Hypothesis

Time‑restricted exercise (TRE) performed during the fasting window of time‑restricted eating synchronizes mTOR oscillations, priming senescent cells for senolytic‑induced synthetic lethality through lysosomal priming and heightened autophagic flux.

Mechanistic Rationale

Senolytics such as dasatinib+quercetin (D+Q) trigger apoptosis in senescent cells that depend on anti‑apoptotic pathways (e.g., BCL‑2 family). Recent work shows that transient mTOR inhibition during fasting upregulates autophagy and lysosomal biogenesis, increasing the capacity to degrade apoptotic debris (source). Exercise acutely activates AMPK, which transiently suppresses mTORC1 and amplifies lysosomal cathepsin activity. When TRE‑aligned exercise coincides with the nadir of mTOR activity, senescent cells experience a dual stress: (1) senolytic‑mediated mitochondrial damage and (2) heightened lysosomal readiness to execute apoptosis‑associated autophagy. This creates a synthetic lethal interaction that is absent when either intervention is applied alone.

Testable Predictions

1. Mice receiving D+Q plus TRE‑aligned high‑intensity interval training (HIIT) will show a greater reduction in p16^Ink4a^‑positive cells in liver and adipose tissue than D+Q alone or TRE‑aligned exercise without senolytics.
2. The combinatorial regimen will increase lysosomal markers (LAMP1, cathepsin B) specifically in senescent cells, measurable by flow cytometry‑based lysosomal activity assays.
3. Physical frailty indices (grip strength, gait speed) will improve by >30% over baseline after 8 weeks, exceeding the improvement seen with either monotherapy.
4. Pharmacologic blockade of lysosomal acidification (e.g., with bafilomycin A1) will abrogate the synergistic benefit, confirming lysosomal dependence.

Experimental Approach

• Animals: 24‑month-old C57BL/6 mice, n=10 per group (Control, D+Q only, TRE‑aligned HIIT only, D+Q+TRE‑HIIT).

• TRE protocol: 8‑hour feeding window aligned with the dark phase; food access restricted accordingly.

• Exercise: Treadmill HIIT (1 min at 20 m/min, 1 min rest, 8 repeats) performed 30 min before the end of the fasting window, 3×/week.

• Senolytic: Dasatinib (5 mg/kg) + quercetin (50 mg/kg) i.p., administered on two consecutive days each week.

• Readouts (week 0, 4, 8):

  • Senescent burden: p16^Ink4a^ immunostaining, SA‑β‑gal activity.
  • Lysosomal function: LAMP1 immunofluorescence, Magic Red cathepsin B assay in isolated hepatocytes/adipocytes.
  • Autophagic flux: LC3‑II/I ratio with chloroquine chase.
  • Functional tests: grip strength, rotarod, treadmill endurance.
  • Serum biomarkers: SASP factors (IL‑6, MCP‑1) via ELISA.

• Pharmacologic control: Subset receiving D+Q+TRE‑HIIT plus weekly bafilomycin A1 (1 mg/kg i.p.) to test lysosomal necessity.

Expected Outcomes and Falsifiability

If the hypothesis is correct, the D+Q+TRE‑HIIT group will demonstrate a statistically significant (p<0.05) greater reduction in senescent cell burden and enhanced lysosomal activity relative to all other groups, accompanied by superior functional improvement. Failure to observe these differences—especially if lysosomal inhibition does not diminish the effect—would falsify the proposed mechanism. Conversely, a lack of added benefit despite confirmed lysosomal engagement would suggest that senolytic efficacy is not limited by lysosomal capacity under these conditions, directing focus toward alternative synergistic pathways (e.g., NAD+‑SIRT1 signaling).