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

backgroundCurrent 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.

methodsPublicly 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.

resultsEnteric glial cells were significantly increased in HSCR mouse samples, mainly as an SPP1

conclusionGSK3β upregulation promotes NOTCH signaling pathway activation, which in turn regulates CS polarization and potentially exacerbates postnatal ENS dysfunction and aggravates HSCR-associated complications.