Hypothesis

Chronic low‑grade SASP from senescent adipose tissue primes microglia, leading to heightened NLRP3 inflammasome activity in the hippocampus and suppressed neurogenesis. A single senolytic dose clears a fraction of these cells, but residual SASP continues to drive microglial priming unless mTORC1 activity is transiently inhibited shortly after clearance. We hypothesize that administering rapamycin 24 h after a dasatinib + quercetin (D+Q) senolytic session will synergistically reduce hippocampal IL‑1β, restore BDNF‑dependent neurogenesis, and improve spatial memory in aged mice.

Mechanistic Rationale

• Senescent adipocytes secrete IL‑6, CCL2, and MMP‑9 that cross the blood‑brain barrier and engage microglial TLR4/CCR2 receptors [Senolytics selectively eliminate senescent cells].
• This chemokine surge primes microglia for a second hit; subsequent D+Q‑induced cell death releases DAMPs that trigger microglial NLRP3 inflammasome activation, increasing IL‑1β and TNF‑α production [Transplanting senescent cells causes dysfunction].
• Rapamycin inhibits mTORC1, suppressing the translation of SASP components and attenuating NLRP3 inflammasome assembly [Rapamycin inhibits mTORC1 to suppress SASP].
• Temporal separation allows senolytic‑induced debris to be cleared before mTOR inhibition blocks the SASP rebound, thereby preventing chronic microglial activation.

Predictions

1. Biochemical – In 20‑month‑old mice, the D+Q → rapamycin sequence will lower hippocampal IL‑1β and caspase‑1 activity by >40 % compared with D+Q alone or rapamycin alone (measured by ELISA and western blot).
2. Cellular – Neuroblast density (DCX⁺ cells) in the dentate gyrus will increase by ≥30 % relative to controls, matching levels seen in young adult mice.
3. Behavioral – Treated mice will show reduced escape latency in the Morris water maze (≥20 % improvement) and increased novel object recognition discrimination index.

Experimental Design

• Groups (n=12 per group): vehicle, D+Q only, rapamycin only, D+Q followed 24 h later by rapamycin, rapamycin followed 24 h later by D+Q (reverse order).
• Interventions: D+Q (5 mg/kg dasatinib + 50 mg/kg quercetin, i.p., single dose); rapamycin (6 mg/kg, i.p., single dose).
• Readouts at 7 days post‑last treatment: hippocampal IL‑1β, caspase‑1, BDNF, DCX⁺ cell counts; behavioral tests.
• Statistical analysis: One‑way ANOVA with Tukey post‑hoc; significance set at p<0.05.

Potential Outcomes & Falsifiability

• If the sequential D+Q → rapamycin group fails to show significant reductions in hippocampal IL‑1β or improvements in neurogenesis and behavior relative to the best single‑agent group, the hypothesis is falsified.
• Conversely, a significant improvement only in the reverse order (rapamycin → D+Q) would suggest that mTOR inhibition must precede senolysis, challenging the proposed timing.

This framework directly tests whether precise temporal coupling of senolysis with mTOR inhibition can uncouple SASP‑driven microglial priming from cognitive decline, offering a translatable strategy for combinatorial aging therapeutics.