NOTCH-mediated glial CXCL9:SPP1 polarization by GSK3β exacerbates postnatal enteric nervous system dysfunction in Hirschsprung disease.

BACKGROUND: Current surgical treatment of Hirschsprung disease (HSCR), a rare congenital intestinal disorder, is frequently complicated by defecation dysfunction. The role of glycogen synthase kinase 3 beta (GSK3β), aberrantly highly expressed in HSCR tissues, in the HSCR pathogenesis remains unclear. METHODS: Publicly available scRNA-seq data of HSCR were analyzed. Histopathological characteristics of clinical samples were observed from HSCR children using Hematoxylin-Eosin staining. Regulation of the CXCL9:SPP1 (CS) polarization of glial cells by the GSK3β-NOTCH axis was assessed in both clinical samples and Schwann cells through immunofluorescence (IF), qRT-PCR, and Western blot. Regulation of neuronal development by the CS polarization was assessed in a co-culture system of Schwann cells and mouse dorsal root ganglion neurons using TUNEL staining, ELISA, IF, and patch-clamp techniques. In an Ednrb knockout mouse model, we verified in vivo that GSK3β regulated CS polarization of enteric glial cells via the NOTCH signaling pathway, which exacerbated developmental abnormalities of the enteric nervous system (ENS) and accelerated the progression of HSCR in vivo. RESULTS: Enteric glial cells were significantly increased in HSCR mouse samples, mainly as an SPP1+ subpopulation where GSK3β was highly expressed and the NOTCH signaling pathway was significantly enriched. GSK3β knockdown significantly attenuated CS polarization and blocked the NOTCH pathway. Mechanistically, GSK3β promoted enteric glial cell CS polarization by activating the NOTCH signaling pathway and aggravated the developmental abnormalities of the ENS, ultimately exacerbating postnatal ENS dysfunction and thereby potentially worsening HSCR-associated complications. CONCLUSION: GSK3β upregulation promotes NOTCH signaling pathway activation, which in turn regulates CS polarization and potentially exacerbates postnatal ENS dysfunction and aggravates HSCR-associated complications.