Functional Analysis of the Gamma-Aminobutyric Acid Type A Receptor Subunit Alpha-1 (GABRA1) Gene During Zebrafish Development

The GABRA1 gene encodes for the alpha-1 (α1) subunit of the Gamma-Aminobutyric acid type A receptor (GABAAR), which are the primary modulators of synaptic inhibition in the central nervous system (CNS). Alpha-1 subunits are essential for maintaining the normal function of native receptors and contribute to over 60% of all GABAARs in the CNS. Remarkably, a broad spectrum of neurodevelopmental and epilepsy-associated disorders have been linked with mutations in the GABRA1 gene. However, the developmental, behavioral, and molecular mechanisms underlying GABRA1-associated epileptic disorders remain to be fully understood. Hence, the overarching goal of this dissertation is to investigate the behavioral and molecular mechanisms associated with GABRA1 deficiency during brain development. Two independent experimental approaches were taken using zebrafish as a model system to address this goal. Our first study describes the identification of a de novo missense variant of GABRA1 and the in vivo functional analysis of gabra1 using morpholino-mediated knockdown. We developed a behavioral paradigm using the Zebrabox technology to investigate the behavioral consequences of gabra1 loss of function. Interestingly, transient knockdown of gabra1 resulted in hypomotility at early larval stages. This hypomotile phenotype was correlated with altered expression of genes that encode for other GABAAR subunits. Mainly we show downregulation of the β2 and γ2 transcripts, which are the primary assembly partners of α1, and upregulation of the homologous α6b subunit. Despite these molecular defects in the expression of major GABAAR subunits, morphant larvae exhibited a positive response to a potent GABAAR antagonist, pentylenetetrazol (PTZ), indicating the presence of an active receptor despite the deficient expression of gabra1. Our findings contrast with those described in a zebrafish germline mutant of gabra1 generated previously. The distinct behavioral phenotypes between knockdown and germline mutation of gabra1 prompted us to characterize an additional gabra1 germline mutant. Therefore, our second unpublished study describes the behavioral and molecular analysis of an uncharacterized nonsense mutant of gabra1 (sa43718 allele) generated by the Wellcome Sanger Institute as part of the zebrafish mutation project in 2016. Behavioral analysis of this allele resulted in lightinduced hyperactive locomotion resembling generalized seizures. Targeted mRNA analysis revealed that this behavioral phenotype was associated with reduced expression of gabra1 and upregulation of gabra4, which encodes the a4 subunit. Mutant larvae were treated with PTZ to examine whether gabra1 mutants had an active receptor. Notably, mutant larvae exhibited reduced response compared to controls, suggesting that a GABAAR with reduced functionality is present. Subsequent proteomic analysis of gabra1 mutants revealed abnormal expression of proteins essential for synaptic vesicle transport, mitochondrial function, and potassium and sodium homeostasis regulation. Collectively, we highlight the essential role of gabra1 in early development and epileptic phenotypes