Hypothesis

In aged cells, lysosomal cholesterol esterification drives a persistent inhibition of the ULK1 complex independent of mTORC1, thereby actively suppressing autophagy as a compensatory response to membrane stress.

Mechanistic Basis

• Lysosomal cholesterol accumulates with age due to diminished activity of acid lipase (LAL) and increased NPC1 dysfunction, leading to cholesterol ester storage within lysosomes6.
• Elevated lysosomal cholesterol promotes the recruitment of SREBP2 to the lysosomal membrane, where it undergoes proteolytic activation and translocates to the nucleus7.
• Nuclear SREBP2 represses transcription of key autophagy genes (e.g., ATG5, ATG7, LC3) by competing with TFEB for binding to coordinated lysosomal expression and regulation (CLEAR) elements8.
• Concurrently, cholesterol-rich lysosomal membranes hinder the assembly of the ULK1-ATG13-FIP200 complex, reducing its kinase activity even when AMPK is active and mTORC1 is inhibited9.

This creates a dual-lock: transcriptional down-regulation of autophagy machinery and direct biochemical inhibition of the initiation complex, explaining why autophagy can be reactivated by young plasma (which delivers functional LAL)5 but remains resistant to mTORC1 inhibition alone1.

Testable Predictions

1. In aged mouse liver, lysosomal cholesterol ester levels will correlate inversely with autophagic flux, and genetic reduction of lysosomal cholesterol (via LAL overexpression) will increase LC3-II turnover without altering mTORC1 phosphorylation.
2. Pharmacological inhibition of SREBP2 cleavage (using fatostatin) will restore ATG5 and ATG7 mRNA expression in aged fibroblasts and rescue autophagy after rapamycin treatment fails.
3. Exposing young cells to exogenous cholesterol-rich liposomes will recapitulate the aged autophagy-suppressed phenotype, which can be reversed by cyclodextrin-mediated cholesterol depletion.

Experimental Design

• Measure lysosomal cholesterol ester using filipin staining coupled with ELISA in liver samples from young (3 mo) and old (24 mo) mice; correlate with GFP-LC3 puncta and mCherry-GFP-LC3 tandem reporter flux.
• Generate liver-specific LAL transgenic aged mice; assess autophagic flux, p62 levels, and lifespan.
• Treat aged primary hepatocytes with fatostatin or siRNA against SREBP2; quantify ATG5/ATG7 mRNA (qPCR) and autophagic flux after torin1 treatment.
• In vitro, incubate HEK293 cells with LDL-derived cholesterol liposomes; monitor ULK1 complex formation by co-immunoprecipitation and autophagosome formation.

If cholesterol-mediated ULK1 inhibition and SREBP2-driven transcriptional repression are central to age-related autophagy suppression, targeting lysosomal cholesterol export or SREBP2 activity should reactivate autophagy more effectively than mTORC1 inhibition alone, providing a falsifiable route to therapeutic intervention.