A zebrafish model of NEXMIF-related encephalopathy identified drivers of hyperlocomotion and provides a scalable platform for drug discovery

Abstract

X-linked Intellectual Disability type 98 (XLID-98) is a neurodevelopmental disorder with a broad phenotypic spectrum caused by pathogenic variants in the neurite extension and migration factor ( NEXMIF ) gene, formerly known as KIAA2022, KIDLIA , or XPN (OMIM 300524). To investigate the pathogenic mechanisms of XLID-98, a double knock-out zebrafish model ( nexmifa/b −/− ) was generated using CRISPR/Cas9 technology. Mutant larvae displayed limited morphological abnormalities, the most notable being an increase in total body length. They also exhibited a significant hyperlocomotor phenotype in the visual-startle OFF assay, which was attenuated by treatment with sodium valproate (VPA) and topiramate, thereby supporting the pharmacological validity of the model. Transcriptomic analysis of larval heads at seven dpf revealed 414 differentially expressed genes enriched in mitogen-activated protein kinase (MAPK) signaling, cell adhesion, and sodium transport pathways. Functional analyses demonstrated that simultaneous CRISPR/Cas9-mediated disruption of the upregulated genes entpd3 (ectonucleoside triphosphate diphosphohydrolase 3) and ddx43 (DEAD-box helicase 43) exacerbated the hyperlocomotor phenotype, suggesting that these genes may participate in a compensatory response, whereas reduced kbtbd7 (kelch repeat and BTB domain-containing protein 7) activity may contribute to phenotype severity. A targeted screen of 87 natural compounds identified kahweol acetate, veratridine, and rauwolscine as potent inducers of entpd3 . However, despite robust transcriptional activation, rauwolscine failed to improve behavioral outcomes, suggesting that modulation of a single molecular target may be insufficient for phenotypic rescue. Collectively, these findings establish the first zebrafish model of NEXMIF-related encephalopathy, identify entpd3 , ddx43 , and kbtbd7 as candidate modulators of this behavioral phenotype, and provide a scalable platform for targeted therapeutic discovery in XLID-98.