SASP‑Driven STAT3/TFEB Axis Spatially Constrains Autophagy in Aging Tissue

Hypothesis

In aged tissues, the senescence‑associated secretory phenotype (SASP) creates a local cytokine milieu that actively suppresses autophagy in neighboring cells through STAT3‑mediated inhibition of TFEB nuclear translocation, thereby coupling inflammatory signaling to a spatial gradient of autophagic repression.

Mechanistic Reasoning

1. SASP factors such as IL‑6 and TGF‑β activate JAK/STAT3 signaling in paracrine targets.
2. Phosphorylated STAT3 interacts with TFEB, retaining it in the cytoplasm and preventing lysosomal gene expression.
3. Cytoplasmic TFEB reduces transcription of ATG genes and lysosomal enzymes, lowering autophagic flux.
4. This suppression is not a passive decay but an active, reversible brake that limits excessive self‑digestion in an inflammatory milieu, protecting cells from inadvertent death while allowing damage to accumulate.
5. Consequently, autophagy genes show reduced expression specifically around senescent cell niches, forming a spatial gradient that correlates with SASP intensity.

Testable Predictions

• In aged mouse liver, regions within ~50 µm of p16^Ink4a^‑positive senescent cells will exhibit lower ATG5, ATG7, and LAMP1 mRNA and protein levels compared with distal regions.
• Concurrently, these pericellular zones will show elevated p‑STAT3 and reduced nuclear TFEB.
• Neutralizing IL‑6 or TGF‑β locally (e.g., with antibodies or genetic knock‑down) will rescue TFEB nuclear localization and restore autophagy gene expression without altering senescent cell burden.
• Genetic ablation of STAT3 in parenchymal cells will abolish the autophagy‑suppressive gradient, leading to heightened autophagic flux and reduced accumulation of ubiquitinated proteins in aged tissue.

Experimental Approach

1. Generate spatial transcriptomics (10x Visium or Slide‑seqV2) and immunofluorescence maps of ATG family genes, TFEB, p‑STAT3, and lysosomal markers in livers from young (3 mo) and aged (24 mo) mice.
2. Overlay senescent cell maps (p16^Ink4a^ or SA‑β‑gal) to compute expression gradients as a function of distance from SASP sources.
3. Perform paracrine blockade: administer IL‑6R antibody (MR16‑1) or TGF‑β neutralizing antibody to aged mice for 2 weeks, then repeat spatial analyses.
4. Use lineage‑specific STAT3 knockout mice (Alb‑Cre;Stat3^fl/fl) to test cell‑autonomy.
5. Quantify autophagic flux via LC3‑II turnover and p62 levels, and assess damage markers (γH2AX, protein carbonylation).

Falsifiability

If spatial transcriptomics reveals no significant reduction of autophagy genes near senescent cells, or if SASP neutralization fails to alter TFEB localization or autophagy readouts, the hypothesis that SASP actively suppresses autophagy via STAT3/TFEB would be refuted, favoring a passive decline model.

SASP upregulation in aged tissues Autophagy inhibition alters SASP Impaired autophagy promotes DNA damage and senescence MS lesion autophagy decrease and mTORC1 elevation Aged blastema metabolic remodeling without autophagy gene changes