Dissecting GPCR Contributions to Gαo-Dependent Motor Dysfunction in GNAO1-Related Disorders Using Caenorhabditis...

Background/Objectives: Pathogenic variants in GNAO1, encoding the inhibitory G protein subunit Gαo, cause severe neurodevelopmental disorders that remain largely refractory to pharmacological treatments. Gαo transduces inhibitory signals downstream of multiple G protein-coupled receptors (GPCRs) involved in motor control. Here, we used gene-edited Caenorhabditis elegans models carrying goa-1 variants, the ortholog of GNAO1, to investigate GPCR contributions to Gαo-dependent locomotor phenotypes. Methods: We combined pharmacological screening of dopamine- and cannabinoid-targeting ligands in goa-1 mutants with structural analysis of ligand-binding pocket conservation and genetic perturbation of receptor function using RNAi and knockout approaches. Results: Pharmacological modulation of GPCR signaling produced non-linear and context-dependent effects. Compounds predicted to further increase excitability may instead promote phenotypic improvement, consistent with compensatory network rebalancing. Structural analyses revealed substantial divergence in ligand-binding pocket conservation for several GPCR-ligand pairs, suggesting that altered binding affinity and selectivity may also contribute to the observed phenotypic outcome. Pharmacological experiments performed in GPCR-depleted mutants allowed for the correlation of structural findings with functional effects for selected receptor-ligand pairs. Finally, genetic reduction in GPCRs coupled to stimulatory G proteins ameliorated hyperactive locomotion in goa-1 mutants, whereas reduction in GPCRs coupled to inhibitory G proteins is largely insufficient to induce or exacerbate locomotor defects. Conclusions: Our findings identify excessive excitatory GPCR input as a key modulator of motor dysfunction in the context of impaired Gαo signaling. They also show that structural conservation is a necessary but not sufficient condition to predict functional responses. Overall, this study establishes C. elegans as a suitable platform to dissect GPCR-mediated signaling and highlights the value of integrating pharmacological and genetic approaches to guide target selection in GNAO1-related disorders.