Abstracts

Rationale: KCNT1 encodes a K⁺-selective ion channel found in multiple organs and tissues including the brain. Mutations in KCNT1 are associated with a group of rare, infant-onset epilepsies. The most common and severe of these is Epilepsy of Infancy with Migrating Focal Seizures (EIMFS). EIMFS is characterized by seizure onset during the first months of life, focal seizures migrating from one cortical region to another, marked pharmacoresistance, severe cognitive long-term disability, and premature mortality. However, other phenotypes observed in patients with KCNT1 mutations include psychiatric disease (e.g., depression, anxiety, attention-deficit/hyperactivity disorder), irritability, gastrointestinal slowing, and motor control issues. Models of all aspects of KCNT1 epilepsy, including non-seizure phenotypes, are crucial for studies aimed at developing more effective treatments. To this end, we have engineered and investigated the neurobehavioral phenotype of a mouse, Kcnt1^A913T, bearing a mutation homologous to one of the most common KCNT1 epilepsy mutations in humans, A934T.    

Methods: Crispr/CAS9 genome editing generated the Kcnt1^A913T mutation on a C57BL/6J background (Jackson, #000064). Kcnt1 phenotype comparisons were made between WT, heterozygous and homozygous littermates. General appearance, health and locomotor activity were investigated utilizing a modified Irwin battery of assays. Elevated zero maze, three-chamber sociability task, and prepulse inhibition of the startle response (PPI) assays were performed to examine changes in anxiety-like behaviors, sociability, and sensorimotor gating, respectively. Testing was performed on animals between 7 and 10 weeks old (n≥11/sex/genotype). Spontaneous seizure activity was monitored via wireless telemetry between 10 and 12 weeks of age. Two-channel intracranial electrodes (Data Sciences International) were implanted (n≥3/sex/genotype) and mice underwent continuously EEG paired with video monitoring for 7-14 days. Data were acquired using Ponemah Physiology Platform software (DSI) and analyzed offline with NeuroScore (DSI) by a blinded observer.

Results: Kcnt1^A913T mutation was generated and subsequently back-crossed for three generations without obvious off-target events. Kcnt1^+/A913T and Kcnt1^A913T/A913T exhibited a significant decrease in latency to fall over three rotarod trial days (p< 0.0001). Additionally, Kcnt1^+/+ traveled significantly further during a one-hour locomotor assay (p< 0.05) compared to heterozygous and homozygous littermates. We detected reduced PPI in both Kcnt1^+/A913T and Kcnt1^A913T/A913T animals in a gene dose-dependent manner (p< 0.05). EEG from Kcnt1 mutants depicted epileptiform spiking, including spike-wave discharges and isolated sharp waves.