Stiff,AGE‑Crosslinked ECM Reprograms Autophagy to Spare Phagocytosed Glycated Matrix While Prioritizing Cytosolic Clearance

Aging tissues develop a dual stress state: extracellular matrix becomes 3‑4 fold stiffer due to AGE crosslinks [2] and lysosomes receive increasing loads of undegradable glycated peptides [5]. We hypothesize that under these conditions autophagy does not globally increase bulk degradation but instead rewires its selectivity—diverting autophagosomes toward cytosolic proteins (e.g., damaged mitochondria) while actively deprioritizing the clearance of phagocytosed AGE‑modified ECM fragments. This substrate triage preserves short‑term energy homeostasis at the cost of lysosomal clogging by glycated peptides, which in turn impairs lysosomal acidification and blocks TFEB‑driven transcription of MMPs, explaining the paradox of elevated MMP activity without net matrix turnover [3,6].

Key mechanistic steps: (1) integrin‑β1 signaling on stiff AGE‑collagen activates FAK‑Src, which phosphorylates and inhibits the Ragulator‑Rag GTPase complex, attenuating mTORC1 recruitment to lysosomes specifically on the phagocytic arm of autophagy. (2) Concurrently, glycated peptides bind to lysosomal LAMP2A and competitively inhibit chaperone‑mediated autophagy, shifting cargo preference toward ubiquitin‑dependent, p62‑mediated sequestration of cytosolic proteins. (3) Accumulated undegraded AGE peptides raise lysosomal pH, reducing cathepsin activity and preventing TFEB nuclear translocation, thereby suppressing MMP gene expression despite elevated basal MMP transcription from stress pathways.

Testable predictions: (i) Fibroblasts cultured on tunable hydrogels mimicking young (0.5 kPa) versus aged (5 kPa) stiffness, supplemented with fluorescently labeled AGE‑collagen, will show increased LC3‑II colocalization with MitoTracker but decreased colocalization with the AGE‑collagen signal after 6 h of starvation (flux assay with bafilomycin A1). (ii) Lysosomal pH measured with LysoSensor Yellow/Blue will be significantly higher in the stiff+AGE condition, correlating with reduced DQ‑collagen degradation. (iii) TFEB nuclear fraction (immunofluorescence) will drop under stiff+AGE conditions, and MMP‑1 secretion (ELISA) will be suppressed despite unchanged mRNA levels; rescuing lysosomal acidification with low‑dose chloroquine will restore MMP secretion without altering autophagic flux toward mitochondria. (iv) Genetic disruption of integrin‑β1 or FAK will prevent the shift in autophagic cargo preference, confirming the mechanosensing link.

Falsification: If autophagic flux toward phagocytosed AGE‑collagen increases proportionally with cytosolic flux under stiff+AGE conditions, or if lysosomal pH remains unchanged and MMP secretion rises, the hypothesis is refuted. This framework directly links matrix mechanics, glycation load, and autophagic substrate selection to the observed failure of matrix remodeling in aged tissue.