We hypothesize that timed delivery of a NAD+‑boosting probiotic strain rescues microglial circadian NAD+ salvage, thereby preventing phenylacetic acid (PAA)‑induced endothelial senescence and preserving cognitive function in aged mice. Circadian disruption lowers microglial NAMPT expression, reducing NAD+ levels and impairing SIRT1‑mediated deacetylation of BMAL1, which weakens the clock’s ability to gate microglial inflammatory responses. Concurrently, age‑shifted gut microbiota elevate circulating PAA, which enters the brain and drives endothelial cell senescence via DNA damage signaling. NAD+‑dependent SIRT1 activation not only reinforces microglial PER/CRY transcriptional rhythms but also upregulates astrocytic expression of the PAA transporter SLC5A8, facilitating microbial metabolite clearance. By administering a riboside‑producing Lactobacillus strain at the peak of microglial Bmal1 transcription (circadian time 6), we predict restoration of NAD+ pools, synchronization of microglial immune oscillations, and reduced PAA‑induced vascular senescence.

Testable predictions:

1. Aged mice receiving the chronobiotic will show a 2‑fold increase in hippocampal NAD+ levels compared with arrhythmic or vehicle controls, measured by LC‑MS at CT6 and CT18.
2. Microglial isolated from chronobiotic‑treated mice will exhibit restored rhythmicity of Nampt, Sirt1, and Bmal1 mRNA (qPCR, 4‑hour sampling over 24 h) with amplitudes matching young adult levels.
3. Plasma PAA concentrations will decrease by 30 % in chronobiotic‑treated aged mice relative to untreated aged controls, while fecal Lactobacillus abundance rises.
4. Endothelial senescence markers (p16^INK4a, γH2AX) in cerebral microvessels will be halved in the chronobiotic group, as shown by immunofluorescence and flow cytometry.
5. Behavioral assays (novel object recognition, Morris water maze) will reveal rescued spatial memory performance, equivalent to that of young adult mice.

Falsifiability: If chronobiotic administration fails to elevate hippocampal NAD+ at CT6, does not restore microglial clock gene rhythms, or does not reduce PAA levels and endothelial senescence, the hypothesis is refuted. Conversely, if NAD+ elevation occurs without circadian timing (e.g., constant‑dose riboside) yet still improves outcomes, the timing component would be nonessential, challenging the mechanistic claim.

Experimental approach: Use 20‑month‑old C57BL/6J mice subjected to 12‑hour light/dark cycles. Treat groups with (i) vehicle, (ii) arrhythmic Lactobacillus riboside producer (continuous dosing via drinking water), (iii) chronobiotic strain delivered only at CT6 via timed gavage, and (iv) a positive control of time‑restricted feeding. Sample tissues at four circadian points over 48 h after two weeks of intervention. Measure NAD+, metabolomics, transcriptomics, cellular senescence, and cognition.

This hypothesis integrates the circadian‑microbiota axis, NAD+ biology, and vascular senescence into a single, testable framework. Success would demonstrate that restoring temporal fidelity of microglial NAD+ salvage is a potent geroprotective strategy that specifically neutralizes a deleterious microbial metabolite, thereby preserving brain vasculature and cognition.