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Glycine receptors (GlyRs) are ligand-gated chloride channels mediating inhibitory neurotransmission in the brain stem and spinal cord. They function as pentamers composed of alpha and beta subunits for which 5 genes have been identified in human (GLRA1, GLRA2, GLRA3, GLRA4, GLRB). Several in vitro studies showed that the pentameric subtype composition as well as its stoichiometry influence the distribution and the molecular function of the receptor. Moreover, mutations in some of these genes are involved in different human conditions ranging from tinnitus to epilepsy and hyperekplexia, suggesting distinct functions of the different subunits. Although the beta subunit is essential for synaptic clustering of the receptor, the specific role of each alpha subtype is still puzzling in vivo. The zebrafish genome encodes for five glycine receptor alpha subunits (glra1, glra2, glra3, glra4a, glra4b) thus offering a model of choice to investigate the respective role of each subtype on general motor behaviour. After establishing a phylogeny of GlyR subunit evolution between human and zebrafish, we checked the temporal expression pattern of these transcripts during embryo development. Interestingly, we found that glra1 is the only maternally transmitted alpha subunit. We also showed that the expression of the different GlyR subunits starts at different time points during development. Lastly, in order to decipher the role of each alpha subunit on the general motor behaviour of the fish, we knocked out individually each alpha subunit by CRISPR/Cas9-targeted mutagenesis. Surprisingly, we found that knocking out any of the alpha2, 3, a4a or a4b subunit did not lead to any obvious developmental or motor phenotype. However, glra1-/- (hitch) embryos depicted a strong motor dysfunction from 3 days, making them incapable to swim and thus leading to their premature death. Our results infer a strong functional redundancy between alpha subunits and confirm the central role played by glra1 for proper inhibitory neurotransmission controlling locomotion. The genetic tools we developed here will be of general interest for further studies aiming at dissecting the role of GlyRs in glycinergic transmission in vivo and the hitch mutant (hic) is of specific relevance as a new model of hyperekplexia., Competing Interests: ES and PD are co-founders of DanioDesign Inc. ES is a co-founder of Modelis Inc. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The commercial affiliations did not play any role in this study; in particular they did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Glycine encephalopathy (GE), or nonketotic hyperglycinemia (NKH), is a rare recessive genetic disease caused by defective glycine cleavage and characterized by increased accumulation of glycine in all tissues. Here, based on new case reports of GLDC loss-of-function mutations in GE patients, we aimed to generate a zebrafish model of severe GE in order to unravel the molecular mechanism of the disease. Using CRISPR/Cas9, we knocked out the gldc gene and showed that gldc-/- fish recapitulate GE on a molecular level and present a motor phenotype reminiscent of severe GE symptoms. The molecular characterization of gldc-/- mutants showed a broad metabolic disturbance affecting amino acids and neurotransmitters other than glycine, with lactic acidosis at stages preceding death. Although a transient imbalance was found in cell proliferation in the brain of gldc-/- zebrafish, the main brain networks were not affected, thus suggesting that GE pathogenicity is mainly due to metabolic defects. We confirmed that the gldc-/- hypotonic phenotype is due to NMDA and glycine receptor overactivation, and demonstrated that gldc-/- larvae depict exacerbated hyperglycinemia at these synapses. Remarkably, we were able to rescue the motor dysfunction of gldc-/- larvae by counterbalancing pharmacologically or genetically the level of glycine at the synapse.

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., (© 2018 The Authors. Epilepsia published by Wiley Periodicals, Inc. on behalf of International League Against Epilepsy.)

Mutations in DEPDC5 are causal factors for a broad spectrum of focal epilepsies, but the underlying pathogenic mechanisms are still largely unknown. To address this question, a zebrafish depdc5 knockout model showing spontaneous epileptiform events in the brain, increased drug-induced seizure susceptibility, general hypoactivity, premature death at 2-3 weeks post-fertilization, as well as the expected hyperactivation of mTOR signaling was developed. Using this model, the role of DEPDC5 in brain development was investigated using an unbiased whole-transcriptomic approach. Surprisingly, in addition to mTOR-associated genes, many genes involved in synaptic function, neurogenesis, axonogenesis, and GABA network activity were found to be dysregulated in larval brains. Although no gross defects in brain morphology or neuron loss were observed, immunostaining of depdc5 -/- brains for several GABAergic markers revealed specific defects in the fine branching of the GABAergic network. Consistently, some defects in depdc5 -/- could be compensated for by treatment with GABA, corroborating that GABA signaling is indeed involved in DEPDC5 pathogenicity. Further, the mTOR-independent nature of these neurodevelopmental defects was demonstrated by the inability of rapamycin to rescue the GABAergic network defects observed in depdc5 -/- brains and, conversely, the inability of GABA to rescue the hypoactivity in another genetic model showing mTOR hyperactivation. This study hence provides the first in vivo evidence that DEPDC5 plays previously unknown roles apart from its canonical function as an mTOR inhibitor. Moreover, these results propose that defective neurodevelopment of GABAergic networks could be a key factor in epileptogenesis when DEPDC5 is mutated., (Copyright © 2018 Elsevier Ltd. All rights reserved.)