Xu N, Wang S, Yang T, Yu M, Sun Y, Zhan Y, Yu Y

Variants in OTUD5 are associated with neurodevelopmental disorders (NDDs), yet the underlying molecular mechanisms remain unclear. This study aimed to investigate the pathogenicity of a novel OTUD5 variant (c.697G?>?A, p.Val233Met) and elucidate its regulatory role in neural progenitor cell (NPC) proliferation and differentiation, thereby uncovering the function of OTUD5 in ...

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neurodevelopment.The OTUD5 variant was identified in two NDD patients via exome sequencing. Patient-derived induced pluripotent stem cells (iPSCs) and CRISPR/Cas9-corrected isogenic controls were generated. NPC proliferative activity was assessed by Ki67 immunofluorescence staining, cell-cycle distribution was analyzed by flow cytometry, and neuronal differentiation was evaluated by Tuj1/MAP2 immunofluorescence staining. Substrate screening was conducted in HEK293T cells using co-immunoprecipitation (Co-IP) and mass spectrometry. Deubiquitination capacity and protein stability were validated through ubiquitination assays and cycloheximide (CHX) chase experiments.The p.Val233Met variant, located within the catalytic OTU domain, induced a marked conformational alteration in the OTUD5 protein. Functionally, the variant caused aberrant NPC proliferation (1.8-fold increase in Ki67+ cells, accompanied by release of G1 arrest) and impaired neuronal differentiation (60% reduction in Tuj+ cells). Mechanistically, wild-type OTUD5 stabilized GSK3? by removing K48-linked ubiquitin chains, whereas the mutant isoform exhibited diminished deubiquitinase activity, accelerating GSK3? degradation and shortening its half-life by 40%.This study establishes a novel disease mechanism whereby OTUD5 mutations disrupt NPC homeostasis through GSK3? destabilization, highlighting the critical role of ubiquitination regulation in neurodevelopment. Our iPSC model provides a platform for testing GSK3?-targeted therapies in OTUD5-related NDDs.

Date: Mar. 18, 2026

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