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Pharmacological chaperones restore proteostasis of epilepsy-associated GABA_A receptor variants.

ABSTRACT: Recent advances in genetic diagnosis identified variants in genes encoding GABA_A receptors as causative for genetic epilepsy. Here, we selected eight disease-associated variants in the α1 subunit of GABA_A receptors causing mild to severe clinical phenotypes and showed that they are loss of function, mainly by reducing the folding and surface trafficking of the α1 protein. Furthermore, we sought client protein-specific pharmacological chaperones to restore the function of pathogenic receptors. Applications of positive allosteric modulators, including Hispidulin and TP003, increase the functional surface expression of the α1 variants. Mechanism of action study demonstrated that they enhance the folding and assembly and reduce the degradation of GABA_A variants without activating the unfolded protein response in HEK293T cells and human iPSC-derived neurons. Since these compounds cross the blood-brain barrier, such a pharmacological chaperoning strategy holds great promise to treat genetic epilepsy in a GABA_A receptor-specific manner.

SUBMITTER: Wang YJ

PROVIDER: S-EPMC10153171 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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Pharmacological chaperones restore proteostasis of epilepsy-associated GABAA receptor variants.

Wang Ya-Juan YJ Seibert Hailey H Ahn Lucie Y LY Schaffer Ashleigh E AE Mu Ting-Wei TW

bioRxiv : the preprint server for biology 20230419

Recent advances in genetic diagnosis identified variants in genes encoding GABAA receptors as causative for genetic epilepsy. Here, we selected eight disease-associated variants in the α1 subunit of GABAA receptors causing mild to severe clinical phenotypes and showed that they are loss of function, mainly by reducing the folding and surface trafficking of the α1 protein. Furthermore, we sought client protein-specific pharmacological chaperones to restore the function of pa ...[more]

PMID: 37131660

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Project description:ObjectiveGABAA 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 GABAA receptor variants using the model organism, Caenorhabditis elegans.MethodsCRISPR-Cas9 gene editing was used to create a complete deletion of unc-49, a C. elegans GABAA 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 GABAA receptor. All newly created strains were analyzed for phenotype and compared against existing unc-49 mutations.ResultsNematodes 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 GABAA 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.SignificanceThese findings establish C. elegans as a genetic model to investigate GABAA receptor mutations and delineate a platform for validating associated variants in any epilepsy-associated gene.Plain language summaryEpilepsy 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.

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Pharmacological chaperones restore proteostasis of epilepsy-associated GABA_A receptor variants.

Publications

Pharmacological chaperones restore proteostasis of epilepsy-associated GABA<sub>A</sub> receptor variants.

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