We hypothesize that a brief NAD+ boosting phase before intermittent dasatinib+quercetin (D+Q) treatment aligns G1 and G2 senescent subpopulations, increasing their susceptibility to clearance and breaking the SASP‑CD38‑NAD+ feedback loop.

Rationale

Recent work shows that senescent cells are not uniform; DNA content stratification separates G1 and G2 subpopulations with distinct senolytic sensitivity[6]. Simultaneously, senescent cells drive CD38 expression in neighbors, consuming NAD+ and suppressing AMPK activity[3]. NAD+ replenishment activates AMPK, which inhibits mTORC1, promotes autophagy, and can push cells toward a uniform metabolic state[4]. We propose that a short course of an NAD+ precursor (e.g., nicotinamide riboside) transiently elevates AMPK activity, reduces mTORC1 signaling, and synchronizes the cell‑cycle distribution of senescent cells, making both G1 and G2 cohorts equally accessible to D+Q.

Testable Predictions

1. In aged mice, NAD+ priming for 48 h followed by intermittent D+Q will lower the proportion of G1 and G2 senescent cells more evenly than D+Q alone, as measured by DNA‑content flow cytometry[6].
2. The combined regimen will reduce SASP‑mediated CD38 upregulation in neighboring non‑senescent cells, resulting in higher tissue NAD+ levels compared with D+Q alone[3].
3. Functional readouts (grip strength, treadmill endurance) will improve significantly more with the sequential approach than with either monotherapy, reflecting broader senescent‑cell burden reduction.

Experimental Design

• Animals: 20‑month‑old C57BL/6J mice, n=10 per group.
• Groups: (1) Vehicle control, (2) NAD+ precursor (NR 300 mg/kg/day oral) for 2 days, (3) Intermittent D+Q (5 mg/kg dasatinib + 50 mg/kg quercetin, i.p., once weekly for 3 weeks), (4) NAD+ priming (2 days) → D+Q schedule.
• Readouts: (a) Flow cytometry of p16^Ink4a^+ cells stained with DAPI to quantify G1 vs G2 DNA content; (b) ELISA for SASP cytokines (IL‑6, IL‑1α) and CD38 activity in plasma; (c) Tissue NAD+ quantification via LC‑MS; (d) Physical performance tests.
• Analysis: Two‑way ANOVA with post‑hoc Tukey; significance set at p<0.05.

Potential Outcomes and Interpretation

If NAD+ priming synchronizes senescent subpopulations, group 4 will show a significant reduction in the disparity between G1 and G2 p16^Ink4a^+ fractions (p<0.01 vs group 3) and a larger drop in SASP‑driven CD38 activity. Failure to observe these changes would falsify the hypothesis, indicating that metabolic state does not dictate cell‑cycle heterogeneity or that NAD+‑mediated AMPK activation does not influence senolytic accessibility.

Broader Impact

Positive results would support a simple, low‑cost adjuvant strategy to enhance existing senolytic protocols, address the biomarker gap by providing a pharmacodynamic readout (NAD+/CD38 ratio), and inform clinical trial design for diseases where senescent‑cell burden contributes to pathology.