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

Variants in the polynucleotide kinase-phosphatase (PNKP) gene leads to a severe neurological disease characterized by microcephaly, intractable seizures, and developmental delay (MCSZ). Despite its clinical impact, the underlying pathological mechanisms remain unclear. In this work, we utilized CRISPR/Cas9 technology to generate a zebrafish model with a pnkp knockout (pnkp-/-), overcoming limitations seen in prior mouse models that suffered from postpartum death. This zebrafish model faithfully recapitulated phenotypic traits observed in human PNKP deficiency, facilitating an in-depth exploration of the molecular mechanisms involved. The study revealed that pnkp deficiency disrupts mitochondria function, leading to mitochondrial apoptosis and inhibition of autophagy. Notably, the anti-seizure drug lamotrigine (LTG) demonstrated particularly promising efficacy in rescuing the hyperactive behavior. RNA-seq data analysis revealed downregulation of Gamma-aminobutyric acid (GABA) receptor-related gene, with the GABAA receptor agonist muscimol (MUSC) partially rescuing the hyperactive behavior in pnkp-/- zebrafish, suggesting potential link between GABAergic signaling and seizure. This work provides evidence supporting the role of damaged mitochondrial function, increased apoptosis and reduced autophagy as potential pathological mechanisms associated with PNKP variants. Furthermore, we propose this zebrafish-based model as a valuable platform for drug screening, holding promise for potential therapeutic interventions in MCSZ caused by PNKP variants.