Hypothesis

We propose that metabolic priming with NAD+ replenishment and AMPK activation reprograms the lipid metabolism and lysosomal capacity of G2-arrested senescent cells, rendering them more susceptible to senolytic-triggered ferroptosis.

Rationale

Senescent cells are heterogeneous; G2-arrested subpopulations resist conventional senolytics due to elevated anti-oxidant defenses and altered mitochondrial function[8]. NAD+ restoration boosts SIRT3 activity, which deacetylates and activates SOD2, lowering mitochondrial ROS but simultaneously increases the pool of acetyl-CoA available for fatty acid synthesis[5]. AMPK activation inhibits mTORC1, upregulates TFEB-driven lysosomal biogenesis, and enhances autophagic flux[6]. Together, these shifts increase intracellular labile iron and phospholipid PUFA content, key determinants of ferroptosis sensitivity, while preserving enough lysosomal capacity to senolytic uptake.

Predictions

1. In aged mice, combined NR (nicotinamide riboside) and AICAR (AMPK activator) pretreatment will increase ferroptosis biomarkers (lipid ROS, ACSL4 upregulation, GPX4 downregulation) specifically in G2-arrested senescent cells identified by p21^high^/phospho-H3^high^.
2. The same pretreatment will lower the effective dose of dasatinib+quercetin required to achieve >=50% reduction of G2-arrested senescent cells in liver and adipose tissue, without affecting G1-arrested cells.
3. Ferroptosis inhibition (liproxstatin-1) will abolish the synergistic senolytic effect, confirming ferroptosis as the mechanistic link.

Experimental Design

• Animals: 20-month-old C57BL/6 mice, n=5 per group.
• Groups: (1) Vehicle control; (2) Dasatinib+quercetin (D+Q) alone; (3) NR + AICAR pretreatment; (4) NR + AICAR followed by D+Q.
• Treatment: NR 400 mg/kg/day oral, AICAR 250 mg/kg/day i.p. for 3 days, then D+Q 5 mg/kg dasatinib + 50 mg/kg quercetin i.p. once daily for 2 days.
• Readouts: Flow cytometry for p21, phospho-H3, SA-β-gal; lipid ROS (C11-BODIPY); ACSL4 and GPX4 western blot; lysosomal marker LAMP1 immunofluorescence; serum SASP cytokines (IL-6, CCL2).
• Ferroptosis rescue: Subset receives liproxstatin-1 10 mg/kg i.p. with D+Q.

Potential Outcomes and Interpretation

If NR + AICAR pretreatment significantly augments lipid ROS and reduces GPX4 only in G2-arrested cells, and this correlates with enhanced senolytic clearance that is blocked by ferroptosis inhibition, the hypothesis is supported. Conversely, if pretreatment fails to alter ferroptosis markers or does not improve senolytic efficacy beyond D+Q alone, the hypothesis is refuted, indicating that metabolic priming does not sensitize G2-arrested senescent cells via ferroptosis.

This framework directly links NAD+/AMPK signaling to the lipid-peroxidation axis of ferroptosis, offering a testable route to overcome senolytic resistance in specific senescence subpopulations.