CB1 receptor inhibition in fragile X syndrome mice impacts alternative splicing alterations in hippocampal synaptoneurosomal transcriptome

de los Reyes-Ramirez, L.; Ciaran-Alfano, L.; Reixachs-Sole, M.; Bergada-Martinez, A.; Galera-Lopez, L.; Martinez-Torres, S.; Navarro-Romero, A.; Maldonado, R.; Eyras, E.; Ozaita, A.

doi:10.1101/2025.07.01.662539

bioRxiv · 10.1101/2025.07.01.662539

CB1 receptor inhibition in fragile X syndrome mice impacts alternative splicing alterations in hippocampal synaptoneurosomal transcriptome

de los Reyes-Ramirez, L.·Ciaran-Alfano, L.·Reixachs-Sole, M.·Bergada-Martinez, A.·Galera-Lopez, L.·Martinez-Torres, S.·Navarro-Romero, A.·Maldonado, R.·Eyras, E.·Ozaita, A.

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Abstract

BackgroundFragile X syndrome (FXS) conveys the most frequent heritable genetic cause of intellectual disability and autism. It is caused by a CGG repeat expansion in FMR1 gene that leads to the loss of fragile X messenger ribonucleoprotein 1 (FMRP). FMRP is highly abundant in synapses, where regulates mRNAs to maintain synaptic plasticity. Treatments under development significantly ameliorate neurological and behavioral landmarks in the mouse model of the disorder, the Fmr1 knockout (FX) mouse. Specifically, previous studies revealed that pharmacological and genetic inhibition of cannabinoid type-1 receptor (CB1R) restored phenotypic traits in FX mice. However, the molecular hallmarks associated with this experimental therapeutic intervention are largely unknown. MethodsFirst, we aimed to evaluate the validity of synaptoneurosomes preparations to investigate specific mRNA modifications at synapses. Afterwards, combining in silico high-throughput analysis and biochemical determinations, we analyzed the hippocampal synaptoneurosomal transcriptome after pharmacological inhibition of CB1R with the specific antagonist/inverse agonist rimonabant in FX male mice ResultsWe verified that synaptoneurosomes provide an accurate representation of synaptic composition and function. Then, we found that rimonabant treatment had a limited impact at gene expression level but produced significant modifications in transcript expression. Indeed, detailed analysis of alternative splicing events revealed a relevant number of events in which splicing was reverted from the FX form to the WT form by the treatment. LimitationsWe demonstrated that rimonabant treatment alters the AS landscape in FX hippocampal synaptoneurosomes; however, further studies are needed to elucidate if other neural components also contribute to the modifications and whether the findings are specific to rimonabant treatment or to CB1R inhibition at synapses. In addition, additional research could be required to clarify whether the changes in AS events could be affected by interindividual variability or technical protocols. ConclusionsWe determined that the AS landscape is modified in FX hippocampal synaptoneurosomes and that these changes are sensitive to rimonabant treatment which could explain the beneficial effects of this experimental therapeutic approach in FXS. Altogether, our results reveal a new level of complexity in the effect of pharmacological treatment to improve symptoms in the context of FXS.

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de los Reyes-Ramirez, L., Ciaran-Alfano, L., Reixachs-Sole, M., Bergada-Martinez, A., Galera-Lopez, L., Martinez-Torres, S., Navarro-Romero, A., Maldonado, R., Eyras, E., Ozaita, A.. 2025-07-04. CB1 receptor inhibition in fragile X syndrome mice impacts alternative splicing alterations in hippocampal synaptoneurosomal transcriptome. https://doi.org/10.1101/2025.07.01.662539

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