Glial Cells Turn Against Gut Neurons in Hirschsprung Disease, Study Finds

Hirschsprung disease, a congenital gut disorder that leaves infants unable to pass stool properly, has long been understood as a story about missing nerve cells. Now, a new study published in iScience suggests that another cast member deserves far more attention: the enteric glial cells that surround and support the gut’s nervous system. Researchers led by Yanyi Li and Zebing Zheng report that a redox-regulating enzyme called TXNRD1 drives enteric glia into a pathological state that actively undermines the neurons they are supposed to nurture, offering a fresh mechanistic explanation for why the diseased bowel malfunctions even beyond its missing nerve cells.

Hirschsprung disease arises when enteric neural crest cells fail to migrate and differentiate properly during development, leaving the distal intestine devoid of the myenteric and submucosal nerve plexuses that normally coordinate peristalsis. The result is abdominal distension and refractory constipation, typically treated by surgically removing the aganglionic segment and reconstructing the bowel. Yet a substantial proportion of patients continues to suffer long-term complications, including enterocolitis, anastomotic strictures, fecal soiling, persistent constipation, and the need for reoperation. These persistent problems have intensified the search for the molecular mechanisms underlying the disease, and the new work points squarely at the glial microenvironment.

Enteric glial cells are the most abundant non-neuronal component of the enteric nervous system, and they are far more than passive scaffolding. They maintain epithelial integrity, coordinate neuro-immune signaling, and support neuronal survival. Recent single-cell RNA sequencing studies have revealed that glia exist in multiple transcriptionally distinct states, some of which resemble reactive microglia in the brain. In tumor-associated macrophages, researchers have described a CXCL9-to-SPP1 polarization axis that separates anti-inflammatory from pro-disease functional states. Whether enteric glia undergo a comparable polarization program in Hirschsprung disease had never been established, and that question became the starting point for the investigation.

The team began with an integrated single-cell transcriptomic analysis of colonic tissue from a mouse model of Hirschsprung disease based on loss of the endothelin receptor B gene, Ednrb, alongside wild-type controls. Among the five transcriptionally distinct glial subclusters they identified, one stood out: a subcluster enriched for both TXNRD1 and SPP1 that expanded markedly in disease samples, while a subcluster marked by Apoe shrank. Enrichment analyses of this expanded population highlighted extracellular matrix organization, cell adhesion, focal adhesion, and PI3K-Akt and MAPK signaling programs. Computational modeling of ligand-receptor interactions further showed that this subcluster engaged in the strongest predicted communication with enteric neurons of any glial population, including a predicted VCAM1-NCAM1 signaling axis.

To probe the regulatory logic behind this glial remodeling, the researchers turned to network-based virtual perturbation using the scTenifoldNet framework, which simulates gene knockout or overexpression within an inferred gene regulatory network. Both virtual knockout and virtual overexpression of TXNRD1 converged on regulatory programs involving redox control, ERK/MAPK-associated immediate-early response genes, and stress-associated glial features. Virtual overexpression produced a more focused pathological shift, with SPP1 emerging as the most prominently perturbed gene. These in silico results positioned TXNRD1, a central regulator of intracellular redox homeostasis, as a plausible upstream driver of the disease-associated glial state.

Human tissue evidence followed. Proteomic profiling of paired proximal dilated and stenotic colonic segments from children undergoing pull-through surgery revealed marked TXNRD1 upregulation in the stenotic, aganglionic tissue, together with significant enrichment of the MAPK signaling pathway. Validation in eight paired patient samples using quantitative PCR, western blotting, and immunofluorescence confirmed elevated TXNRD1 alongside increased phosphorylation of Raf, MEK, and ERK, while the stress kinases p38 and JNK showed no significant changes, indicating selective activation of the ERK branch of the MAPK cascade. Immunofluorescence staining also showed that SPP1 was increased and CXCL9 reduced within S100β-positive enteric glia in the stenotic segments, mirroring the single-cell findings in human tissue.

