Epilepsy and neurobehavioral abnormalities in mice with aKCNB1pathogenic variant that alters conducting and non-conducting functions of K_V2.1

Abstract

Developmental and epileptic encephalopathies (DEE) are a group of severe epilepsies that usually present with intractable seizures, developmental delay and are at a higher risk for premature mortality. Numerous genes have been identified as a monogenic cause of DEE, including KCNB1. The voltage-gated potassium channel K V 2.1, encoded by KCNB1, is primarily responsible for delayed rectifier potassium currents that are important regulators of excitability in electrically excitable cells, including neurons and cardiomyocytes. The de novo pathogenic variant KCNB1-p.G379R was identified in an infant with epileptic spasms, atonic, focal and tonic-clonic seizures that were refractory to treatment with standard antiepileptic drugs. Previous work demonstrated deficits in potassium conductance, but did not assess non-conducting functions. To determine if the G379R variant affected clustering at endoplasmic reticulum-plasma membrane junctions K V 2.1-G379R was expressed in HEK293T cells. K V 2.1-G379R expression did not induce formation of endoplasmic reticulum-plasma membrane junctions, and coexpression of K V 2.1-G379R with K V 2.1-WT lowered induction of these structures relative to K V 2.1-WT alone, suggesting a dominant negative effect. To model this variant in vivo, we introduced Kcnb1 G379R into mice using CRISPR/Cas9 genome editing. We characterized neurological and neurobehavioral phenotypes of Kcnb1 G379R/+ (Kcnb1 R/+ ) and Kcnb1 G379R/G379R (Kcnb1 R/R ) mice, and screened for cardiac abnormalities. Immunohistochemistry studies on brains from Kcnb1 +/+ (WT), Kcnb1 R/+ and Kcnb1 R/R mice revealed genotype-dependent differences in the levels and subcellular localization of K V 2.1, with reduced plasma membrane expression of the K V 2.1-G379R protein, consistent with in vitro data. Kcnb1 R/+ and Kcnb1 R/R mice displayed profound hyperactivity, repetitive behaviors, impulsivity and reduced anxiety. In addition, both Kcnb1 R/+ and Kcnb1 R/R mice exhibited abnormal interictal EEG abnormalities, including isolated spike and slow waves. Spontaneous seizure events were observed in Kcnb1 R/R mice during exposure to novel environments and/or handling, while both Kcnb1 R/+ and Kcnb1 R/R mutants were more susceptible to induced seizures. Kcnb1 R/+ and Kcnb1 R/R mice exhibited prolonged rate-corrected QT interval on surface ECG recording. Overall, the Kcnb1 G379R mice recapitulate many features observed in individuals with DEE due to pathogenic variants in KCNB1. This new mouse model of KCNB1 associated DEE will be valuable for improving the understanding of the underlying pathophysiology and will provide a valuable tool for the development of therapies to treat this pharmacoresistant DEE.