Enteric cholinergic decline as a driver of epigenetic field cancerization via mucosal inflammation and mismatch repair suppression

Aging of the enteric nervous system (ENS) specifically reduces cholinergic neurotransmission, weakening the vagal anti‑inflammatory reflex that normally keeps mucosal immune activation in check. This loss permits microbiota‑derived LPS and secondary bile acids to chronically stimulate TLR4 and FXR pathways in colonic epithelium, elevating NF‑κB signaling and ROS production. Persistent oxidative stress accelerates DNA methylation drift in Lgr5+ stem cells, creating the patchwork epigenetic fields observed in nearly all colorectal cancers. Simultaneously, inflammation‑induced nitric oxide suppresses the expression of MLH1 and MSH2, directly impairing mismatch repair (MMR) activity. Thus, ENS cholinergic decline provides a mechanistic hub linking microbiome aging, epithelial epigenetic instability, and MMR deficiency—three processes that independently increase cancer risk but converge when neural regulation fails.

Testable predictions:

1. In aged mice, conditional ablation of ChAT+ enteric neurons will cause a measurable drop in colonic acetylcholine (measured by HPLC) coinciding with increased mucosal IL‑6, TNF‑α, and 8‑oxo‑dG levels.
2. These mice will show earlier onset of methylation‑age acceleration in isolated crypt stem cells (using the Horvath clock) and a higher frequency of MLH1/MSH2 loss‑of‑function patches compared with age‑matched controls.
3. Restoring cholinergic tone via systemic administration of a selective α7‑nAChR agonist (e.g., PNU‑282987) or via probiotic supplementation with acetylcholine‑producing Lactobacillus strains will normalize mucosal cytokine levels, reduce ROS, decelerate epigenetic drift, and rescue MMR protein expression.
4. Human colon biopsies from patients with early‑onset colorectal cancer will exhibit reduced choline acetyltransferase immunoreactivity in the myenteric plexus relative to cancer‑free age‑matched controls, and this deficit will correlate with local methylation‑age acceleration and MMR deficiency scores.

Falsifiability: If ENS cholinergic loss does not precede or correlate with mucosal inflammation, epigenetic drift, or MMR decline in the above models, the hypothesis would be refuted. Conversely, demonstration that cholinergic restoration mitigates all three downstream phenomena would support a causal role for ENS aging in field cancerization.