Role of eEF1A isoforms in neuritogenesis and epilepsy
Abstract
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.