Hypothesis

Aged cells actively suppress autophagy through a NAD+-sensitive SIRT1/TFEB axis that is further locked in by inflammasome‑driven caspase‑1 cleavage of TFEB and lysosomal membrane proteins, creating a self‑reinforcing loop that preserves damaged macromolecules as a stress‑adaptation reserve.

Mechanistic Basis

• NAD+ decline reduces SIRT1 deacetylase activity, limiting TFEB nuclear translocation and lysosomal biogenesis[https://pmc.ncbi.nlm.nih.gov/articles/PMC12986615/].
• Persistent mTORC1 activity phosphorylates ULK1 (Ser758), blocking autophagosome initiation even under starvation[https://pmc.ncbi.nlm.nih.gov/articles/PMC6398527/].
• Chronic inflammation and mtROS activate the NLRP3 inflammasome, leading to caspase‑1–mediated cleavage of TFEB at Asp215, generating a truncated TFEB that cannot drive lysosomal gene expression[https://www.nature.com/articles/s41467-020-15478-9].
• Cleaved TFEB acts as a dominant‑negative isoform, sequestering full‑length TFEB in the cytoplasm and reducing transcription of Atg genes[https://www.cell.com/molecular-cell/fulltext/S1097-2765(20)00428-5].
• The resulting autophagy insufficiency fuels further mtROS and inflammasome activation, closing a feedback loop[https://www.nature.com/articles/nrm.2020.30].

Novel Insight

We propose that the inflammasome‑TFEB cleavage is the decisive switch that converts reversible mTORC1‑mediated suppression into a semi‑stable, senescence‑associated autophagy block. Unlike mTOR inhibition alone, blocking caspase‑1 should restore TFEB flux even when NAD+ remains low, because it removes the dominant‑negative TFEB species.

Testable Predictions

1. In aged mouse lung fibroblasts, caspase‑1 activity will correlate inversely with TFEB nuclear levels and LC3‑II flux.
2. Pharmacological inhibition of caspase‑1 (e.g., VX‑765) or genetic knockout of Nlrp3 will increase TFEB nuclear translocation and autophagosome formation without altering NAD+ or mTORC1 activity.
3. Expression of a cleavage‑resistant TFEB mutant (D215A) in aged hepatocytes will rescue lysosomal biogenesis and reduce p62 aggregates, even when SIRT1 activity remains low.
4. Conversely, overexpressing a caspase‑1‑cleaved TFEB fragment in young cells will suppress autophagy and induce senescence markers (SA‑β‑gal, p16) independent of mTORC1 status.

Experimental Approach

• isolate primary fibroblasts from young (3 mo) and old (24 mo) mice; measure NAD+, SIRT1 activity, phospho‑ULK1, caspase‑1 activity, TFEB cleavage (western blot with TFEB‑N and TFEB‑C antibodies), nuclear TFEB (immunofluorescence), LC3‑II flux (with bafilomycin A1).
• treat aged cells with caspase‑1 inhibitor VX‑765 (10 µM) or vehicle; assess rescue of autophagy flux and lysosomal mass (LysoTracker).
• generate adenoviral vectors expressing WT TFEB, cleavage‑resistant TFEB(D215A), or cleaved TFEB fragment; transduce aged liver in vivo; monitor Atg mRNA, p62 levels, inflammation markers, and fibrosis scores.
• use Nlrp3−/− aged mice as genetic control; compare autophagy markers to wild‑type aged littermates.

Falsifiability

If caspase‑1 inhibition fails to increase TFEB nuclear localization or autophagic flux in aged cells despite verified target engagement, or if cleavage‑resistant TFEB does not ameliorate lysosomal dysfunction, the hypothesis would be refuted, indicating that inflammasome‑mediated TFEB cleavage is not a critical node in age‑related autophagy suppression.