Functional analysis of epilepsy-associated GABAA receptor mutations using Caenorhabditis elegans

Abstract

Objective: GABA_A receptor subunit mutations pose a significant risk for genetic generalized epilepsy; however, there are over 150 identified variants, many with unknown or unvalidated pathogenicity. We aimed to develop in vivo models for testing GABA_A receptor variants using the model organism, Caenorhabditis elegans.

Methods: CRISPR-Cas9 gene editing was used to create a complete deletion of unc-49, a C. elegans GABA_A receptor, and to create homozygous epilepsy-associated mutations in the endogenous unc-49 gene. The unc-49 deletion strain was rescued with transgenes for either the C. elegans unc-49B subunit or the α1, β3, and γ2 subunits for the human GABA_A receptor. All newly created strains were analyzed for phenotype and compared against existing unc-49 mutations.

Results: Nematodes with a full genetic deletion of the entire unc-49 locus were compared with existing unc-49 mutations in three separate phenotypic assays-coordinated locomotion, shrinker frequency and seizure-like convulsions. The full unc-49 deletion exhibited reduced locomotion and increased shrinker frequency and PTZ-induced convulsions, but were not found to be phenotypically stronger than existing unc-49 mutations. Rescue with the unc-49B subunit or creation of humanized worms for the GABA_A receptor both showed partial phenotypic rescue for all three phenotypes investigated. Finally, two epilepsy-associated variants were analyzed and deemed to be loss of function, thus validating their pathogenicity.

Significance: These findings establish C. elegans as a genetic model to investigate GABA_A receptor mutations and delineate a platform for validating associated variants in any epilepsy-associated gene.

Plain language summary: Epilepsy is a complex human disease that can be caused by mutations in specific genes. Many possible mutations have been identified, but it is unknown for most of them whether they cause the disease. We tested the role of mutations in one specific gene using a small microscopic worm as an animal model. Our results establish this worm as a model for epilepsy and confirm that the two unknown mutations are likely to cause the disease.

Keywords: convulsion; locomotion; seizure; shrinker; unc‐49.

FIGURE 1

Generation and locomotor analysis of a complete unc‐49 gene deletion. (A) Cartoon schematic outline of the unc‐49 gene structure and existing and new mutations used in this study. Three distinct unc‐49 subunits (UNC‐49A, UNC‐49B, and UNC‐49C) are assembled from a common N‐terminus combined with either the A, B, or C fragment at the C‐terminus. The e407 allele is a nonsense mutation affecting all three subunits, whereas e382 (missense) and tm5487 (small deletion) affect UNC‐49B only. We generated a complete unc‐49 deletion (unc‐49 null) excising all coding fragments and introduced novel transgenic mutations of Cys167 and Gly254 into the endogenous gene. (B) Locomotion was quantified by counting thrashes per minute of worms in solution. In comparison to Bristol N2 wild types, our new unc‐49 null mutant as well as the unc‐49 e407, unc‐49 e382, and unc‐49 tm5487 alleles had significantly defective locomotion (indicated by *). None of the unc‐49 alleles were significantly different from each other. Comparisons made by one‐way ANOVA with Tukey post hoc comparisons (p < 0.01, N = 30 for each strain). Data shown as mean ± SEM.

FIGURE 2

Analysis of shrinker and convulsion frequencies of a complete unc‐49 deletion. (A) Shrinker frequency was quantified by recording a lack of backward movement to a gentle tap to the animals head. All unc‐49 mutants had a significant increase (*) in shrinker frequency in comparison with Bristol N2 wild types, but were not different from each other. Comparisons made by Fisher's exact test (p < 0.01, N = 30 for each strain). Data shown as mean ± SEM. (B) Convulsion frequency was quantified following a 15‐min exposure to 7 mg/mL PTZ by scoring the number of head‐bobbing convulsions over a 30‐s period. All unc‐49 mutants had a significant increase (*) in convulsion frequency in comparison with Bristol N2 wild types. The tm5487 allele also showed less convulsions than either the unc‐49 null and e407 alleles. Comparisons made by one‐way ANOVA with Tukey post hoc comparisons (p < 0.01, N = 30 for each strain). Data shown as mean ± SEM.

FIGURE 3

Transgenic expression of unc‐49B or the human GABA_A receptor partially rescues the unc‐49 null phenotypic deficits. (A) Locomotion was quantified by counting thrashes per minute of worms in solution. Transgenic expression of either unc‐49B or the human GABA_A receptor in the unc‐49 null background significantly increased locomotion (*); however, locomotion was lower than that seen for wild types. Comparisons made by one‐way ANOVA with Tukey post hoc comparisons (p < 0.01, N = 30 for each strain). Data shown as mean ± SEM. (B) Shrinker frequency was quantified by recording a lack of backward movement to a gentle tap to the animals head. Transgenic expression of either unc‐49B or the human GABA_A receptor in the unc‐49 null background significantly decreased shrinker frequency (*); however, shrinker frequency was greater than that seen for wild types. Comparisons made by Fisher's exact test (p < 0.01, N = 30 for each strain). Data shown as mean ± SEM. (C) Convulsion frequency was quantified following a 15‐min exposure to 7 mg/mL PTZ by scoring the number of head‐bobbing convulsions over a 30‐s period. Transgenic expression either unc‐49B or the human GABA_A receptor in the unc‐49 null background significantly decreased convulsion frequency (*); however, convulsion frequency was greater than that seen for wild types. Comparisons made by one‐way ANOVA (p < 0.01, N = 30 for each strain). Data shown as mean ± SEM.

FIGURE 4

Epilepsy‐linked GABA_A receptor point mutations are functionally null. (A) Locomotion was quantified by counting thrashes per minute of worms in solution. Similar to the unc‐49 nulls, either a C167W or G254D point mutation in the C. elegans unc‐49 gene (orthologous to human C166W and G251D, respectively) significantly reduced nematode locomotion (*) in comparison with wild‐type worms. Comparisons made by one‐way ANOVA with Tukey post hoc comparisons (p < 0.01, N = 30 for each strain). Data shown as mean ± SEM. (B) Shrinker frequency was quantified by recording a lack of backward movement to a gentle tap to the animals head. Similar to the unc‐49 nulls, the C167W or G254D point mutations in unc‐49 significantly increased shrinker frequency (*) in comparison with wild‐type worms. Comparisons made by Fisher's exact test (p < 0.01, N = 30 for each strain). Data shown as mean ± SEM. (C) Convulsion frequency was quantified by following a 15‐min exposure to 7 mg/mL PTZ by scoring the number of head‐bobbing convulsions over a 30‐s period. In comparison to wild‐type worms, the C167W or G254D point mutations in unc‐49 significantly increased shrinker frequency (*) to a level indistinguishable from unc‐49 null. Comparisons made by one‐way ANOVA (p < 0.01 N = 30 for each strain). Data shown as mean ± SEM.