Hypothesis

Urolithin A (UA) triggers an ER‑to‑mitochondria calcium flux that is required for mitophagy initiation. We hypothesize that this mitochondrial calcium rise also modulates the AMPK/mTORC1 signaling hub at mitochondria‑lysosome membrane contact sites, leading to TFEB‑driven lysosomal biogenesis. The resulting increase in lysosomal capacity couples mitochondrial damage sensing to organelle renewal, creating a feed‑forward loop that sustains UA‑induced mitophagy.

Mechanistic Reasoning

• UA‑dependent calcium release relies on ITR‑1/IP3R and MCU‑1, as shown in C. elegans (UA induces mitochondrial calcium release).
• Elevated mitochondrial calcium can activate AMPK locally via calcium‑sensing kinases (CAMKK2) and inhibit mTORC1 through TSC2 phosphorylation, a mechanism documented for other organelle contacts (AMPK‑ULK1‑mTORC1 axis).
• AMPK phosphorylation of ULK1 initiates phagophore formation, while AMPK‑mediated mTORC1 suppression relieves the cytosolic retention of TFEB, allowing its nuclear translocation and lysosomal gene expression (mTORC1‑TFEB Crosstalk).
• UA also stimulates SIRT1‑dependent deacetylation of PGC‑1α, promoting mitochondrial biogenesis that works in tandem with lysosomal expansion (Distinct roles of UA and spermidine).
• Together, these pathways create a calcium‑dependent signaling node at ER‑mitochondria‑lysosome junctions that couples fission, autophagosome formation, and lysosomal clearance.

Testable Predictions

1. Lysosomal calcium chelation (e.g., with BAPTA‑AM targeted to lysosomes) will block UA‑induced TFEB nuclear translocation and reduce mitophagy flux (LC3‑II/PINK1 colocalization) despite normal mitochondrial calcium spikes.
2. Pharmacological activation of AMPK (AICAR) in MCU‑1 knockdown worms will rescue UA‑mediated lifespan extension, indicating that AMPK can bypass the mitochondrial calcium step.
3. Overexpression of the tethering protein VAPB‑PTPIP51 will amplify UA‑driven lysosomal biogenesis and extend healthspan beyond UA alone in mammalian cells.
4. Microbiome‑defined mice lacking UA‑producing strains will show absent mitochondrial calcium signal and consequently no AMPK/mTORC1/TFEB response, confirming the gut‑metabolite origin of the signaling hub.

Falsifiability

If lysosomal calcium chelation or TFEB knockout fails to diminish UA‑stimulated mitophagy or lysosomal biogenesis, the proposed calcium‑dependent AMPK/mTORC1‑TFEB coupling at mitochondria‑lysosome contacts is refuted. Conversely, confirmation would position UA as a modulator of inter‑organelle calcium signaling that integrates mitochondrial quality control with lysosomal renewal, offering a mechanistic basis for combinatorial postbiotic strategies.