PAX3 deficiency is associated with NLRP3-related inflammatory signaling and hearing loss in a mouse model of Waardenburg syndrome

Abstract

PAX3 gene defects are a major cause of Waardenburg syndrome (WS), a genetic disorder characterized by pigmentation abnormalities and sensorineural hearing loss. However, the molecular mechanisms by which PAX3 deficiency contributes to hearing impairment remain incompletely understood, particularly the potential involvement of NLRP3-related inflammatory signaling. In this study, a heterozygous Pax3 knockout [Pax3(+/-)] mouse model was established using CRISPR-Cas9 technology, and cochlear morphology was examined by HE staining. Immunofluorescence staining, immunohistochemistry, western blotting, quantitative PCR, transcriptomic analysis, methylation-specific PCR, and TUNEL staining were performed to evaluate melanocyte-lineage dysfunction, cochlear inflammatory signaling, and apoptosis. Pax3(+/-) mice showed pigmentation abnormalities, elevated ABR thresholds, reduced Kcnj10 and Dct expression, increased NLRP3, IL-1β, and IL-18 expression, and enhanced cochlear apoptosis, without obvious gross cochlear malformation under HE staining. In PIG1 human melanocytes, PAX3 knockdown induced NLRP3 inflammasome activation, as evidenced by increased NLRP3 expression, caspase-1 activation, IL-1β and IL-18 release, GSDMD-related pyroptosis-associated changes, reduced cell viability, and increased apoptosis. MCC950 or S-adenosylmethionine partially attenuated the inflammatory and apoptotic changes in vitro. These findings support a model in which PAX3 deficiency is associated with melanocyte-lineage dysfunction, impaired strial homeostasis, and activation of an NLRP3-related inflammatory pathway. This pathway may contribute to WS-related hearing impairment, but further in vivo rescue studies are required to establish causality and therapeutic efficacy.