• A novel OTUD5 missense variant (p.Val233Met) was identified in two NDD patients, causing conformational changes in the catalytic OTU domain.
• The variant disrupts neural progenitor cell homeostasis by increasing proliferation (1.8-fold) and impairing neuronal differentiation (60% reduction).
• Mechanistically, wild-type OTUD5 stabilizes GSK3β by removing K48-linked ubiquitin chains; the mutant shows reduced deubiquitinase activity, accelerating GSK3β degradation.
• This study provides a patient-derived iPSC model for testing GSK3β-targeted therapies in OTUD5-related NDDs.