Endogenous epitope tagging of eEF1A2 in mice reveals early embryonic expression of eEF1A2 and subcellular compartmentalisation of neuronal eEF1A1 and eEF1A2.

All vertebrate species express two independently-encoded forms of translation elongation factor eEF1A. In humans and mice eEF1A1 and eEF1A2 are 92 % identical at the amino acid level, but the well conserved developmental switch between the two variants in specific tissues suggests the existence of important functional differences. Heterozygous mutations in eEF1A2 result in neurodevelopmental disorders in humans; the mechanism of pathogenicity is unclear, but one hypothesis is that there is a dominant negative effect on eEF1A1 during development. The high degree of similarity between the eEF1A proteins has complicated expression analysis in the past; here we describe a gene edited mouse line in which we have introduced a V5 tag in the gene encoding eEF1A2. Expression analysis using anti-V5 and anti-eEF1A1 antibodies demonstrates that, in contrast to the prevailing view that eEF1A2 is only expressed postnatally, it is expressed from as early as E11.5 in the developing neural tube. Two colour immunofluorescence also reveals coordinated switching between eEF1A1 and eEF1A2 in different regions of postnatal brain. Completely reciprocal expression of the two variants is seen in post-weaning mouse brain with eEF1A1 expressed in oligodendrocytes and astrocytes and eEF1A2 in neuronal soma. Although eEF1A1 is absent from neuronal cell bodies after development, it is widely expressed in axons. This expression does not appear to coincide with myelin sheaths originating from oligodendrocytes but rather results from localised translation within the axon, suggesting that both variants are transcribed in neurons but show completely distinct subcellular localisation at the protein level. These findings will form an underlying framework for understanding how missense mutations in eEF1A2 result in neurodevelopmental disorders.

Recapitulation of the EEF1A2 D252H neurodevelopmental disorder-causing missense mutation in mice reveals a toxic gain of function.

Heterozygous de novo mutations in EEF1A2, encoding the tissue-specific translation elongation factor eEF1A2, have been shown to cause neurodevelopmental disorders including often severe epilepsy and intellectual disability. The mutational profile is unusual; ~50 different missense mutations have been identified but no obvious loss of function mutations, though large heterozygous deletions are known to be compatible with life. A key question is whether the heterozygous missense mutations operate through haploinsufficiency or a gain of function mechanism, an important prerequisite for design of therapeutic strategies. In order both to address this question and to provide a novel model for neurodevelopmental disorders resulting from mutations in EEF1A2, we created a new mouse model of the D252H mutation. This mutation causes the eEF1A2 protein to be expressed at lower levels in brain but higher in muscle in the mice. We compared both heterozygous and homozygous D252H and null mutant mice using behavioural and motor phenotyping alongside molecular modelling and analysis of binding partners. Although the proteomic analysis pointed to a loss of function for the D252H mutant protein, the D252H homozygous mice were more severely affected than null homozygotes on the same genetic background. Mice that are heterozygous for the missense mutation show no behavioural abnormalities but do have sex-specific deficits in body mass and motor function. The phenotyping of our novel mouse lines, together with analysis of molecular modelling and interacting proteins, suggest that the D252H mutation results in a gain of function.

Face-valid phenotypes in a mouse model of the most common mutation in EEF1A2-related neurodevelopmental disorder.

De novo heterozygous missense mutations in EEF1A2, encoding neuromuscular translation-elongation factor eEF1A2, are associated with developmental and epileptic encephalopathies. We used CRISPR/Cas9 to recapitulate the most common mutation, E122K, in mice. Although E122K heterozygotes were not observed to have convulsive seizures, they exhibited frequent electrographic seizures and EEG abnormalities, transient early motor deficits and growth defects. Both E122K homozygotes and Eef1a2-null mice developed progressive motor abnormalities, with E122K homozygotes reaching humane endpoints by P31. The null phenotype is driven by progressive spinal neurodegeneration; however, no signs of neurodegeneration were observed in E122K homozygotes. The E122K protein was relatively stable in neurons yet highly unstable in skeletal myocytes, suggesting that the E122K/E122K phenotype is instead driven by loss of function in muscle. Nevertheless, motor abnormalities emerged far earlier in E122K homozygotes than in nulls, suggesting a toxic gain of function and/or a possible dominant-negative effect. This mouse model represents the first animal model of an EEF1A2 missense mutation with face-valid phenotypes and has provided mechanistic insights needed to inform rational treatment design.

Modelling epilepsy in the mouse: challenges and solutions.

In most mouse models of disease, the outward manifestation of a disorder can be measured easily, can be assessed with a trivial test such as hind limb clasping, or can even be observed simply by comparing the gross morphological characteristics of mutant and wild-type littermates. But what if we are trying to model a disorder with a phenotype that appears only sporadically and briefly, like epileptic seizures? The purpose of this Review is to highlight the challenges of modelling epilepsy, in which the most obvious manifestation of the disorder, seizures, occurs only intermittently, possibly very rarely and often at times when the mice are not under direct observation. Over time, researchers have developed a number of ways in which to overcome these challenges, each with their own advantages and disadvantages. In this Review, we describe the genetics of epilepsy and the ways in which genetically altered mouse models have been used. We also discuss the use of induced models in which seizures are brought about by artificial stimulation to the brain of wild-type animals, and conclude with the ways these different approaches could be used to develop a wider range of anti-seizure medications that could benefit larger patient populations.