Emodin shifts senescent autophagy hierarchy to prioritize mitochondrial SASP cargo via p62 oligomerization

Hypothesis

Emodin reprograms the autophagy hierarchy in senescent cells by promoting p62 oligomerization, which redirects autophagic cargo toward mitochondrial components and SASP‑maintaining protein assemblies, converting a pro‑survival flux into a senolytic signal.

Mechanistic Rationale

• Senescent cells rely on basal autophagy to degrade damaged organelles while preserving SASP drivers Autophagy sustains senescent viability.
• Emodin inhibits EGFR/MAPK and suppresses STAT3 Emodin EGFR MAPK, STAT3 suppression, pathways known to relieve mTORC1 inhibition and induce autophagy EGFR inhibition induces autophagy.
• However, autophagy induction alone does not guarantee selectivity; the key lies in altering the selectivity machinery. p62 acts as a hub that oligomerizes to bind ubiquitinated cargo and LC3 p62 oligomerization mitophagy.
• We propose that emodin, through its electrophilic quinone moiety, modifies cysteine residues on p62, enhancing its oligomerization state and shifting its cargo preference toward mitochondrial outer‑membrane proteins (e.g., TOM20) and SASP regulators such as cGAS‑STING adapters.
• This shift would cause preferential degradation of mitochondria that fuel SASP production and of signaling platforms that sustain the inflammatory secretome, thereby triggering senolysis despite unchanged overall LC3‑II levels.

Testable Predictions

1. In emodin‑treated senescent fibroblasts, p62 will show increased high‑molecular‑weight oligomers detectable by native PAGE or crosslinking assays.
2. Immunofluorescence will reveal increased co‑localization of LC3 with mitochondrial markers (TOM20, COXIV) and with SASP‑related proteins (cGAS, STING) but not with lysosomal markers such as LAMP1 in early treatment windows.
3. CRISPR‑mediated mutation of p62’s oligomerization domain (ΔPB1) will abolish emodin‑induced mitochondrial clearance and rescue cells from senolysis, while leaving general autophagy flux (measured by bafilomycin‑sensitive LC3‑II turnover) unchanged.
4. Pharmacological blockade of p62 phosphorylation (using CK2 inhibitors) will mimic the oligomerization‑defective phenotype, confirming a post‑translational mechanism.
5. Metabolomic profiling will show a selective decline in mitochondrial ROS and SASP cytokines (IL‑6, IL‑8) preceding loss of viability, indicating that cargo reprogramming precedes cell death.

Experimental Approach

• Cell model: Human IMR‑90 fibroblasts rendered senescent by irradiation (10 Gy) or oncogenic RAS.
• Treatment: Emodin (25 µM) for 6‑24 h; include controls (vehicle, chloroquine, EGFR inhibitor).
• Readouts:
  • Native PAGE and Western blot for p62 oligomers.
  • Proximity ligation assay (PLA) for p62‑LC3 and p62‑TOM20 interactions.
  • Mitochondrial flux (Seahorse OCR) and SASP cytokine ELISA.
  • Viability (Annexin V/PI) and clonogenic survival.
• Genetic tools: p62 WT, ΔPB1, and cysteine‑to‑serine mutants overexpressed via lentivirus; CRISPR knockout of p62 rescued with WT or mutant.
• Expected outcome: Only WT p62, not oligomerization‑defective mutants, will enable emodin‑driven mitochondrial SASP cargo clearance and senolysis.

Falsification

If emodin induces senescence cell death without altering p62 oligomerization status or without shifting LC3 mitochondrial/SASP co‑localization, the hypothesis is falsified. Likewise, if p62 oligomerization mutants do not affect emodin’s senolytic potency, the proposed mechanism is incorrect.