Hypothesis

Aging‑induced loss of CB1 signaling in the small intestine triggers nuclear accumulation of YAP in enterocytes, which drives a senescence‑associated secretory phenotype (SASP) rich in IL‑6, TNF‑α and matrix metalloproteinases (MMP‑3/9). These SASP factors cleave occludin (but not ZO‑1) at the extracellular loop, increasing paracellular permeability specifically in the small intestine while sparing the colon. The resulting microbial translocation and circulating DAMPs amplify systemic inflammaging. Restoring CB1 activity or inhibiting YAP‑dependent SASP will prevent occludin cleavage, preserve barrier integrity, and reduce systemic inflammatory markers.

Mechanistic Rationale

CB1 receptors normally couple to Gi/o proteins that inhibit cAMP/PKA and suppress YAP translocation via LATS1/2 activation (see CB1‑ZO‑1 mechanism)【https://www.frontiersin.org/journals/endocrinology/articles/10.3389/fendo.2023.1241097/full】. In aged enterocytes, ↓CB1 relieves this inhibition, allowing YAP to enter the nucleus. Nuclear YAP cooperates with TEAD transcription factors to upregulate the miR‑17~92 cluster, which targets the mRNA of the senescence suppressor p21^CIP1, thereby promoting p16^INK4a‑dependent senescence【https://doi.org/10.1093/gerona/glt106】. Senescent enterocytes secrete a SASP that includes MMP‑3 and MMP‑9, proteases known to cleave occludin’s extracellular domain, disrupting its homophilic binding without affecting ZO‑1 scaffolding【https://pmc.ncbi.nlm.nih.gov/articles/PMC9427709/】. Occludin loss preferentially increases permeability to bacterial LPS and flagellin, while ZO‑1 remains relatively preserved, explaining the region‑specific pattern observed in primate colon versus small intestine【https://doi.org/10.1093/gerona/glt106】. Colon epithelial cells retain higher basal expression of miR‑200c, which represses YAP and MMP transcription, protecting occludin and accounting for the subset of healthy elderly with unchanged colonic barrier function.

Predictions and Experimental Design

1. Spatial YAP activation – Immunofluorescence of young vs. aged mouse intestine will show increased nuclear YAP in small‑intestinal villi but not in colonic crypts.
2. SASP‑dependent occludin cleavage – Isolated intestinal epithelial cells from aged mice will exhibit elevated MMP‑3/9 activity and occludin fragments in supernatants; pharmacological MMP inhibition (SB‑3CT) will restore occludin levels without altering ZO‑1.
3. CB1 rescue – Villus‑targeted AAV‑CB1 transduction in aged mice will reduce nuclear YAP, lower SASP cytokines, prevent occludin cleavage, decrease FITC‑dextran permeability, and lower serum IL‑6/TNF‑α and LPS‑binding protein.
4. YAP inhibition – Pharmacologic YAP‑TEAD disruption (verteporfin) will mimic CB1 rescue effects, confirming YAP as the downstream effector.
5. Falsifiability – If CB1 restoration or YAP inhibition fails to reduce occludin cleavage, SASP, or systemic inflammaging despite restoring ZO‑1, the hypothesis is refuted.

Potential Outcomes and Falsifiability

• Support: Significant reduction in circulating IL‑6/TNF‑α (≥30%), decreased plasma LPS, and improved tight‑junction integrity (↑TEER, ↓paracellular flux) in aged mice after CB1 or YAP targeting, coupled with occludin preservation.
• Refute: No change in systemic inflammaging markers or occludin status despite verified CB1/YAP modulation, indicating that barrier loss is not the primary driver of inflammaging in this model.

This hypothesis integrates CB1 signaling, YAP‑mediated senescence, protease‑dependent occludin remodeling, and regional miRNA buffering to explain heterogeneous gut barrier aging and offers a clear, testable route to link gut‑targeted interventions with systemic inflammaging outcomes.