γ‐Aminobutyric acid receptor alpha 1 subunit loss of function causes genetic generalized epilepsy by impairing inhibitory network neurodevelopment

Abstract

Summary Objective In humans, mutations of the γ‐aminobutyric acid receptor subunit 1 (GABRA1) cause either mild or severe generalized epilepsy. Although these epilepsy‐causing mutations have been shown to disrupt the receptor activity in vitro, their in vivo consequences on brain development and activity are not known. Here, we aim at unraveling the epileptogenesis mechanisms of GABRA1 loss of function. Methods We generated a gabra1−/− zebrafish mutant line displaying highly penetrant epileptic seizures. We sought to identify the underlying molecular mechanisms through unbiased whole transcriptomic assay of gabra1−/− larval brains. Results Interestingly, mutant fish show fully penetrant seizures at juvenile stages that accurately mimic tonic–clonic generalized seizures observed in patients. Moreover, highly penetrant seizures can be induced by light stimulation, thus providing us with the first zebrafish model in which evident epileptic seizures can be induced by nonchemical agents. Our transcriptomic assay identified misregulated genes in several pathways essential for correct brain development. More specifically, we show that the early development of the brain inhibitory network is specifically affected. Although the number of GABAergic neurons is not altered, we observed a drastic reduction in the number of inhibitory synapses and a decreased complexity of the GABAergic network. This is consistent with the disruption in expression of many genes involved in axon guidance and synapse formation. Significance Together with the role of GABA in neurodevelopment, our data identify a novel aspect of epileptogenesis, suggesting that the substratum of GABRA1‐deficiency epilepsy is a consequence of early brain neurodevelopmental defects, in particular at the level of inhibitory network wiring.

Abnormal expression of GABAA receptor sub-units and hypomotility upon loss of gabra1 in zebrafish

“…Over the course of development the expression of gabra1 became more restricted to the midbrain-hindbrain regions (Fig. 2) consistent with previously published work (Monesson-Olson et al, 2018; Samarut et al, 2018).…”

“…These data are consistent with Samarut et. al., who demonstrated that mutation of gabra1 results in hypomotility, albeit at a later stage in development that would be equivalent to a juvenile onset (Samarut et al, 2018). In contrast to Samarut et.…”

“…The gene expression changes we observe are strongly supported by previous conclusions in mice with mutations in the Gabra1 gene (Arain et al, 2015; Zhou et al, 2015). Recent work in zebrafish has demonstrated that the homozygous nonsense mutation of gabra1 , does not disrupt overall brain structure or the total number of GABAergic cells, but does influence the brain transcriptome (Samarut et al, 2018). Collectively, these data raise the possibility that other alpha sub-units may compensate for the loss of gabra1, ultimately producing unique compositions of the GABA A R. The function of these receptors is unknown.…”

Glia-neuron interactions underlie state transitions to generalized seizures

“…Using simultaneous electrical recordings, we confirmed that genetically encoded calcium indicators in combination with twophoton imaging can reliably report epileptic activity, especially on the lower frequency ranges related to preictal and ictal events. Hence, we propose that two-photon calcium imaging is complementary to commonly used high-throughput behavioral assays in zebrafish for studying mechanism underlying seizure generation (Afrikanova et al, 2013;Baraban et al, 2005;Samarut et al, 2018).…”

Defective excitatory/inhibitory synaptic balance and increased neuron apoptosis in a zebrafish model of Dravet syndrome

“…Dravet syndrome (DS) is a catastrophic childhood epilepsy with multiple comorbidities, which include severe intellectual disability, impaired social development, persistent drug-resistant seizures and high risk of sudden unexpected death in epilepsy (SUDEP) [1], [33]. In recent years, thanks to vertebrate genome conservation, full genome sequencing and readily available molecular tools and techniques, the zebrafish has become a powerful and versatile animal model for studying genetic forms of epilepsy and also to screen for novel anti-epileptic drugs [8], [10], [22]. Among epilepsy mutants, zebrafish larvae with loss of function of the scn1Lab gene, one of the two zebrafish orthologs of SCN1A, was instrumental to identify drugs alleviating seizures in refractory epilepsy [8], [10].…”