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Background <p>Variants in <i>OTUD5</i> are associated with neurodevelopmental disorders (NDDs), yet the underlying molecular mechanisms remain unclear. This study aimed to investigate the pathogenicity of a novel <i>OTUD5</i> variant (c.697G &gt; A, p.Val233Met) and elucidate its regulatory role in neural progenitor cell (NPC) proliferation and differentiation, thereby uncovering the function of <i>OTUD5</i> in neurodevelopment.</p> Methods <p>The <i>OTUD5</i> 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.</p> Results <p>The p.Val233Met variant, located within the catalytic OTU domain, induced a marked conformational alteration in the <i>OTUD5</i> protein. Functionally, the variant caused aberrant NPC proliferation (1.8-fold increase in Ki67<sup>+</sup> cells, accompanied by release of G1 arrest) and impaired neuronal differentiation (60% reduction in Tuj<sup>+</sup> cells). Mechanistically, wild-type <i>OTUD5</i> stabilized GSK3β by removing K48-linked ubiquitin chains, whereas the mutant isoform exhibited diminished deubiquitinase activity, accelerating GSK3β degradation and shortening its half-life by 40%.</p> Conclusion <p>This study establishes a novel disease mechanism whereby <i>OTUD5</i> mutations disrupt NPC homeostasis through GSK3β destabilization, highlighting the critical role of ubiquitination regulation in neurodevelopment. Our iPSC model provides a platform for testing GSK3β-targeted therapies in <i>OTUD5</i>-related NDDs.</p> Graphical Abstract <p></p>

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A novel OTUD5 variant disrupts neural progenitor cell homeostasis: mechanistic insights from HEK293T cell-based analyses

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年3月18日

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  • Na Xu,
  • Shihao Wang,
  • Tingting Yang,
  • Meiping Yu,
  • Yu Sun,
  • Yongkun Zhan,
  • Yongguo Yu

摘要

Background

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 neurodevelopment.

Methods

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.

Results

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 GSK3β by removing K48-linked ubiquitin chains, whereas the mutant isoform exhibited diminished deubiquitinase activity, accelerating GSK3β degradation and shortening its half-life by 40%.

Conclusion

This study establishes a novel disease mechanism whereby OTUD5 mutations disrupt NPC homeostasis through GSK3β destabilization, highlighting the critical role of ubiquitination regulation in neurodevelopment. Our iPSC model provides a platform for testing GSK3β-targeted therapies in OTUD5-related NDDs.

Graphical Abstract