Developmental and epileptic encephalopathies (DEE) are a group of severe epilepsies that usually present with intractable seizures, developmental delay, and often have elevated 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. In addition to its canonical role as a voltage-gated potassium conductance, K v 2.1 also serves a highly conserved structural function organizing endoplasmic reticulum-plasma membrane junctions clustered in the soma and proximal dendrites of neurons. The de novo pathogenic variant KCNB1 -p.G379R was identified in an infant with epileptic spasms, and 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 K v 2.1 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 co-expression of K v 2.1-G379R with K v 2.1-wild-type lowered induction of these structures relative to K v 2.1-WT alone, consistent with a dominant negative effect. To model this variant in vivo, we introduced Kcnb1 G379R into mice using CRISPR/Cas9 genome editing. We characterized neuronal expression, neurological and neurobehavioral phenotypes of Kcnb1 G379R/+ ( Kcnb1 R/+ ) and Kcnb1 G379R/G379R ( Kcnb1 R/R ) mice. Immunohistochemistry studies on brains from Kcnb1 +/+ , Kcnb1 R/+ and Kcnb1 R/R mice revealed genotype-dependent differences in the expression levels of K v 2.1 protein, as well as associated K v 2.2 and AMIGO-1 proteins. Kcnb1 R/+ and Kcnb1 R/R mice displayed profound hyperactivity, repetitive behaviors, impulsivity and reduced anxiety. Spontaneous seizures were observed in Kcnb1 R/R mice, as well as seizures induced by exposure to novel environments and/ or handling. Both Kcnb1 R/+ and Kcnb1 R/R ...

Objective: Dravet syndrome is a severe developmental and epileptic encephalopathy (DEE) most often caused by de novo pathogenic variants in SCN1A.Individuals with Dravet syndrome rarely achieve seizure control and have significantly elevated risk for sudden unexplained death in epilepsy (SUDEP).Heterozygous deletion of Scn1a in mice (Scn1a +/− ) recapitulates several core phenotypes, including temperature-dependent and spontaneous seizures, SUDEP, and behavioral abnormalities. Furthermore, Scn1a +/− mice exhibit a similar clinical response to standard anticonvulsants. Cholesterol 24-hydroxlase (CH24H) is a brain-specific enzyme responsible for cholesterol catabolism. Recent research has indicated the therapeutic potential of CH24H inhibition for diseases associated with neural excitation, including seizures.Methods:In this study, the novel compound soticlestat, a CH24H inhibitor, was administered to Scn1a +/− mice to investigate its ability to improve Dravet-like phenotypes in this preclinical model. Results: Soticlestat treatment reduced seizure burden, protected against hyperthermia-induced seizures, and completely prevented SUDEP in Scn1a +/− mice. Video-electroencephalography (EEG) analysis confirmed the ability of soticlestat to reduce occurrence of electroclinical seizures.Significance: This study demonstrates that soticlestat-mediated inhibition of CH24H provides therapeutic benefit for the treatment of Dravet syndrome in mice and has the potential for treatment of DEEs.

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.

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