Deficiency of pnkp in zebrafish causes microcephaly, seizures, and developmental delay through mitochondrial dysfunction.

Variants in PNKP cause a severe neurodevelopmental disorder characterized by microcephaly, seizures, and developmental delay (MCSZ). Despite clear clinical significance, the pathological mechanisms underlying this condition remain incompletely understood. In this study, CRISPR/Cas9 genome editing was applied to generate a zebrafish pnkp knockout model ( pnkp -/- ), overcoming limitations associated with previously reported mouse models that exhibited postnatal lethality. The pnkp -/- zebrafish faithfully recapitulated key phenotypic traits observed in human PNKP deficiency, facilitating in-depth exploration of the molecular mechanisms contributing to disease. Loss of PNKP function resulted in mitochondrial DNA damage, accompanied by disruption of mitochondrial ultrastructure and bioenergetic function, increased apoptosis, and reduced autophagic activity, collectively leading to pronounced cerebral neurodevelopmental abnormalities. Transcriptomic analysis based on RNA sequencing identified marked down-regulation of gamma-aminobutyric acid (GABA) receptor-related genes. Pharmacological activation of GABA A receptors with muscimol partially rescued hyperactive behavior in pnkp -/- zebrafish, suggesting a potential link between GABAergic signaling and seizure-related phenotypes. Drug screening performed using the pnkp -/- zebrafish model further identified lamotrigine as a comparatively effective compound for seizure control associated with PNKP mutations. These findings clarify mechanistic links between PNKP deficiency and MCSZ pathology, identify candidate therapeutic agents, and provide an experimental framework for investigation of disorders associated with defective DNA repair.