Hypothesis

Aged airway club cells lose the ability to generate transient, high‑amplitude mTORC1 spikes after injury because declining CYP450‑mediated lipid metabolism disrupts the lysosomal amino‑acid sensing mechanism that gates mTOR activation. Restoring circadian‑timed CYP450 activity—or supplying its specific oxysterol products—will re‑establish pulsatile mTORC1 signaling, thereby rescuing regenerative proliferation without triggering chronic senescence.

Mechanistic Basis

• In young club cells, injury triggers a rapid rise in intracellular phospholipids that are metabolized by CYP2F2/CYP4B1 to bioactive oxysterols (e.g., 24‑S‑hydroxycholesterol). These oxysterols bind to SCAP/SREBP complexes, promoting lysosomal efflux of Rag GTPases and enabling a brief, nutrient‑dependent mTORC1 activation burst that drives proliferation [1][4][5].
• With age, CYP450 expression falls, oxysterol production drops, and lysosomal Ragulator‑Rag signaling becomes constitutively low‑active. This creates a permissive environment for chronic, low‑level mTORC1 signaling driven by basal amino‑acid pools and mitochondrial ROS, which pushes cells toward senescence and EMT [2][3].
• The loss of the oxysterol‑dependent “on‑switch” uncouples mTORC1 from injury cues, while the persistent ROS‑mediated “off‑switch” failure keeps the dial stuck at an intermediate position that supports neither homeostasis nor repair.

Testable Predictions

1. Pharmacological rescue – Treating aged mouse airway explants with a CYP450‑derived oxysterol (e.g., 24‑S‑hydroxycholesterol) will produce a transient mTORC1 phosphorylation peak (p‑S6K) within 15 min of mechan injury, matching the amplitude seen in young tissues.
2. Genetic rescue – Club‑cell‑specific overexpression of CYP2F2 in aged mice will restore injury‑induced p‑S6K pulses and increase Ki67+ progenitor cells by 2‑fold compared with controls.
3. Circadian gating – Administering the oxysterol at the peak of endogenous CYP450 expression (subjective night) yields a stronger and more sustained proliferative response than dosing at the trough, indicating that timing matters.
4. Falsification – If oxysterol supplementation fails to produce a transient mTORC1 spike or instead sustains basal p‑S6K levels, the hypothesis that CYP450‑derived lipids gate pulsatile mTORC1 is invalid.

Experimental Approach

• Model: Use 18‑month‑old C57BL/6 mice; isolate tracheal club cells via FSC‑high/CD24+ sorting.
• Injury: Apply naphthalene (10 mg/kg, i.p.) to induce club cell ablation; harvest lungs at 0, 2, 6, 12, 24 h post‑injury.
• Readouts: Western blot for p‑S6K (Thr389) and total S6K; immunofluorescence for Ki67 and SCGB1A1; ROS measurement with MitoSOX.
• Interventions: (a) Intratracheal delivery of 24‑S‑hydroxycholesterol (1 µM); (b) Adeno‑associated virus club‑cell promoter‑driven CYP2F2; (c) Vehicle controls.
• Analysis: Quantify the area under the p‑S6K curve (AUC) to distinguish transient versus chronic activation; correlate AUC with proliferation indices.

If oxysterol‑restored mTORC1 pulsatility improves regeneration without elevating senescence markers (p16^INK4a^, SA‑β‑gal), we will have demonstrated that the aging‑induced “dial” malfunction stems from a loss of ligand‑gated switching, not from mTORC1 activity per se. This reframes longevity interventions: instead of blunt mTOR inhibition, we aim to repair the signal‑generation machinery that lets cells toggle between survival and civilization modes.