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.