Hypothesis

It's known that with age, lysosomal free cholesterol accumulates and activates SREBP2, which in turn represses TFEB transcriptional activity and promotes expression of Rubicon and mTORC1 regulators, creating a self‑reinforcing brake that suppresses autophagy at initiation, autophagosome formation, and lysosomal fusion. This cholesterol‑SREBP2‑TFEB axis explains why multiple autophagy checkpoints are inhibited simultaneously and why removing single brakes (e.g., Rubicon knockdown) only partially restores flux unless lysosomal cholesterol is also lowered.

Mechanistic Model

• Lysosomal cholesterol buildup → SREBP2 cleavage → nuclear SREBP2.
• Nuclear SREBP2 binds TFEB promoter regions, reducing TFEB mRNA and protein levels.
• Reduced TFEB diminishes transcription of lysosomal and autophagy genes, lowering VPS34‑ULK1 complex activity.
• SREBP2 also upregulates RUBCN (Rubicon) transcription and enhances mTORC1 signaling via increased lysosomal amino acid sensing.
• Elevated Rubicon blocks autophagosome‑lysosome fusion; hyperactive mTORC1 phosphorylates TFEB, retaining it in the cytoplasm.
• The combined effect stalls autophagy at three checkpoints: initiation (ULK1), phagophore expansion (LC3 lipidation via ATG3/7), and fusion.

Testable Predictions

1. It's expected that aged tissues will show higher lysosomal free cholesterol, increased nuclear SREBP2, and decreased TFEB protein compared with young controls.
2. It's predicted that pharmacological depletion of lysosomal cholesterol (using cyclodextrin or NPC1 overexpression) will reduce SREBP2 activation, increase TFEB activity, and lower Rubicon and phospho‑mTORC1 levels.
3. Combining cholesterol depletion with Rubicon siRNA should restore autophagic flux to a greater extent than either intervention alone in aged fibroblasts.
4. Mice with lysosomal‑specific NPC1 overexpression are likely to exhibit delayed age‑related motor decline and reduced neurodegeneration, despite normal Rubicon expression.

Experimental Approach

• We'll measure lysosomal cholesterol using filipin staining and Amplex Red cholesterol assay in young vs. old mouse brain and muscle.
• We'll perform Western blot and immunofluorescence for nuclear SREBP2, TFEB, Rubicon, and phospho‑S6K (mTORC1 read‑out) in the same samples.
• We'll treat aged primary neurons with 2‑hydroxypropyl‑β‑cyclodextrin (HPβCD) or adenoviral NPC1, then assess LC3‑II turnover with bafilomycin A1 and p62 degradation.
• We'll conduct RNAi knockdown of Rubicon in aged fibroblasts ± cholesterol depletion, quantify autophagic flux via mCherry‑GFP‑LC3 reporter.
• We'll generate NPC1 overexpression mice driven by LysM‑Cre; monitor lifespan, rotarod performance, and hippocampal neuronal survival.

If lysosomal cholesterol is a upstream coordinator, lowering it should simultaneously attenuate multiple autophagy brakes, supporting the idea that age‑related autophagy suppression is a regulated program rather than a collection of independent failures.

1 2 3 4 5