Fasting-Induced TFEB Activation Selectively Clears Chronically Senescent Cells with High Lysosomal Dependence While Preserving Transient Negotiator Phenotype

Hypothesis: Intermittent fasting triggers TFEB‑mediated lysosomal biogenesis that preferentially eliminates senescent cells exhibiting a chronic SASP profile and high basal mTORC1 activity, while sparing transiently senescent hostage negotiators that maintain low mTORC1 signaling and rely on alternative survival pathways.

Rationale: Chronic senescent cells accumulate lysosomal damage and become dependent on lysosomal‑mediated autophagy for survival ([4]). Their SASP is enriched in IL‑6, CCL2 and MMPs, correlating with sustained mTORC1 signaling that drives TFEB cytoplasmic retention. In contrast, transient senescent fibroblasts in acute wounds display a PDGF‑AA‑rich SASP, low p‑S6K, and retain nuclear TFEB, allowing lysosomal priming without degradation ([1], [5]). A 20+ hour fasting window lowers systemic glucose and insulin, reducing mTORC1 activity via AMPK activation and promoting TFEB nuclear translocation (established). Nuclear TFEB upregulates lysosomal genes (LAMP1, CTSD) and enhances autophagic flux. We predict that chronic senescent cells, already primed for lysosomal dependence, will undergo lysosomal membrane permeabilization and caspase‑independent death upon TFEB‑driven lysosomal overload, whereas transient negotiators, with lower lysosomal load and intact TFEB nuclear residency, will resist this stress and continue to secrete PDGF‑AA to support repair.

Testable predictions:

1. In murine excisional wound models, mice subjected to 24‑hour fasts prior to injury will show a selective reduction of p16+/p21+ fibroblasts expressing IL‑6 and CCL2 (chronic SASP) but unchanged numbers of PDGF‑AA‑positive senescent fibroblasts compared with ad libitum controls (flow cytometry and immunofluorescence).
2. Pharmacologic inhibition of TFEB (using siRNA or the small‑molecule inhibitor CPT-1A) will abolish the fasting‑induced loss of chronic senescent cells without affecting PDGF‑AA+ populations.
3. Lysosomal immunostaining (LAMP1) will reveal increased lysosomal size and cathepsin D activity specifically in IL‑6+/CCL2+ senescent cells from fasted wounds, whereas PDGF‑AA+ senescent cells will show unchanged lysosomal metrics.
4. Functional readout: wound closure rates will improve in fasted wild‑type mice but not in TFEB‑deficient mice, linking selective senescent clearance to enhanced repair.

Potential confounders: fasting may also affect macrophage clearance; we will control by clodronate liposome depletion of macrophages to isolate the direct effect on senescent cells.

If validated, this hypothesis reframes senolytics not as blunt eliminators but as metabolic timers that distinguish pathogenic from beneficial senescence through lysosomal readiness, offering a route to harness fasting or TFEB activators as context‑specific senotherapies.