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

The review, led by Jim Jacob and Federica Sarno of the University of Groningen’s University Medical Center Groningen, together with Maria Zernova, Inge M. Strating, Barbara van Munster, and Monika Trzpis, set out to answer a deceptively simple question. If delirium arises from an interaction between predisposing vulnerabilities—advanced age, neurodegeneration, frailty—and acute precipitating factors such as inflammation, surgery, or critical illness, could methylation changes be part of the molecular wiring that connects the two? The team systematically screened the literature and identified 1,110 potentially relevant articles. After applying their inclusion criteria, only nine studies survived. That attrition alone tells a story: despite a strong theoretical rationale, the empirical record linking DNA methylation to delirium is thin, and the authors conclude that robust evidence remains lacking.

The nine included studies split into two broad families. Six examined differential DNA methylation at candidate CpG sites—the cytosine–guanine dinucleotides where methyl groups are most commonly attached and where methylation is known to influence transcription. Three took a different tack, assessing age-related methylation patterns stratified by delirium status, exploiting the fact that the methylation state of many sites across the genome shifts predictably with chronological and biological age. Only four of the nine studies performed true genome-wide differential methylation analysis, the kind of unbiased survey that can, in principle, discover methylation signatures no one thought to look for in advance. The remaining studies were hypothesis-driven, focusing on genes selected for their plausibility.

Among the four genome-wide studies, two independent investigations each identified a single CpG site that was hypomethylated in patients who experienced delirium. The first site, cg21295729, annotated to the gene LDLRAD4, was found in blood and involved a comparison of 43 delirium cases against 44 controls. The second, cg16526133, annotated to ADAMTS9, emerged in brain tissue, comparing 11 cases with 25 controls. Hypomethylation at a CpG site typically suggests increased accessibility of DNA to transcriptional machinery, hinting—though only hinting—that these genes might behave differently in people who develop the syndrome. Notably, LDLRAD4 and ADAMTS9 have no obvious shared functional storyline, and the two studies pointed to different tissues, different cohorts, and different design logic. The other two genome-wide studies found no statistically significant methylation differences at all, a result the review does not shy away from reporting.

Perhaps the most intriguing signal in the review involves inflammation. Gene-specific analyses showed that methylation levels at CpG sites annotated to the TNF gene—the canonical inflammatory cytokine tumor necrosis factor—correlated negatively with age in peripheral blood mononuclear cells and in whole blood samples taken from delirious patients. In plain terms, the older the delirious patient, the lower the methylation at these TNF-associated sites, a pattern consistent with progressively disinhibited inflammatory gene expression in a vulnerable host. Critically, this age-dependent pattern was absent in saliva and buccal samples, and absent in control subjects. That tissue specificity matters enormously. It suggests that the association is not a generic artifact of aging, but something particular to blood-derived immune cells in patients who actually went through a delirious episode. Blood, of course, carries the immune cells believed to orchestrate the systemic inflammatory response that many theories place at the center of delirium pathophysiology.

A complementary pattern emerged for genes involved in neurotrophic signaling. CpG sites annotated to BDNF, GDNF, and NR4A2—genes encoding factors that support neuronal survival, plasticity, and synaptic resilience—showed a positive correlation with age in delirious patients, meaning methylation increased as patients got older. Because increased promoter methylation is often, though not invariably, associated with reduced gene expression, the finding raises the possibility of an age-related epigenetic tightening of the brain’s own maintenance systems. In a patient facing an acute inflammatory or metabolic insult, the combination of a revved-up immune program and a dampened neurotrophic program is precisely the kind of double hit that theories of delirium have long hypothesized: the brain, already running on diminished reserves, is simultaneously provoked and deprived of support at the moment of the insult.

The reviewers are careful, and admirably so, about what such signals can and cannot support. Methylation measured in blood may or may not reflect what is happening in neurons or glia; the brain-tissue study was small, with just 11 cases; and methylation is highly sensitive to cell-type composition, meaning that shifts in the proportions of immune cell subsets could masquerade as differential methylation. The studies also varied widely in sample size, tissue type, statistical thresholds, and whether analyses corrected for the many confounders—age, medication, illness severity, smoking, diet—that are known to sculpt the methylome. Two of the four genome-wide studies reported nothing significant, and the authors emphasize that the existing literature is limited both in number and in methodological rigor, constraining firm conclusions about whether methylation is a cause, a consequence, or merely a correlate of the syndrome.

