CRISPR/Cas9-Corrected Isogenic iPSC Lines for Functional Characterization of Two Novel UBQLN2 Variants Identified in an FTD Patient

Pathogenic UBQLN2 variants have been implicated in neurodegenerative proteinopathies, including frontotemporal dementia (FTD). We previously identified altered protein homeostasis, oxidative stress, mitochondrial polarization, and antioxidant pathways in neural stem cells (NSCs) derived from a patient carrying two novel UBQLN2 variants. Here, we tested whether these variants drive the disease-associated phenotypes by generating CRISPR/Cas9-corrected isogenic iPSC lines and differentiating them into NSCs CRISPR/Cas9 editing generated independent isogenic iPSC clones carrying the corrected UBQLN2 sequence. Two clones were selected and characterized for pluripotency, karyotype, and STR profiling to confirm their identity and parental origin, and subsequently differentiated into NSCs. NSC identity was confirmed by established neural stem cell markers. Protein abundance, mitochondrial membrane potential, and ROS levels were assessed by immunoblotting, immunofluorescence, and flow cytometry. Compared with the parental patient-derived cells, the isogenic NSCs showed reduced UBQLN2 protein aggregation, restored mitochondrial membrane potential, and reduced ROS levels These findings provide functional evidence supporting a causal contribution of the UBQLN2 variants to the observed cellular phenotypes and establish patient-derived and CRISPR/Cas9-corrected isogenic iPSC models for investigating the cellular and molecular mechanisms underlying UBQLN2-associated neurodegeneration.