Rapamycin-induced autophagy masks gut barrier leak without restoring dynamic ZO-1 resealing, leading to withdrawal‑dependent exacerbation of inflammaging
Hypothesis
Chronic mTORC1 inhibition with rapamycin reduces intestinal permeability by boosting autophagic clearance of damaged tight‑junction components, but it does not reinstate the active, mTORC1‑dependent actomyosin contractility required for rapid ZO‑1‑mediated purse‑string seals during epithelial cell extrusion. Consequently, the barrier remains structurally fragile; upon rapamycin withdrawal, the accumulated deficit in dynamic resealing overwhelms the transient autophagic benefit, causing a rebound increase in LPS translocation and systemic inflammaging.
Mechanistic Rationale
- mTORC1 regulates RhoA/ROCK‑driven actomyosin tension that powers the apical ZO‑1 redistribution observed 15 min before cell extrusion 4. Rapamycin suppresses this pathway, diminishing the mechanical force needed to form a functional seal even if total ZO‑1 protein levels are preserved.
- Autophagy removes misfolded or oxidized junctional proteins 2, lowering leak read‑outs in steady‑state assays, yet it does not replace lost proteins or rebuild the cortical actin‑myosin network that underlies rapid resealing.
- Epithelial proliferation, also mTORC1‑dependent, supplies new cells to replace extruded ones 3. Chronic rapamycin blocks this regenerative input, thinning the epithelium over time and increasing reliance on the compromised sealing mechanism.
When rapamycin is present, the combined effect of reduced leak (via autophagy) and lowered proliferative demand masks the underlying sealing defect. Upon drug cessation, autophagy activity falls back to baseline, proliferative capacity remains suppressed, and the defective actomyosin‑driven resealing becomes rate‑limiting, producing a net increase in permeability.
Testable Predictions
- In vivo withdrawal assay – Aged mice treated with rapamycin for 8 weeks will show reduced FITC‑dextran permeability during treatment; after a 2‑week washout, permeability will exceed baseline levels of age‑matched controls.
- Live imaging of ZO‑1 dynamics – In intestinal organoids, rapamycin will decrease the speed and apical accumulation of ZO‑1 during induced cell extrusion (measured by laser‑ablation‑triggered extrusion) without altering total ZO‑1 fluorescence.
- Rescue experiment – Co‑treatment with a low‑dose ROCK activator (e.g., CN03) during rapamycin exposure will restore normal ZO‑1 resealing kinetics and prevent the post‑withdrawal permeability surge, despite continued mTORC1 inhibition.
- Inflammaging read‑out – Serum LPS‑binding protein and IL‑6 will rise significantly after washout in rapamycin‑only mice but remain unchanged in the rapamycin + ROCK activator group.
Falsifiability
If washout does not produce a permeability increase above baseline, or if ZO‑1 resealing kinetics are unchanged by rapamycin, the hypothesis is refuted. Similarly, if ROCK activation fails to ameliorate the withdrawal effect, the proposed mTORC1‑actomyosin link would be insufficient.
Broader Implication
This reframes rapamycin’s geroprotective action in the gut as a temporary metabolic camouflage rather than a true rejuvenation of barrier architecture. Lifespan extensions observed under chronic mTOR inhibition may therefore depend on continuous drug exposure, raising questions about the suitability of intermittent dosing strategies for long‑term healthspan.