Hypothesis

Intermittent fasting (IF) activates a TFEB‑dependent lysosomal program that selectively targets senescent cells exhibiting a maladaptive SASP while sparing transient, wound‑healing senescent cells, thereby preserving their chaperone functions.

Mechanistic Rationale

1. TFEB as a fasting sensor – Prolonged fasting elevates intracellular AMPK and reduces mTORC1 activity, driving nuclear translocation of TFEB, the master regulator of lysosomal biogenesis and autophagy[1]. Enhanced lysosomal capacity increases the degradation flux of autophagosomes.
2. Selective cargo recognition – Senescent cells with a chronic SASP up‑regulate surface uPAR and secrete soluble factors that bind the autophagy adaptor p62/SQSTM1 via ubiquitin‑like motifs. TFEB up‑regulation increases p62 expression, promoting preferential engulfment of these SASP‑rich senescent cells.
3. Preservation of transient senescent cells – Acute wound‑induced senescent fibroblasts display a limited SASP (low IL‑6, high TGF‑β1) and lack sustained uPAR exposure. Their lysosomal membrane remains less permeabilized, making them resistant to p62‑mediated sequestration under IF‑induced autophagy.
4. Outcome – IF thus shifts the senescent cell pool toward a protective phenotype, reducing inflammatory burden while maintaining senescence‑dependent tissue repair signals.

Testable Predictions

• Prediction 1: In young mice subjected to a standardized dermal wound, 24‑hour IF cycles (alternating 16 h fasting/8 h refeeding) will increase nuclear TFEB and lysosomal LAMP1 levels in wound tissue compared with ad libitum fed controls[2].
• Prediction 2: Flow cytometry of wound‑derived p16^high^ cells will show a higher proportion of uPAR^low^/p62^low^ senescent cells (preserved transient phenotype) and a reduced uPAR^high^/p62^high^ fraction (cleared maladaptive senescent cells) after IF versus controls.
• Prediction 3: SASP profiling of wound homogenates will reveal decreased IL‑1α, IL‑6, and MMP‑3 concentrations, while TGF‑β1 and PDGF‑AA levels remain unchanged or elevated after IF.
• Prediction 4: Genetic knockdown of TFEB in fibroblasts will abolish the differential clearance, resulting in uniform reduction of both uPAR^low^ and uPAR^high^ senescent cells and impaired wound closure.

Experimental Approach

1. Animal model: C57BL/6 mice, 8 weeks old, receive full‑thickness excisional wounds on the dorsum.
2. Intervention: IF group (16 h fast/8 h feed) for 5 days post‑wounding; control group fed ad libitum.
3. Readouts (day 5 and day 10):
   • Immunofluorescence for nuclear TFEB and LAMP1.
   • Flow cytometry for p16, uPAR, and p62.
   • Multiplex ELISA for SASP cytokines.
   • Histological assessment of re‑epithelialization and collagen deposition.
   • Optional: TFEB‑flox;Cre‑ER fibroblasts transplanted into wounds to test cell‑autonomy.

Falsification Criteria

If IF fails to increase nuclear TFEB/lysosomal markers, or if the uPAR^high^/p62^high^ senescent cell fraction is not selectively reduced while total p16^+^ cell numbers drop uniformly, the hypothesis is falsified. Similarly, if TFEB loss does not impair the selective senescence clearance or wound repair advantage, the proposed mechanism is invalid.

Broader Implications

This hypothesis reframes IF not as a blunt senolytic but as a conditional modulator that exploits the inherent heterogeneity of senescent cells. It suggests that nutritional interventions could be timed to augment the beneficial chaperone role of senescent cells during tissue repair while mitigating their chronic deleterious effects—an approach testable in aging, fibrosis, and cancer models where senescence dynamics are pivotal.

[1] https://www.fightaging.org/archives/2024/07/the-useful-functions-of-senescent-cells/ [2] https://doi.org/10.1101/2025.06.08.658533 [3] https://doi.org/10.1038/s41586-021-03547-7 [4] https://pmc.ncbi.nlm.nih.gov/articles/PMC5873888/ [5] https://pulse.cedars-sinai.org/news/are-senolytic-supplements-right-for-me [6] https://doi.org/10.1038/nm.4324