Hypothesis

Aged cells accumulate the lysosomal lipid bis(monoacylglycero)phosphate (BMP) due to heightened acid sphingomyelinase (ASM) activity in senescent cells. BMP enrichment stiffens lysosomal membranes and hinders the function of the calcium channel TRPML1, blunting lysosomal Ca2+ efflux. Reduced cytosolic Ca2+ lowers calcineurin activity, keeping TFEB phosphorylated and cytoplasmic even when mTORC1 is inhibited. Consequently, autophagy is actively suppressed not only by mTORC1‑TFEB axis dysregulation but also by a parallel lysosomal Ca2+‑calcineurin blockade. Exosomal BMP released from senescent cells can transfer this lipid defect to nearby healthy cells, propagating a tissue‑wide autophagy block that reinforces the SASP‑driven vicious cycle.

Mechanistic Rationale

• BMP as a membrane‑rigidifying lipid: BMP’s conical shape promotes negative curvature, which in excess creates microdomains that impede TRPML1 gating (see structural studies of lysosomal channels) {2}.
• TRPML1‑Ca2⁺‑calcineurin‑TFEB axis: Lysosomal Ca2+ release activates calcineurin, which dephosphorylates TFEB at Ser142, promoting nuclear translocation {3}. In aging, diminished TRPML1 activity reduces this signal, leaving TFEB cytoplasmic.
• Senocrine propagation: Senescent cells secrete exosomes enriched in ASM‑generated BMP {4}; uptake by neighboring cells recapitulates the lysosomal lipid shift, spreading autophagy suppression.
• Feedback with mTORC1: BMP‑laden lysosomes also sequester Rag GTPases, sustaining mTORC1 localization to the lysosomal surface, thereby reinforcing the primary suppression pathway {1}.

Testable Predictions

1. Biochemical: Lysosomal BMP levels will be significantly higher in liver and brain of 24‑month‑old mice vs 3‑month‑old controls, correlating with decreased TRPML1‑mediated Ca2+ flux (measured by Lysosomal‑GCaMP).
2. Genetic: Lysosome‑specific overexpression of TRPML1 in aged mice will restore TFEB nuclear localization and autophagic flux (LC3‑II turnover) despite persistent mTORC1 activity (phospho‑S6K).
3. Pharmacological: Inhibition of ASM with amitriptyline or exogenous BMP‑scavenging liposomes will reduce lysosomal BMP, increase TRPML1 activity, and rescue autophagy in primary hepatocytes from old mice.
4. Paracrine: Co‑culture of senescent fibroblasts with young epithelial cells will transfer BMP‑rich exosomes, lowering TRPML1 activity in the recipients; neutralizing exosomes with GW4869 will prevent this effect.
5. Falsification: If BMP reduction fails to increase TFEB nuclear entry or autophagic flux when mTORC1 is genetically inhibited (RagA/B knockout), the hypothesis is refuted, indicating that BMP acts upstream of mTORC1 rather than as a parallel suppressor.

Experimental Outline

• Lipidomics: Isolate lysosomes from young/old tissues, quantify BMP by LC‑MS/MS.
• Imaging: Live‑cell lysosomal Ca2+ imaging using GP4‑based sensors; TFEB‑GFP nuclear/cytoplasmic ratio.
• Interventions: Treat aged mice with amitriptyline (ASM inhibitor) or TRPML1 agonist (ML‑SA1) for 2 weeks; assess p62/SQSTM1 accumulation, lysosome number, and SASP cytokines.
• Readouts: Autophagic flux (bafilomycin A1‑challenged LC3‑II), mTORC1 signaling (p‑S6K), and functional outcomes (grip strength, cognitive testing).

By linking a specific lysosomal lipid alteration to both the mTORC1‑TFEB axis and calcium‑dependent TFEB regulation, this hypothesis provides a reversible, upstream node that explains why autophagy appears “actively suppressed” in aging and offers distinct intervention points that can be empirically validated or falsified.