Intestinal epithelial ion channel TMEM16A deficiency impairs enteroendocrine cell differentiation and epithelial homeostasis and enhances anxiety-like behavior in mice

Transmembrane member 16A (TMEM16A) is involved in intestinal epithelial ion transport and secretory homeostasis, but its effects on enteroendocrine cell (EEC) function and gut-brain axis-related behaviors remain unclear. This study aims to investigate the effects of intestinal epithelial TMEM16A deficiency on the differentiation phenotype of chromogranin A (CgA)-positive EECs, related gut-brain signaling molecules, hippocampal inflammatory signals and anxiety-like behavior, and to analyze the role of the vagal afferent pathway in this process.

Male intestinal epithelial TMEM16A conditional knockout mice (KO) and littermate control mice (wild type, WT), aged 8 to 10 weeks and weighing 24 to 26 g, were subjected. Terminal ileal mucosal tissues from WT mice were treated with DMSO, T16A_inh-A01 or CaCC_inh-A01, and Ca²⁺-activated Cl^- secretion were measured using short-circuit current recording. Intestinal epithelial TMEM16A expression between the WT and KO mice was compared with Western blotting and immunofluorescence staining. Behavioral parameters were compared between the WT and KO mice using open field, elevated plus maze and light-dark box tests. The WT and KO mice were treated with cholecystokinin-saporin conjugate (CCK-SAP) or blank saporin to establish WT+Blnk-SAP, WT+CCK-SAP, KO+Blnk-SAP and KO+CCK-SAP groups (n=10), and to evaluate the involvement of CCK-sensitive vagal afferent pathways. The differentiation phenotype of CgA⁺ EEC in intestinal tissues between the 2 types of mice was compared by immunohistochemistry and qPCR. WT- and KO-derived organoids were divided into untreated and bone morphogenetic protein 4 (BMP4)-treated groups to detect organoid differentiation phenotypes and levels of 5-hydroxytryptamine (5-HT), tryptophan hydroxylase 1 (TPH1), glucagon-like peptide-1 (GLP-1) and peptide YY (PYY). Immunohistochemical staining and Western blotting were performed to detect the expression of intestinal epithelial renewal-related molecules and those in inflammatory signals in the hippocampal CA3 region.

Compared with the DMSO group, TMEM16A inhibitors reduced Ca²⁺-activated Cl^- secretion in intestinal epithelium (P<0.0001). Compared with the WT group, the KO group exhibited significantly decreased intestinal epithelial TMEM16A expression (P<0.0001), and weakened TMEM16A immunofluorescence signals in intestinal villus and crypt epithelium; decreased total distance traveled, reduced time in the center, and lower center entry frequency in the open field test (P<0.0001); decreased total distance traveled and open-arm time (P=0.0171, P<0.0001) and increased closed-arm time (P=0.0460) in the elevated plus maze test; and decreased light-box entries and shortened time spent in the light box in the light-dark box (P<0.0001), but no significant change was observed in nose-poke frequency. Compared with the KO+Blnk-SAP group, the KO+CCK-SAP group showed an increase in open-arm time from 1.75% to 13.16% (P<0.0001), decreased closed-arm time (P=0.0045), increased total distance traveled and center-zone time in the open field test (P=0.0002, P=0.0238), and increased light-box time and entries (P=0.0023, P=0.0055). Compared with the WT mice, the KO mice showed decreased numbers of intestinal CgA⁺ EECs and CgA mRNA expression (P<0.0001), reduced proportion of mature budding organoids from 77.80% in the WT group to 23.60% in the KO group (P<0.0001), and decreased 5-HT and TPH1 levels (P<0.0001). BMP4 treatment increased 5-HT, TPH1, GLP-1 and PYY levels in WT organoids (P<0.0001, P<0.0001, P<0.0001, P=0.0060), whereas no significant changes were observed in the KO+BMP4 group. In the KO group, intestinal CgA, Lgr5 and Ascl2 expression levels were decreased (P<0.0001, P<0.0001, P=0.0016), whereas Ki67, Wnt3a, β-catenin, Dll1 and EGF expression levels were increased (P<0.0001), and NLRP3 and IL-1β expression levels in the hippocampal CA3 region were enhanced (P<0.0001).

Intestinal epithelial TMEM16A deficiency attenuates the differentiation phenotype of CgA⁺ EECs, reduces 5-HT, TPH1, GLP-1 and PYY levels, and is accompanied by enhanced NLRP3/IL-1β inflammatory signals in the hippocampal CA3 region and anxiety-like behavior. The CCK-sensitive vagal afferent pathway is involved in TMEM16A deficiency-related anxiety-like behavior.