The hypothesis is that eliminating senescent cells in the gut epithelium and myenteric plexus, while simultaneously restoring Wnt signaling in intestinal stem cells, will reduce microbiota‑derived phenylacetic acid (PAA) production, repair barrier integrity, and attenuate hippocampal neuroinflammation, thereby improving cognitive function in aged animals. This prediction follows from three observations: (1) age‑related shifts in the gut microbiome elevate PAA, which directly induces endothelial cell senescence [https://doi.org/10.1101/2023.11.17.567594]; (2) enteric cholinergic neurons are more vulnerable to age‑dependent degeneration than central neurons, suggesting a local loss of regulatory control over mucosal immunity and permeability [https://pubmed.ncbi.nlm.nih.gov/23537898/]; (3) ACCA epigenetic drift hypermethylates Wnt pathway genes in intestinal stem cells, diminishing tissue repair and promoting a pro‑inflammatory niche [https://idw-online.de/de/news862472]. If gut senescence drives dysbiosis rather than merely reflecting it, then clearing those senescent cells should normalize microbial metabolism and lower PAA levels. Conversely, if dysbiosis precedes senescence, senolysis alone will not reduce PAA unless the epithelial regenerative capacity is also rescued. Combining senolysis with Wnt activation tests both directions of causality.

To test this, aged (24‑month) C57BL/6 mice will receive either (a) vehicle, (b) the senolytic dasatinib + quercetin (D+Q) administered intermittently, (c) a Wnt agonist such as RSPO1‑Fc fusion protein delivered via osmotic pump, or (d) the combination of D+Q plus RSPO1 for eight weeks. Fecal samples will be collected weekly for PAA quantification by LC‑MS. Gut barrier function will be assessed using FITC‑dextran permeability assays and zonulin staining. Colonic tissue will be analyzed for senescence markers (p16^INK4a^, SA‑β‑gal) and cholinergic neuron density (ChAT immunostaining). Hippocampal sections will be probed for microglial activation (Iba1, MHC‑II) and astrocytic reactivity (GFAP). Finally, mice will undergo behavioral testing (novel object recognition, Morris water maze) to gauge cognitive performance.

Falsifiable outcomes: (1) If PAA levels fall significantly only in the combination group, and this correlates with restored barrier integrity, reduced hippocampal inflammation, and improved memory, the hypothesis is supported—indicating that gut senescence and Wnt drift jointly sustain a dysbiotic, neurotoxic milieu. (2) If D+Q alone lowers senescence markers but PAA and neuroinflammation remain unchanged, while RSPO1 alone improves barrier function without affecting senescence, the data suggest that each arm addresses only one half of the loop, refuting the idea that a single intervention suffices. (3) If neither intervention alters PAA, barrier metrics, or brain outcomes despite clear modulation of gut senescence or Wnt signaling, the hypothesis is falsified, implying that the microbiota‑senescence axis is not a dominant driver of age‑related neuroinflammation in this model.

This experimental design directly tests causality in the bidirectional gut‑brain axis, leveraging the very complexity that has been overlooked. By measuring both microbial metabolites and host senescence phenotypes, it moves beyond correlative gut‑or‑brain‑only studies and offers a clear, mechanistic route to either validate a novel anti‑aging strategy or redirect focus to other pathways.