Novel Gain of Function Mouse Model of KCNT1-Related Epilepsy

KCNT1 -related epilepsy is an autosomal dominant neurodevelopmental disorder resulting from de novo pathogenic variants in KCNT1 which encodes a sodium-gated potassium channel. These variants are associated with Epilepsy of Infancy with Migrating Focal Seizures (EIMFS) and Autosomal Dominant Nocturnal Frontal Lobe Epilepsy (ADNFLE). At least 64 human KCNT1 variants have been reported, the majority are gain-of-function and each has unique electrophysiological and epileptic characteristics. A multi-disciplinary collaboration generated a novel mouse model (C57BL/6- Kcnt1 em1Bryd ) that carries the G269S variant corresponding to the human G288S variant located within the coding region of the channel pore. We characterized network excitability of cultured cortical neurons from all three genotypes, since many KCNT1 mouse models require homozygosity for seizure detection. Kcnt1 +/G269S neurons exhibited sustained hyperexcitability and hypersynchronous bursting versus Kcnt1 +/+ controls, while Kcnt1 G269S/G269S neurons showed early excessive bursting followed by network collapse by 28 days-in-vitro, suggesting excitotoxicity. Therefore, behavioral comparisons were conducted between Kcnt1 +/G269S and Kcnt1 +/+ mice including developmental milestones, motor and cognitive abilities, respiratory physiology, and seizure susceptibility. Kcnt1 +/G269S displayed no abnormal gross motor abilities, poor coordination, erratic breathing, and increased apneas, as functional outcome measures. Critically, Kcnt1 +/G269S were more susceptible to thermal-induced seizures in early life. In summary, these data: (i) provide a novel mouse model of KCNT1-related epilepsy, (ii) provide strong in vitro evidence of neuronal hyperexcitability, (iii) illustrate early-life seizures as a functional outcome measure, and (iv) lay the groundwork for future analysis of neural activity in vivo and modeling circuit level dynamics in vitro , in vivo, and in silico . Gain-of-function mutations in the sodium-gated potassium channel KCNT1 have been linked to pediatric epilepsy of varying severity. The human KCNT1 variant G288S (G269S in mice) is linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy (ADNFLE), Epilepsy of Infancy with Migrating Focal Seizures (EIMFS), and other severe developmental epileptic encephalopathies. There are currently no therapeutics to prevent the progression of KCNT1 - related epilepsy, therefore, the scientific community requires a novel mouse model that is well characterized, in vitro and in vivo, to screen and assess targeted therapeutics. Herein, we engineered a novel mouse to assess developmental and adult phenotypes resulting from the G288S/G269S variant, in vitro and in vivo , to advance translation toward therapeutic testing for individuals with KCNT1 -related epilepsy.