Modeling epilepsy by loss-of-function of the CUG-binding protein Elav-like family member 2 in zebrafish with multi-omics analysis

Abstract Background: The CUG-binding protein Elav-like family member 2 ( CELF2 ) gene has been linked to the pathogenesis of epilepsy, but its precise role remains unclear. This study aimed to investigate the pathogenic mechanisms of CELF2 mutation in epilepsy, utilizing zebrafish models to explore its molecular pathways and biological impact. Methods: Whole-exome sequencing was performed to identify CELF2 mutations associated with epilepsy. CELF2 zebrafish model were generated using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-related protein 9 technology and morpholinos, followed by behavioral and electroencephalographic analyses to confirm epileptic phenotypes. Proteomic and metabolomic analyses were conducted to examine the impact of CELF2 deficiency on metabolic pathways, and single-cell sequencing was used to assess alterations in neuronal cell populations. Results: An infant with infantile epileptic spasms syndrome associated with a CELF2 ( p.Pro520Arg ) gene mutation was reported. We established zebrafish models with celf2 gene knockout and knockdown and found that zebrafish with celf2 mutations exhibited epilepsy-like behaviors, which could be rescued by injection of CELF2 wild-type mRNA. Significant changes were observed in crucial marker genes associated with the nervous system in the celf2 +/− group, including FOS , BDNF , NPAS4 , GABRA1 , GABRG2 , and PYYA . Disruptions in lipid metabolism, heat shock protein 90 beta1 (Hsp90b1), were identified in proteomic and metabolomic analyses. Single-cell sequencing showed changes in nucleosome localization, nucleosome DNA binding, arginine and proline metabolic pathways, gonadotropin-releasing hormone signaling pathway, and nucleotide-binding oligomerization domain receptor signaling pathway. Conclusions: Our study has revealed a promising association between defects in the CELF2 gene and epilepsy using a zebrafish model, suggesting that CLEF2 is a causative gene in epilepsy. These findings not only indicate the potential impact on the biological process influenced by the CELF2 gene defect but also offer hopeful insights into the pathogenesis of epilepsy and potential therapeutic targets.