Bottom‑up longevity: testing whether central cholinergic activation reshapes gut enteroendocrine signaling to extend mammalian lifespan

Hypothesis

Central cholinergic enhancement via piracetam + choline increases vagal efferent output, driving acetylcholine release onto intestinal muscarinic M3 receptors on enteroendocrine L‑cells. This triggers GLP‑1 and PYY secretion, improves gut barrier function, shifts the microbiome toward a longevity‑promoting profile, reduces systemic inflammaging, and ultimately extends lifespan. If vagal efferent signaling or gut M3 receptors are blocked, the lifespan benefit will be abolished, falsifying the hypothesis.

Mechanistic Rationale Beyond Cited Work

In C. elegans, cholinergic motor neurons switch from ACR‑6‑mediated lifespan shortening to GAR‑3‑mediated extension via HSF‑1 activation. We propose an analogous temporal receptor switch in mammalian gut: early‑life M2 autoreceptor activation limits acetylcholine release and gut motility, whereas sustained central cholinergic tone later in life favors M3‑β‑arrestin signaling that activates epithelial HSF‑1 and upstream AKT‑mTORC1 pathways, promoting stress resistance and autophagy. This dual‑receptor model explains why indiscriminate cholinergic agonists have failed to extend lifespan—without the proper temporal pattern they may exacerbate M2‑mediated inhibitory tone.

Experimental Design

Subjects: 20‑month‑old C57BL/6J mice (n=10 per group). Groups: 1) Vehicle control; 2) Piracetam + choline (P+C) 300 mg/kg + 100 mg/kg daily; 3) P+C + atropine (muscarinic antagonist, 1 mg/kg i.p.); 4) P+C + subdiaphragmatic vagotomy; 5) P+C + GLP‑1 receptor antagonist (exendin‑9, 100 µg/kg). Readouts (baseline, 3 mo, 6 mo):

• Vagal efferent activity: in vivo electrophysiology of dorsal motor nucleus and c‑Fos staining.
• Gut luminal acetylcholine (HPLC).
• Enteroendocrine hormone levels (GLP-1, PYY) in plasma and intestinal tissue (ELISA).
• Gut barrier integrity: FITC‑dextran permeability, zonulin, occludin/claudin‑1 Western blot.
• Microbiome composition: 16S rRNA sequencing, SCFA quantification.
• Systemic inflammation: plasma IL‑6, TNF‑α, CRP.
• Tissue stress resistance: HSF‑1 nuclear translocation, HSP70 expression, autophagy markers (LC3‑II/I, p62) in intestinal epithelium.
• Lifespan: survival monitoring until natural death.

Predicted Outcomes

If the hypothesis holds, P+C mice will show ↑ vagal efferent firing, ↑ gut ACh, ↑ GLP‑1/PYY, ↓ permeability, ↑ SCFA‑producing taxa, ↓ inflammatory cytokines, ↑ epithelial HSF‑1/AKT activity, and a significant lifespan extension (~15 %). Groups receiving atropine, vagotomy, or GLP‑1R blockade will abolish these improvements and lifespan benefit, confirming the necessity of vagal‑M3‑GLP‑1 signaling. Failure to observe any of these changes would falsify the proposed bottom‑up mechanism.

Falsifiability

The hypothesis is falsifiable because it makes specific, directional predictions about neural, hormonal, microbial, and physiological readouts that can be independently measured. Demonstrating that central cholinergic elevation does not alter vagal efferent tone, or that gut M3/GLP‑1 signaling is unchanged despite lifespan extension, would refute the model. Conversely, confirming the predicted cascade would strongly support a bottom‑up longevity strategy where the brain primes the gut to drive systemic health.