A Second Pathogenic Protein, PolyGN2C-iso2, Reveals a Dual-Protein Pathology in Neuronal Intranuclear Inclusion Disease.

BACKGROUND

Neuronal intranuclear inclusion disease (NIID) pathogenesis has been strongly linked to uN2CpolyG translated from NOTCH2NLC transcript variant 1. However, emerging evidence suggests that NOTCH2NLC transcript variant 2 may also generate a disease-relevant protein, PolyGN2C-iso2, but isoform-discriminating and antibody-independent evidence remains incomplete.

METHODS

We characterize the aggregation propensity of PolyGN2C-iso2 in vitro, develop isoform-discriminating monoclonal antibodies for its detection in patient tissues, and perform targeted proteomic analysis of laser-microdissected p62-positive lesion cells. An AAV-mediated mouse model expressing PolyG(108×)N2C-iso2 is generated to assess its pathogenic potential, followed by behavioral, imaging, histopathological, proteomic, and functional analyses.

RESULTS

NOTCH2NLC transcript variant 2 generates a distinct protein, PolyGN2C-iso2, which forms aggregates in vitro. Using developed monoclonal antibodies together with targeted proteomics, we provide evidence that PolyGN2C-iso2 is present within the pathognomonic intranuclear inclusions in NIID patient tissues, where it co-localizes with uN2CpolyG. The PolyG(108×)N2C-iso2 mouse model recapitulates key pathological hallmarks of NIID, including white matter abnormalities and cognitive deficits not fully captured by previous models. Mechanistically, PolyGN2C-iso2 expression is found to induce profound mitochondrial dysfunction.

CONCLUSIONS

Our findings support the possibility that NIID involves a dual-protein pathogenic process involving both uN2CpolyG and PolyGN2C-iso2, which may have implications for therapeutic strategies targeting NOTCH2NLC-derived pathogenic proteins.