Time‑Restricted Feeding Primes Senescent Cells for Dasatinib‑Quercetin‑Induced Apoptosis via mTORC1 Suppression and Autophagy‑Dependent Sensitization

Hypothesis: Combining intermittent fasting (time‑restricted feeding, TRF) with the senolytic regimen dasatinib plus quercetin (D+Q) produces synergistic clearance of senescent cells that exceeds the additive effects of each intervention alone. TRF reduces mTORC1 signaling and activates autophagy, which alters the apoptotic threshold of senescent cells by decreasing anti‑apoptotic BCL‑2 family protein expression and increasing mitochondrial priming. This metabolic state renders senescent cells more vulnerable to D+Q, where dasatinib inhibits SRC‑family kinases that sustain survival signaling and quercetin antagonizes BCL‑2/xL, thereby triggering apoptosis. Consequently, the combined treatment yields a greater reduction in senescent cell burden, SASP secretion, and age‑related functional decline than either approach singly, extending healthspan beyond current limits.

Testable predictions:

1. In naturally aged mice (20‑24 months), four groups will be compared: ad libitum control, D+Q alone (weekly oral dosing), TRF alone (16‑hour daily fast), and D+Q + TRF (same D+Q schedule during the fasting window).
2. Senescent cell burden will be quantified by p16Ink4a‑positive immunohistochemistry and SA‑β‑gal activity in liver, kidney, and adipose tissue. We predict the D+Q + TRF group will show a ≥50 % greater reduction than the sum of the individual treatments (additive expectation).
3. SASP cytokine levels (IL‑6, TNF‑α, MMP‑3) in plasma and tissue homogenates will be lowest in the combined group, correlating with senescent cell clearance.
4. Molecular readouts will confirm mechanistic links: TRF will decrease phosphorylated S6K (p‑S6K) and increase LC3‑II/LC3‑I ratio and decrease p62, indicating mTORC1 inhibition and autophagy activation. In senescent cells isolated from these tissues, BCL‑2/xL protein levels will be lower and BIM/BAX higher only in the D+Q + TRF condition.
5. Functional outcomes—grip strength, treadmill endurance, and frailty index—will improve most in the combined group, and median lifespan will be extended beyond the ~36 % increase observed with D+Q alone [1].

Falsifiability: If the D+Q + TRF group does not demonstrate a statistically significant greater reduction in senescent cell burden or SASP than the additive expectation (i.e., the combined effect equals or is less than the sum of D+Q alone and TRF alone), the hypothesis is falsified. Likewise, if autophagy inhibition (e.g., via chloroquine co‑administration) abolishes the enhanced senolytic effect without affecting D+Q or TRF individually, the proposed autophagy‑dependent priming mechanism is refuted.

Sex‑specific analysis will be included, as prior work suggests divergent senolytic responses [3]. By integrating metabolic reprogramming with senescent cell apoptosis, this hypothesis addresses the empiric gap identified in the literature—no peer‑reviewed study has tested D+Q with intermittent fasting protocols [1‑6]—and offers a concrete, falsifiable roadmap to determine whether timed nutrient restriction can unlock the full therapeutic potential of senolytics for healthspan extension.