What makes the field worth watching is the underlying biology. DNA methylation is a dynamic layer of regulation; while many marks are stable, others respond within hours or days to inflammation, hypoxia, and stress hormones, all of which surge during the kinds of acute events that precipitate delirium. Methylation also offers something epidemiology alone cannot: a mechanistic bridge between vulnerability and insult. If older brains carry methylation signatures that favor TNF derepression in immune cells while simultaneously suppressing neurotrophic support, then a surgical trigger or a fever could plausibly flip a network of gene-expression switches that cumulatively destabilize cognitive control circuits. The convergence of the two methylation trends—one pointing toward immune activation, the other toward weakened neuronal maintenance—is conceptually coherent with prevailing models of delirium, even if the empirical evidence has not yet reached the strength that coherence deserves.

The practical implications of the review are correspondingly modest but clear. The authors argue that what the field needs is larger, well-powered, longitudinal cohort studies with genome-wide methylation profiling, careful adjustment for confounders, and ideally paired blood and brain tissue to test whether peripheral signals mirror central ones. Prospective designs, in which methylation is measured before surgery or before an infection takes hold, would help distinguish pre-existing epigenetic vulnerability from methylation changes induced by the episode itself. Standardized covariate adjustment and cell-composition correction would strengthen comparability across studies. Until then, the review stands as both a map and a warning: DNA methylation remains a theoretically compelling contributor to delirium pathophysiology, supported by a handful of suggestive and partially converging findings, but the field has yet to produce the rigorous, replicated evidence required to elevate epigenetics from plausible mechanism to established biology. For clinicians and researchers watching a rapidly aging global population, closing that evidence gap is not an academic luxury; it is the necessary groundwork for the first epigenetically informed approaches to predicting, and perhaps one day preventing, one of medicine’s most disruptive syndromes.

To appreciate why methylation has drawn attention in delirium research, it helps to understand how plastic this mark can be. Cytosine methylation is maintained by enzymes of the DNMT family and actively removed through TET-mediated oxidation pathways, creating a reversible system that responds to environmental and physiological cues. In the immune system, stimulus-responsive methylation changes are well documented: monocytes and T cells remodeled their methylomes within hours of activation, and inflammatory cytokine promoters are among the genomic regions most sensitive to such remodeling. This makes blood an attractive, if imperfect, surrogate tissue for studying the interface between systemic inflammation and brain dysfunction.

Age adds a second layer of complexity. Large consortium analyses of thousands of blood and brain samples have shown that methylation drifts in a highly stereotyped way across the lifespan, and that some loci accelerate or decelerate their drift in the context of disease. The delirium studies reviewed here sit squarely within this tradition, testing whether the syndrome modifies the expected age-methylation relationship rather than producing absolute differences between cases and controls. That analytic framing may be well suited to a condition defined by interactions between age-related vulnerability and acute insult, though it also demands large, age-diverse samples that most existing cohorts cannot provide.

The review also reflects a broader methodological moment. Epigenome-wide association studies in neuropsychiatry have repeatedly shown that small cohorts underpowered to detect the modest effect sizes typical of methylation differences. The field’s experience elsewhere suggests that combining cohorts, harmonizing platforms, and sharing raw data will be prerequisites for any credible replication of the signals highlighted in this work.

Subject of Research: DNA methylation as a candidate molecular mechanism in delirium pathophysiology

Article Title: A systematic review on the contribution of DNA methylation to delirium pathophysiology

Article References: Jacob, J., Zernova, M., Strating, I. M., van Munster, B., Sarno, F., & Trzpis, M. (2026). A systematic review on the contribution of DNA methylation to delirium pathophysiology. Epigenetics Communications. https://doi.org/10.1186/s43682-026-00051-9

Image Credits: AI Generated

DOI: 10.1186/s43682-026-00051-9

Keywords: delirium, DNA methylation, epigenetics, systematic review, TNF, inflammation, aging, neurotrophic signaling, BDNF, genome-wide methylation, neuropsychiatry, blood-based biomarkers