Abstracts

Rationale: De novo mutations in the sodium channel gene SCN8A cause severe epileptic encephalopathy with early seizure onset, developmental delay, cognitive impairment, movement disorders, and elevated risk of sudden death. A knock-in mouse model carrying the patient mutation p.Asn1768Asp (N1768D) recapitulated the seizures and sudden death characteristic of the disorder. The N1768D mouse also exhibited cardiac abnormalities that may contribute to sudden death after seizures. To define the contributions of specific neuronal populations and cardiac cells to the seizure and sudden death phenotypes, we developed a conditional allele of Scn8a that can be activated by CRE recombinase. We chose the recurrent mutation p.Arg1872Trp (R1872W), which has been seen in 14 individuals with particularly severe SCN8A encephalopathy to enable investigation of behavioral and movement phenotypes not present in the original N1768D mouse. Methods: We used TALENs along with a targeting vector to insert two copies of the final Scn8a exon 26 into the endogenous locus. The upstream copy is flanked by loxP sites and encodes wild type Arg1872. Upon exposure to CRE, the upstream copy is removed resulting in expression of the downstream exon containing the R1872W mutation. We crossed mice with the conditional Scn8a allele with mice expressing EIIA-Cre to generate heterozygous mice with global, constitutive expression of the R1872W mutation. We also utilized Nestin-Cre (pan-neuronal), Emx1-Cre (excitatory neurons), and Gad2-Cre (inhibitory neurons) to evaluate contributions of neuronal subpopulations to SCN8A encephalopathy. We evaluated age of seizure onset, survival, video-EEG, and neuronal firing in brain slice recordings. Experiments to investigate cardiac and motor phenotypes using Myltm1-Cre and Hb9-Cre, respectively, are in progress. Results: Global activation of R1872W with EIIA-cre resulted in seizure onset at 2 weeks of age followed by death within 24 hours, often immediately after the first seizure. Brain slice recordings revealed sustained spontaneous firing in cortex and hippocampus, indicative of extreme neuronal hyperexcitability. Pan-neuronal activation by Nestin-Cre also resulted in early seizure onset and sudden death. Activation of R1872W with the excitatory neuron-specific Emx1-Cre was sufficient to cause seizures and sudden death, with onset between 3 weeks and 2 months of age. In contrast, activation of R1872W in inhibitory neurons with Gad2-Cre did not induce seizures or neurological abnormalities measured by EEG. Conclusions: These findings provide insight into the pathogenic mechanism of gain-of-function mutations of SCN8A. This work also underscores the difference between SCN8A encephalopathy and Dravet Syndrome, which is caused by haploinsufficiency of SCN1A in inhibitory neurons. Our observations that pathogenic Scn8a mutations primarily affect excitation identifies excitatory neurons as critical targets for therapeutic intervention. Funding: NIH R01 NS34509