Hypothesis: GLI-Mediated Transcriptional Repression Drives Age-Dependent Autophagy Suppression

Persistent Hedgehog (Hh) ligand release from senescent stromal cells maintains low‑level SMO activity in aged parenchyma, favoring the accumulation of GLI2/GLI3 repressor forms (https://doi.org/10.1016/j.devcel.2015.03.009). These repressor GLIs recruit histone deacetylase complexes (HDAC1/2 and SIRT1) to the TFEB promoter, leading to H3K9 deacetylation and chromatin compaction that diminishes TFEB transcription (https://pmc.ncbi.nlm.nih.gov/articles/PMC4502732/). Concurrently, mTORC1 hyperactivation phosphorylates ULK1/ATG13 to block autophagy initiation (https://pmc.ncbi.nlm.nih.gov/articles/PMC6826502/). The combined transcriptional and post‑translational hits create a coordinated suppression of the autophagy‑lysosome pathway, which is not a simple loss‑of‑function but an active, reversible brake.

Testable predictions

1. In aged mouse liver or human fibroblasts, ChIP‑qPCR will show increased occupancy of GLI2/GLI3 and HDAC1/SIRT1 at the TFEB promoter compared with young controls.
2. Pharmacological inhibition of GLI DNA binding (GANT61) or CRISPRi‑mediated knock‑down of GLI2/GLI3 will raise TFEB mRNA and protein levels, restore nuclear TFEB translocation, and increase LC3‑II turnover in the presence of bafilomycin A1.
3. Restoring autophagy flux via GLI inhibition will reduce senescence‑associated secretory phenotype (SASP) markers (IL‑6, PAI‑1) but, if damage load is high, may sensitize cells to apoptosis, providing a falsifiable outcome: GLI blockade fails to improve viability when DNA damage exceeds a threshold.

Experimental design

• Isolate primary hepatocytes from young (3 mo) and aged (24 mo) mice.
• Treat halves with GANT61 (10 µM) or DMSO control for 24 h.
• Measure: (a) TFEB promoter ChIP for GLI2/GLI3 and HDAC1; (b) TFEB mRNA (qPCR) and protein (Western, cytosolic vs nuclear); (c) autophagic flux (LC3‑II/I ratio with/without bafilomycin); (d) SASP cytokine ELISA; (e) Annexin V/PI apoptosis assay.
• Expected outcome: GANT61 reduces GLI/TFEB promoter binding, raises TFEB activity, and lifts autophagy flux in aged cells, reversing at least 50 % of the age‑related decline seen in untreated aged samples.

If GLI inhibition does not alter TFEB expression or autophagic flux, the hypothesis is falsified, indicating that age‑related autophagy suppression operates independently of Hedgehog‑GLI signaling.