Abstract

Neuronal Intranuclear Inclusion Disease (NIID), caused by GGC repeat expansions in the NOTCH2NLC gene, has a poorly understood molecular pathogenesis. This study aimed to systematically delineate the molecular pathology of NIID for the first time by employing an unbiased proteomic approach in sweat gland tissue. We isolated sweat gland tissue from 20 NIID patients and 6 healthy controls via Laser Capture Microdissection and performed in-depth proteomic analysis using data-independent acquisition mass spectrometry, followed by functional annotation and mechanistic prediction through bioinformatics analyses, including Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Ingenuity Pathway Analysis. A total of 265 differentially expressed proteins were identified. Functional enrichment analysis revealed a pathological network composed of three core dysfunctions: (1) widespread mitochondrial dysfunction, evidenced by the general downregulation of proteins associated with energy metabolism and mitochondrial structure; (2) multidimensional autophagy failure, characterized by autophagic flux blockage (macroautophagy failure) and the predicted inhibition of Chaperone-Mediated Autophagy; and (3) a paradoxical and ineffective oxidative stress response, demonstrating a functional uncoupling between the upstream NRF2 activation signal and the execution of the downstream antioxidant pathway. The cellular validation confirmed that the pathogenic uN2CpolyG protein causes the downregulation of core hub proteins, substantiating the molecular pathology observed in patient tissue. Furthermore, a signal decoupling state was identified in the pivotal PI3K-Akt survival pathway. This study provides the first systematic proteomic view of NIID pathology in sweat gland tissue, substantiating that its core pathology is a self-reinforcing vicious cycle of mitochondrial dysfunction, abnormal autophagy, and oxidative stress imbalance. These findings offer a robust molecular framework for