CHARACTERIZATION OF A NOVEL ANTIEPILEPTIC THERAPY BY TARGETING THE EEF2K/EEF2 PATHWAY

eEF2K (eukaryotic Elongation Factor 2 Kinase) is an ubiquitous Ca++/Calmoduline-dependent kinase that regulates protein translation by catalyzing the phosphorylation of eEF2 (eukaryotic Elongation Factor 2) at Thr56 (Nairn and Palfrey, 1987). In neurons, eEF2K is activated by Ca++ influx mediated by glutamate stimulation (Scheetz et al., 2000; Park et al., 2008), leading to an increased expression of certain protein involved in synapse formation and plasticity (Davidkova and Carroll; 2007; Park et al., 2008; Verpelli et al., 2010), whereas the general protein translation is decreased (Browne and Proud, 2002). We recently demonstrated that eEF2K activity functionally regulates excitation/inhibition ratio in brain. In particular, eEF2K-knockout (eEF2K-/-) mice display enhanced GABAergic transmission and tonic inhibition by the upregulation of protein involved in inhibitory synapses functioning and are less susceptible to epileptic seizures (Heise et al., 2017). Accordingly to these data, we propose eEF2K/eEF2 pathway as possible target for antiepileptic therapies. In a previous work, indeed, we demonstrated that the inhibition of eEF2K was able to rescue the epileptic phenotype in a mouse model of genetic human epilepsy (Synapsin1-/- mice) (Heise et al., 2017). The aim of this project was to validate the effect of the eEF2K inhibition in models of Dravet syndrome, one of the most drug-resistant forms of epilepsy. Dravet syndrome is characterized by febrile/hyperthermia seizures at onset and later development of afebrile seizures, cognitive impairment, elevated mortality, and ataxia (Dravet et al., 2005). About 80% of patients with Dravet Syndrome carry heterozygous missense or truncating mutations in the SCN1A gene encoding the NaV1.1 sodium channel subunit (Claes et al., 2001). This subunit is mostly expressed parvalbumin-positive interneurons and loss of function mutations of NaV1.1 reduces interneurons activity (Ogiwara et al., 2007), leading to the development of a hyperexcitable neuronal network (Liautard et al., 2013). We studied the effect of eEF2K deletion in Scn1a+/- mice, through a genetic and a pharmacological approach. For the genetic approach, we generated a mouse model by crossing Scn1a+/- mice with eEF2K-/- mice. Since the enhancement of the GABAergic transmission has been demonstrated ameliorative for the most common symptoms of Dravet syndrome (epilepsy, ataxia and social behavior defects) in the mouse model of the pathology (Ogiwara et al., 2007; Han et al., 2012), we evaluated the effect of eEF2K depletion on the pathological phenotype of Scn1a+/- mice. First, we found that eEF2K deletion protected Scn1a+/- mice from the onset of epileptic seizures either under basal condition or under thermal stress, a condition known to trigger seizures in Dravet syndrome patients as well as in Scn1a+/- mice (Oakley et al., 2009). Also motor coordination defect, memory impairments and stereotyped behavior were reverted by eEF2K depletion. The analysis of spontaneous inhibitory postsynaptic currents (sIPSCs) suggested that the rescue of the pathological phenotype was driven by the potentiation of the GABAergic synapses. In addition, the analysis of eEF2 phosphorylation in samples from cerebral cortex and hippocampus of Scn1a+/- mice revealed that eEF2K/eEF2 pathway might play a role in the progression of the pathology. For the pharmacological approach, we tested two selective inhibitors of eEF2K: JAN-384 and A-484954. We discovered that JAN-384 could not pass the brain blood barrier and, thus, it cannot be used in our experiments, but A-484954 inhibited efficaciously the activity of eEF2K in mouse brain. This compound will be tested on Scn1a+/- mice in order to validate a possible pharmacological therapy in Dravet syndrome.