kcnb1loss-of-function in zebrafish causes neurodevelopmental and epileptic disorders associated with GABA dysregulation

Abstract

ABSTRACTKEY POINTSkcnb1 is expressed in distinct cell subtypes and various regions of the central nervous system in zebrafishBrain anatomy and neuronal circuits are not disrupted in thekcnb1loss-of-function zebrafish modelLoss ofkcnb1leads to altered behavior phenotype, light and sound-induced locomotor impairmentskcnb1knock-out zebrafish exhibit increased locomotor sensitivity to PTZ and elevated expression of epileptogenesis-related geneskcnb1-/-larvae show spontaneous and provoked epileptiform-like electrographic activity associated with disrupted GABA regulationObjectiveKCNB1encodes an α-subunit of the delayed-rectifier voltage-dependent potassium channel Kv2.1.De novopathogenic variants ofKCNB1have been linked to developmental and epileptic encephalopathies (DEE), diagnosed in early childhood and sharing limited treatment options. Loss-of-function (LOF) ofKCNB1with dominant negative effects has been proposed as the pathogenic mechanism in these disorders. Here, we aim to characterize a knock-out (KO) zebrafish line targetingkcnb1 (kcnb1+/-andkcnb1-/-) for investigating DEEs.MethodsThis study presents the phenotypic analysis of akcnb1knock-out zebrafish model, obtained by CRISPR/Cas9 mutagenesis. Through a combination of immunohistochemistry, behavioral assays, electrophysiological recordings, and neurotransmitter quantifications, we have characterized the expression, function, and impact of thiskcnb1LOF model at early stages of development.ResultsIn wild-type larval zebrafish, kcnb1 was found in various regions of the central nervous system and in diverse cell subtypes including neurons, oligodendrocytes and microglial cells. Bothkcnb1+/-andkcnb1-/-zebrafish displayed impaired swimming behavior and “epilepsy-like” features that persisted through embryonic and larval development, with variable severity. When exposed to the chemoconvulsant pentylenetetrazol (PTZ), both mutant models showed elevated locomotor activity. In addition, PTZ-exposedkcnb1-/-larvae exhibited higher bdnf mRNA expression and activated c-Fos positive neurons in the telencephalon. This same model presents spontaneous and provoked epileptiform-like electrographic activity associated with disrupted GABA regulation. In this KO model, neuronal circuit organization remained unaffected.SignificanceWe conclude thatkcnb1knock-out in zebrafish leads to early-onset phenotypic features reminiscent of DEEs, affecting neuronal functions and primarily inhibitory pathways in developing embryonic and larval brains. This study highlights the relevance of this model for investigating developmental neuronal signaling pathways inKCNB1-related DEEs.