromised function, at least in terms of translation elongation. Whether it has a toxic function ca not yet be known, but the severity of the phenotype in the G70S homozygous animal could suggest a gain of function. In in vitro experiments with exogenous eEF1A2 carrying the epilepsy-causing mutation R423C, protein expression of the mutant construct in immortalised cell lines was significantly higher when cotransfected with the wildtype construct, which mirrors the condition in humans, than when transfected alone, so the mutant protein could be stabilised in the presence of wildtype eEF1A2. I used CRISPR on LUHMES cells to make a mutant neuronal cell line containing the D252H mutation in eEF1A2. Due to time restraints no phenotypic differences between the wild type line and the D252H mutation line have yet been identified, but would form the focus of a future project.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:723884 |
| Date | January 2017 |
| Creators | Davies, Faith Cathryn Joy |
| Contributors | Abbott, Catherine ; Sims, Andrew |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/23588 |
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