Abstracts

Rationale: De novo missense mutations of SCN8A cause infantile epileptic encephalopathy with multiple seizure types, intellectual disability (ID), motor deficits and elevated risk of SUDEP. More than 150 mutations have been identified. The most common pathogenic mechanism is gain-of-function with elevated channel activity (Meisler et al, Epilepsia 2016). A mouse knock-in model of the N1768D mutation recapitulates seizures and early fatality (Wagnon et al, HMG 2015). Excess firing and elevated persistent current was found in neurons of the hippocampus and medial entorhinal cortex, along with arrhythmic firing of cardiac myocytes (Lopez-Santiago et al, PNAS 2017; Frasier et al, PNAS 2016; Ottolini et al, in revision).The more severe recurrent SCN8A mutation p.Arg1872Trp (R1872W) arose de novo in 14 unrelated individuals. Loss of the positively-charged arginine residue in the cytoplasmic C-terminal domain results in impaired channel inactivation (Wagnon et al, ACTN 2016). Clinical features include early onset of refractory seizures, moderate to severe ID, and significant motor impairment ranging from hypertonia to quadriplegia. To model the motor impairment and dissect the roles of specific neurons in pathogenesis, we developed a novel mouse model with conditional expression of the R1872W mutation. Methods: R1872W is located in exon 26, the final coding exon of the Scn8a gene. The TALEN targeting construct contained 2 copies of exon 26, the floxed upstream exon encoding wildtype channel and the downstream exon encoding the R1872W mutant, to produce expression of wildtype Nav1.6 in the absence of Cre recombinase and mutant Nav1.6 in the presence of Cre.  Results: Correct integration of the 2.9 kb targeting fragment into the Scn8a gene was confirmed by Southern blot and Sanger sequencing of junction fragments. The founder transmitted the conditional allele to 50% of offspring. In the absence of Cre, mice with the conditional allele expressed only wildtype channel.To assess the effect of global expression of the R1872W mutant, the conditional mutant was crossed with the ubiquitously expressed EIIa-Cre line. Offspring inheriting the conditional allele with Cre exhibit convulsive seizures and death by 15 days of age. Deletion of the WT exon was confirmed by PCR. Expression of the mutant transcript was confirmed by RT-PCR of brain RNA. Conclusions: We have thus developed a conditional mouse model of SCN8A encephalopathy expressing a severe pathogenic mutation. Expression of the mutant channel is activated by Cre recombinase. We will activate the mutant channel with neuron-specific Cre recombinases to dissect the contributions of specific circuits to epileptogenesis and motor dysfunction. We will also determine whether adult activation of the R1872W channel is sufficient to cause seizures. Activation of the conditional allele at specific developmental time points could result in consistent timing of seizure onset, which would be a practical advantage for future drug screening.  Funding: Supported in part by NINDS R01 NS34509-19