Hypothesis

Clearing senescent gut epithelial cells with senolytics restores vagal afferent signaling, thereby reducing brain‑derived SASP and inflammaging through a succinate‑HDAC4 axis.

Mechanistic Rationale

• Age‑associated gut dysbiosis elevates phenylacetic acid and other metabolites that induce epithelial senescence [1]. Senescent gut cells secrete a SASP rich in succinate, LPS, and IL‑6 [2]; these molecules activate TLR4 and succinate receptors on vagal afferents, driving HDAC4 nuclear translocation and altered gene expression in the nucleus tractus solitarius (NTS) [3].
• Chronic vagal afferent activation propagates inflammatory signaling to the brain, amplifying microglial SASP (IL‑1β, IL‑6) and compromising autonomic control of gut barrier integrity [4].
• Senolytic clearance of gut senescent cells reduces succinate/LPS load, diminishes TLR4‑driven HDAC4 activation, and restores vagal tone. Improved vagal signaling then suppresses brain microglial NF‑κB activity via cholinergic anti‑inflammatory pathways, lowering cerebral SASP [5].
• Concurrently, a healthier gut epithelium supports production of butyrate and other SCFAs that act as HDAC inhibitors, reinforcing epigenetic youthfulness in both gut and brain [6]; this creates a positive feedback loop that stabilizes the microbiome‑host metabolite exchange.

Testable Predictions

1. Geriatric mice treated with dasatinib+quercetin (D+Q) will show a ≥30 % reduction in gut p16^INK4a^+ cells versus vehicle, accompanied by a ↓ in luminal succinate (measured by LC‑MS) and ↓ serum LPS‑binding protein.
2. Vagal electrophysiology (ex vivo vagal nerve recordings) will reveal increased afferent firing rate and heightened heart‑rate variability after D+Q, effects abolished by subdiaphragmatic vagotomy or TLR4 blockade.
3. Brain readouts (Iba1 immunostaining, IL‑1β ELISA, cortical SASP transcriptome) will demonstrate decreased microglial activation and lowered IL‑1β/IL‑6 levels only when gut senescence is cleared and vagal integrity is preserved; vagotomy will rescue gut senescence loss but not brain inflammation.
4. Epigenetic clocks (gut epithelium and hippocampal DNAmAge) will show a reversal of ~1.5 years post‑D+Q, an effect attenuated by HDAC4 over‑expression in the NTS or by butyrate depletion (antibiotics).
5. Behavioral improvements (Morris water maze, novel object recognition) will correlate with vagal firing restoration and will be lost when succinate is exogenously replenished via intraperitoneal injection.

Falsifiability

If D+Q clears gut senescent cells but fails to (a) elevate vagal afferent activity, (b) reduce brain SASP markers, or (c) improve cognitive performance, the hypothesis that gut‑senescence‑driven vagal dysregulation mediates brain inflammaging is falsified. Conversely, if vagotomy abolishes the brain benefits despite effective gut senescence clearance, the causal vagal link is confirmed.

Required Measurements

• Gut tissue: p16^INK4a^, SASP (IL‑6, IL‑1β, succinate) via qPCR/ELISA/LC‑MS.
• Vagal function: ex vivo nerve firing, in vivo heart‑rate variability, c‑Fos in NTS.
• Brain: Iba1, IL‑1β, IL‑6, HDAC4 localization (immunofluorescence), DNAmAge (Horvath clock).
• Microbiome: 16S rRNA sequencing for SCFA producers.
• Behavior: standard cognitive assays.

By integrating senolytics, vagal physiology, and metabolite‑epigenetic signaling, this hypothesis offers a concrete, falsifiable framework to test whether the gut‑brain axis drives aging through a senescence‑succinate‑HDAC4 circuit that can be therapeutically reset.