Hypothesis

Daily supplementation with 500 mg urolithin A (UA) will reduce the relative abundance of gut bacteria that convert dietary phenylalanine/tyrosine to phenylacetic acid (PAA), thereby lowering circulating PAA levels and synergistically enhancing UA‑driven mitophagy in CD8⁺ T cells.

Rationale

UA activates a calcium‑dependent mitophagy cascade that improves mitochondrial fitness and reverses aging signatures in immune cells [[https://pmc.ncbi.nlm.nih.gov/articles/PMC12758194/]]. Conversely, age‑related microbiota shifts increase PAA, a metabolite that induces endothelial senescence via mitochondrial oxidative stress [[https://doi.org/10.1101/2023.11.17.567594]]. Although UA does not directly inhibit PAA synthesis, its ability to alter luminal redox state and promote mucin production could create an environment unfavorable for PAA‑generating taxa (e.g., certain Clostridia and Proteobacteria). Demonstrating that UA reshapes the microbiome to cut a pro‑aging metabolite would link two arms of the gut‑mitochondria axis and explain inter‑individual variability in UA responsiveness.

Predictions

1. After 24 weeks of UA supplementation, participants will show a ≥20 % decrease in the abundance of PAA‑producing bacteria relative to baseline, measured by 16S rRNA shotgun metagenomics.
2. Concurrently, plasma PAA concentrations will drop by ≥15 % compared with placebo.
3. The reduction in PAA will mediate additional improvements in mitophagy markers (phospho‑Parkin Ser65, ULK1) and immune phenotypes (increased youthful CD8⁺ T‑cell frequency, enhanced fatty‑acid oxidation) beyond those observed with UA alone.
4. Participants classified as low‑UA producers at baseline (based on pre‑supplementation urinary urolithin levels) will derive the greatest microbiome‑mediated benefit, narrowing the responder gap.

Experimental Design

A double‑blind, placebo‑controlled, parallel‑group trial will enroll 120 middle‑aged adults (45‑65 y) stratified by baseline UA producer status (high vs. low). Participants receive either UA 500 mg/day or matched placebo for 24 weeks. Stool samples collected at weeks 0, 12, and 24 will undergo metagenomic sequencing to quantify taxa known for phenylalanine/tyrosine fermentation (e.g., Clostridium sporogenes, Proteus mirabilis). Plasma PAA will be quantified by LC‑MS. Mitophagy activity will be assessed in isolated CD8⁺ T cells via phospho‑Parkin flow cytometry and mitochondrial ROS assays. Immune rejuvenation will be evaluated by flow cytometric profiling of naïve/memory CD8⁺ subsets and ex vivo cytokine production after viral antigen stimulation.

Potential Outcomes & Falsifiability

• Supported: Significant reductions in PAA‑producing bacteria and plasma PAA in the UA arm, correlated with greater increases in mitophagy markers and youthful immune phenotypes than placebo; mediation analysis shows PAA change accounts for ≥30 % of the UA effect on immune outcomes.
• Refuted: No meaningful shift in PAA‑related microbiota or plasma PAA despite UA‑induced mitophagy improvements, or changes in PAA do not correlate with enhanced immune markers.

If UA fails to modulate the microbiome‑PAA axis, the hypothesis is falsified, indicating that UA’s benefits act independently of gut‑derived pro‑aging metabolites and that combinatorial strategies targeting PAA producers would be needed for synergistic effects.