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

Abstract

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.

1. Introduction

Neurodevelopmental disorders (NDDs) are a group of conditions characterized by abnormal development of the central nervous system caused by genetic or environmental factors. They present with complex phenotypes such as cognitive deficits, motor impairments, and social dysfunction [1]. These disorders typically manifest early in life, affecting approximately 3% of children worldwide, with over 60% of cases associated with lifelong functional impairments. Due to the high incidence of early-onset neurological dysfunction and long-term care needs, NDDs impose a significant economic and healthcare burden on society [2]. Although hundreds of NDD-associated genes (e.g., CHD8, SYNGAP1) have been identified, the pathogenic mechanisms in approximately 40% of cases remain unclear. In particular, the roles of epigenetic regulators such as deubiquitinases (DUBs) in neurodevelopment require further elucidation.

In recent years, exome sequencing has revealed a large number of pathogenic genes associated with NDDs [3]. Among these, DUBs have attracted increasing attention due to their critical roles in maintaining protein homeostasis via the ubiquitin-proteasome system and dynamically regulating signaling pathways [4]. As a key member of the OTU family, OTUD5 (OTU deubiquitinase 5) plays essential roles in DNA damage repair, cell cycle regulation, and the maintenance of embryonic stem cell pluripotency by specifically cleaving K48- and K63-linked ubiquitin chains from substrate proteins [5]. Clinical studies have linked loss-of-function variants in OTUD5 to multiple developmental syndromes, such as X-linked intellectual disability syndrome (OMIM #301056), which are characterized by severe developmental delay, congenital heart defects, and neuroectodermal dysplasia [6]. OTUD5 has been shown to regulate early embryonic gene transcription by stabilizing chromatin regulators such as ARID1A/B and HDAC2 [5]. However, the spatiotemporal mechanisms by which OTUD5 regulates the dynamic balance between proliferation and differentiation in neural progenitor cells (NPCs) remain undefined.