The mechanistic experiments in cultured rat enteric glial cells tied these observations together. When the researchers overexpressed TXNRD1, the cells selectively increased ERK phosphorylation, elevated their production of SPP1, suppressed CXCL9, accumulated reactive oxygen species, migrated less effectively, and secreted less glial cell line-derived neurotrophic factor, or GDNF. Treatment with the ERK inhibitor SCH772984 partially restored CXCL9 expression and blunted SPP1 upregulation, while the antioxidant N-acetyl-L-cysteine reduced ROS accumulation, dampened ERK activation, and reversed the SPP1-high/CXCL9-low polarization. Notably, ERK inhibition did not normalize ROS levels, suggesting that redox dysregulation acts upstream of ERK/MAPK signaling rather than as a downstream consequence.

The consequences for neurons were striking. In co-culture experiments with primary dorsal root ganglion neurons, TXNRD1-overexpressing glia increased neuronal apoptosis, impaired neuronal migration, reduced phosphorylation of the RET receptor and expression of its co-receptor GFRα1, downregulated the synaptic proteins PSD-95 and synaptophysin, and disrupted intracellular calcium homeostasis. Levels of the neurotrophic factors NGF and BDNF also fell in the co-culture system. Crucially, both ERK inhibition and supplementation with exogenous GDNF partially rescued these defects, implicating suppression of the GDNF/GFRα1/RET signaling axis as a key route by which the remodeled glia damage neuronal development and function.

The in vivo experiments provided the most compelling translational signal. The team generated Ednrb conditional knockout mice, which develop Hirschsprung-like phenotypes, and delivered an adeno-associated virus carrying TXNRD1-targeting shRNA by intraperitoneal injection at postnatal day 5. Knockdown of TXNRD1 markedly ameliorated the disease features, reducing abdominal distension, proximal colonic dilatation, distal stenosis, and fecal retention. Treated mice survived significantly longer, with four animals reaching the study endpoint, and fecal water content rose toward control levels, indicating improved intestinal function. At the molecular level, TXNRD1 suppression decreased SPP1, restored CXCL9, increased the neuronal marker TUJ1 and the neural crest marker p75, and upregulated the communication mediators NCAM1 and VCAM1 in the bowel.

The authors are careful to acknowledge the limitations of their work. The proteomic screen relied on only two paired patient samples, the validation cohorts were small, and the intraperitoneal viral delivery was not restricted to enteric glia, complicating cell-type-specific interpretation. Primary dorsal root ganglion neurons served as a surrogate for enteric neurons, and the temporal origins of the SPP1-high glial state remain unresolved, with lineage tracing and spatial transcriptomics needed to clarify its development. Even so, the study establishes a coherent mechanistic framework linking redox dysregulation, ERK/MAPK activation, pathological glial polarization, and neuronal dysfunction in Hirschsprung disease. By showing that enteric glia are active participants in the disease rather than passive bystanders, and that silencing TXNRD1 alleviates HSCR-like phenotypes in mice, the work nominates TXNRD1 and the glial polarization program it controls as promising targets for future therapies aimed at improving outcomes for children with this challenging disorder.

Subject of Research: The role of TXNRD1-driven enteric glial remodeling in neuronal dysfunction in Hirschsprung disease

Article Title: TXNRD1-driven enteric glial remodeling promotes neuronal dysfunction in Hirschsprung disease

Article References: Li, Y., Wang, Y., Jin, Z., Tang, C., Xia, X., Gong, Y., Du, Q., Huang, L., Li, Z., Liao, Y., He, S., Wang, B., Liu, Y., & Zheng, Z. (2026). TXNRD1-driven enteric glial remodeling promotes neuronal dysfunction in Hirschsprung disease. iScience, 29(10), Article 117636. https://doi.org/10.1016/j.isci.2026.117636

Image Credits: AI Generated

DOI: 10.1016/j.isci.2026.117636

Keywords: Hirschsprung disease, enteric glial cells, TXNRD1, SPP1, CXCL9, ERK/MAPK signaling, redox dysregulation, GDNF, single-cell RNA sequencing, enteric nervous system, Ednrb knockout mice, neurotrophic signaling