Hypothesis

Age‑related shifts in the gut microbiome alter luminal concentrations of short‑chain fatty acids (SCFAs) and tryptophan metabolites, which modulate lysosomal Ca²⁺ release via TRPML1 in intestinal epithelial cells. This change dictates calcineurin‑dependent TFEB dephosphorylation, controlling intestinal autophagy and barrier integrity. Altered TFEB activity then reshapes vagal afferent signaling to the brain, suppressing neuronal TFEB and driving neuro‑degenerative autophagy failure.

Mechanistic Model

1. Microbial metabolite sensing – Declining butyrate and rising indoxyl sulfate in aged microbiota reduce lysosomal Ca²⁺ efflux through TRPML1, limiting calcineurin activation [5].
2. TFEB regulation – Impaired calcineurin fails to dephosphorylate TFEB at S142/S211, leaving mTORC1‑mediated phosphorylation dominant and trapping TFEB in the cytoplasm [2,4]. Cytoplasmic TFEB cannot drive ATG5/ATG7/ATG12 transcription, collapsing intestinal autophagy [3].
3. Barrier and signaling consequences – Loss of autophagic flux increases epithelial permeability, releasing microbial‑associated molecular patterns that activate vagal afferents. Chronic vagal firing shifts neurotransmitter release (e.g., reduced acetylcholine) toward a phenotype that inhibits neuronal calcineurin/TRPML1 signaling, mirroring the gut defect and suppressing neuronal TFEB [6].
4. Feedback loop – Neuronal autophagy failure elevates CNS‑derived metabolites that further dysregulate the microbiome, reinforcing the cycle.

Testable Predictions

• Restoring lysosomal Ca²⁺ flux in aged intestinal epithelia (via TRPML1 agonist ML‑SA1) will increase nuclear TFEB, improve autophagy markers (LC3‑II/p62), and reduce gut permeability.
• Vagal transection or chemogenetic inhibition of vagal afferents will block the intestinal TFEB‑to‑brain TFEB link, preventing neurodegeneration despite gut rescue.
• Microbiota transplantation from young donors into aged mice will normalize fecal butyrate/indoxyl sulfate ratios, rescue intestinal TFEB activity, and ameliorate neuronal autophagy deficits and cognitive decline.
• Pharmacological inhibition of neuronal calcineurin will phenocopy gut‑driven TFEB suppression, confirming the vagal midpoint.

Experimental Approach

• Mouse models: Aged (24‑mo) C57BL/6J receive TRPML1 agonist, vagal deafferentation, or fecal microbiota transplantation (FMT). Controls receive vehicle or sham.
• Readouts: Immunofluorescence for TFEB subcellular localization in intestinal villi and hippocampal neurons; Western blot for LC3‑II, p62, ATG5/7/12; lysosomal Ca²⁺ imaging with Fluo‑4 AM; serum LPS and zonulin for barrier integrity; in‑vivo vagal electroencephalography; behavioral assays (Morris water maze, novel object recognition).
• Microbiota profiling: 16S rRNA sequencing and targeted metabolomics (SCFAs, indoxyl sulfate) to correlate metabolite shifts with TFEB activity.

If the predicted chain of metabolite → TRPML1/calcineurin → TFEB → vagal signaling → neuronal TFEB holds, it establishes a peripheral lever to modulate brain autophagy without crossing the blood‑brain barrier, directly addressing the neglected gut‑brain axis in aging autophagy.