Hypothesis

Aged cells actively suppress autophagy through a PER2‑dependent transcriptional block that is amplified by chronic integrin‑FAK signaling downstream of matrix stiffening. This block reduces lysosomal biogenesis and autophagosome‑lysosome fusion, overriding compensatory BAG3‑CASA upregulation.

Mechanistic Model

1. Circadian repression – In aged fibroblasts, PER2 expression declines [1], leading to reduced transcription of CLEAR network genes (TFEB, LAMP1) that drive lysosomal biogenesis.
2. Mechanotransduction amplification – Persistent ECM stiffness sustains integrin‑FAK‑Src activation [3], which phosphorylates PER2 on serine residues that promote its cytoplasmic retention and proteasomal degradation. This creates a feed‑forward loop: stiff matrix → FAK activation → PER2 loss → weakened circadian autophagy drive.
3. BAG3 compensation limits – Although BAG3 rises with age [2] and can bind TSC1 to partially inhibit mTORC1, its ability to initiate autophagosome formation is insufficient when lysosomal capacity is low due to PER2‑mediated transcriptional deficit.
4. Outcome – Autophagosome formation (LC3‑II) may appear normal or elevated, but flux stalls because lysosomes are scarce or dysfunctional, matching observations of elevated LC3-II alongside impaired degradation in aged neurons [2].

Predictions and Experimental Tests

• Prediction 1: Preventing PER2 phosphorylation (e.g., using a PER2‑S→A mutant or FAK inhibitor) will restore lysosomal gene expression and autophagic flux in aged fibroblasts cultured on stiff matrices, without altering BAG3 levels.
• Prediction 2: Overexpressing TFEB in aged cells will rescue flux only when PER2 activity is concurrently restored, indicating that lysosomal biogenesis is the limiting downstream node.
• Prediction 3: In vivo, conditional knockout of FAK in fibroblasts of aged mice will increase PER2 nuclear localization, boost lysosomal biogenesis, and reduce tissue‑level protein aggregates.

Experimental Approach

• Culture young and senescent human fibroblasts on tunable polyacrylamide gels (soft 0.5 kPa vs stiff 10 kPa). Treat with FAK inhibitor (PF‑573228) or express phospho‑deficient PER2. Measure:
  • PER2 localization (immunofluorescence)
  • mRNA of TFEB, LAMP1, CATHEPSIN D (qPCR)
  • Lysosomal number and activity (LysoTracker, DQ‑BSA)
  • Autophagic flux (LC3‑II turnover with bafilomycin A1)
• Validate in fibroblasts isolated from aged mouse skin with fibroblast‑specific FAK knockout; assess p62 accumulation and collagen remodeling.

Falsifiability

If PER2 phosphorylation status or FAK activity does not influence lysosomal gene expression or autophagic flux under stiff conditions, or if restoring PER2 alone fails to rescue flux without TFEB overexpression, the hypothesis would be refuted. This directly tests whether the circadian‑mechanotransduction axis actively suppresses autophagy rather than merely reflecting passive decline.