Nucleocytoplasmic shuttling of USP15 promotes pathological autophagy that disrupts neural stem cell homeostasis and neurodevelopment.

During organogenesis, stem cells undergo cellular and metabolic remodelling, facilitated by autophagy-mediated turnover of organelles. Autophagy impairment has been linked to human diseases, including neurodevelopmental disorders associated with disrupted neural stem/precursor cell (NPC) homeostasis, but the underlying mechanisms and pathogenic processes governing these connections remain poorly understood. Here, we report three de novo variants of uncertain significance (p.Gly223Asp, p.Gly889Glu, p.Met978Val) in the deubiquitinase USP15 in human probands with diverse clinical features, including a spectrum of brain malformations and metabolic phenotypes. Proband variants differentially altered USP15 activity and nucleocytoplasmic localization. USP15 showed dynamic localization in NPCs of embryonic mouse cerebral cortex. Using a knock-in mouse model carrying the p.Met978Val variant, we showed that aberrant cytoplasmic accumulation of USP15, but not its loss-of-function, impaired NPC self-renewal and differentiation, leading to reduced neuronal output and enlarged lateral ventricles. Mechanistically, USP15 deubiquitinated autophagy regulator ATG16L1, impeded its normal turnover, and impaired autophagy. Concurrently, lipid droplet mobilization and mitochondrial dynamics were attenuated. Reestablishing the ubiquitination-deubiquitination balance restored autophagy activity and normal neurogenesis. Our findings suggest that nucleocytoplasmic shuttling of USP15 creates a switch-like autophagy signal controlling NPC homeostasis, and its disruption may contribute to the pathogenesis of complex neurodevelopmental conditions.