Hypothesis

Aging lysosomes lose calcium release capacity through reduced TRPML1 activity, which blocks autophagosome‑lysosome fusion and sustains mTORC1 activation on the lysosomal surface. This calcium deficit acts downstream of Rubicon‑mediated VPS34 inhibition and upstream of SASP‑driven inflammation, creating a unified hub that actively suppresses autophagy.

Mechanistic Basis

• Rubicon accumulation inhibits VPS34, limiting PI3P generation and autophagosome nucleation 1.
• Senescent cells maintain mTORC1 hyperactivity that phosphorylates and inhibits ULK1, blocking initiation 2.
• SASP cytokines diffuse to neighboring cells, reinforcing inflammatory stress that further impairs lysosomal function 3.
• Lysosomal calcium release via TRPML1 is required for autophagosome‑lysosome fusion and for lysosomal reformation after fusion; its decline with age has been documented but not linked to autophagy suppression 4.
• Low luminal calcium fails to activate calcineurin, leading to increased TFEB phosphorylation and reduced lysosomal biogenesis, while calcium‑sensitive mTORC1 remains active on damaged lysosomes, perpetuating the block.

Thus, lysosomal calcium deficiency could be the convergent node that translates Rubicon elevation, mTORC1 persistence, and SASP exposure into a stable autophagy‑off state.

Predictions

1. In aged tissues, TRPML1 expression or activity will be inversely correlated with Rubicon levels and positively correlated with autophagic flux.
2. Pharmacological activation of TRPML1 (e.g., with ML‑SA1) will restore autophagosome‑lysosome fusion and reduce p62 accumulation in aged cells, even when Rubicon is overexpressed.
3. Genetic knock‑down of TRPML1 in young cells will phenocopy age‑related autophagy suppression, increasing senescence markers and SASP secretion.
4. Chelating lysosomal calcium with BAPTA‑AM will abolish the autophagy‑extending effects of young plasma transfusion in aged rats.

Experimental Approach

• Measure TRPML1‑mediated calcium flux using LysoSensor dyes in hepatocytes from young vs. old mice, correlating with Rubicon immunoblots and LC3‑II turnover.
• Treat aged mouse liver slices with ML‑SA1 (± Rubicon siRNA) and assess autophagic flux via mCherry‑GFP‑LC3 reporter, p62 levels, and lysosome integrity (LAMP1 staining).
• Induce senescence in cultured human fibroblasts with irradiation, then knock down TRPML1 with CRISPRi; quantify SASP IL‑6/IL‑8 secretion and mTORC1 activity (p‑S6K).
• In vivo, administer young plasma to aged TRPML1‑knockout mice and monitor autophagy markers and lifespan; lack of benefit would falsify the calcium‑centric model.

These experiments directly test whether restoring lysosomal calcium can bypass Rubicon‑driven and SASP‑mediated suppression, providing a mechanistic link between organelle ion homeostasis and the active downregulation of autophagy in aging.