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Therapeutic efficacy of the BKCa channel opener chlorzoxazone in a mouse model of Fragile X syndrome

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Abstract

Fragile X syndrome (FXS) is an X-linked neurodevelopmental disorder characterized by several behavioral abnormalities, including hyperactivity, anxiety, sensory hyper-responsiveness, and autistic-like symptoms such as social deficits. Despite considerable efforts, effective pharmacological treatments are still lacking, prompting the need for exploring the therapeutic value of existing drugs beyond their original approved use. One such repurposed drug is chlorzoxazone which is classified as a large-conductance calcium-dependent potassium (BKCa) channel opener. Reduced BKCa channel functionality has been reported in FXS patients, suggesting that molecules activating these channels could serve as promising treatments for this syndrome. Here, we sought to characterize the therapeutic potential of chlorzoxazone using the Fmr1-KO mouse model of FXS which recapitulates the main phenotypes of FXS, including BKCa channel alterations. Chlorzoxazone, administered either acutely or chronically, rescued hyperactivity and acoustic hyper-responsiveness as well as impaired social interactions exhibited by Fmr1-KO mice. Chlorzoxazone was more efficacious in alleviating these phenotypes than gaboxadol and metformin, two repurposed treatments for FXS that do not target BKCa channels. Systemic administration of chlorzoxazone modulated the neuronal activity-dependent gene c-fos in selected brain areas of Fmr1-KO mice, corrected aberrant hippocampal dendritic spines, and was able to rescue impaired BKCa currents recorded from hippocampal and cortical neurons of these mutants. Collectively, these findings provide further preclinical support for BKCa channels as a valuable therapeutic target for treating FXS and encourage the repurposing of chlorzoxazone for clinical applications in FXS and other related neurodevelopmental diseases.

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Fig. 1: Dose-dependent acute effects of chlorzoxazone (CHLOR) in Fmr1-KO mice.
Fig. 2: Acute behavioral effects of chlorzoxazone (CHLOR, 5 mg/kg) compared with gaboxadol (GAB, 0.5 and 3 mg/kg) and metformin (MET, 200 mg/kg).
Fig. 3: Chronic behavioral effects of chlorzoxazone (CHLOR, 5 mg/kg) compared with gaboxadol (GAB, 0.5 and 3 mg/kg) and metformin (MET, 200 mg/kg).
Fig. 4: Effects of chlorzoxazone on neuronal activity and structural plasticity of Fmr1-KO mice.
Fig. 5: Effects of chlorzoxazone (CHLOR) on hippocampal and cortical BKCa currents in Fmr1-KO mice and their WT littermates.

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All original data are available upon request to the corresponding author.

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Acknowledgements

The authors thank Delphine Gonzales and the genotyping facility of Neurocentre Magendie (PUMA), funded by Inserm and LabEX BRAIN ANR-10-LABEX-43, for animal genotyping. The authors also thank Dr Txomin Lalanne and Dr Lauriane Beliez from Ephyx Neuroscience for their technical assistance with electrophysiological recordings. We thank Valeria Petroni for the assistance in animal testing, Elodie Poinama for animal care, Marie-Laure Rousseau, and Thierry Lafon for administrative and technical support.

Funding

This research was funded by FONDATION POUR L’AUDITION, grant number FPA-RD-2020-8, to SP. SP and CF received additional financial support from “Association Autour de Williams” and “Federation Williams France”, as well as from CNRS and Bordeaux University. BB received financial support from the French government in the framework of the University of Bordeaux’s IdEx “Investments for the Future” program (GPR BRAIN_2030) and from the “Agence Nationale pour la Recherche” (ANR-23-CE37-0013-02, project “Thalagram”).

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Author notes

  1. These authors contributed equally: Celeste Ferraguto, Marion Piquemal-Lagoueillat.

Authors and Affiliations

  1. Univ. Bordeaux, CNRS, EPHE, INCIA, UMR 5287, Bordeaux, France

    Celeste Ferraguto, Marion Piquemal-Lagoueillat, Valerie Lemaire, Sandrine S. Bertrand, Bruno Bontempi & Susanna Pietropaolo

  2. Univ. Bordeaux, INSERM, Neurocentre Magendie, U1215, Bordeaux, France

    Maïté M. Moreau

  3. Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy

    Stefania Trazzi, Beatrice Uguagliati & Elisabetta Ciani

  4. Assetsup, Le Boulinier, Rueil-Malmaison, France

    Eric Louette

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Contributions

Experimental activity, data collection, and analysis: CF, MPL, VL, MMM, ST, BU, EC, SSB, and SP. Study design: EL, BB, and SP. Project supervision: SP. Manuscript writing: CF, BB, and SP. All authors have reviewed and approved the manuscript.

Corresponding author

Correspondence to Susanna Pietropaolo.

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Competing interests

VL, EL, and SP are inventors on the patent: “Methods of treatment and/or prevention of disorders and symptoms related to BKCa and/or SK channelopathies” (N°. EP20775228.8 and N°. US17760612) owned by CNRS. The remaining authors have nothing to disclose.

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Ferraguto, C., Piquemal-Lagoueillat, M., Lemaire, V. et al. Therapeutic efficacy of the BKCa channel opener chlorzoxazone in a mouse model of Fragile X syndrome. Neuropsychopharmacol. (2024). https://doi.org/10.1038/s41386-024-01956-6

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