Hypothesis

We propose that supplying NAD+ precursors at the circadian nadir of hepatic NAD+ restores mitophagic flux and mitigates age‑associated mitochondrial DNA deletions, independent of core clock gene amplitude.

Mechanistic Rationale

Circadian NAD+ oscillations drive SIRT3 activity, which deacetylates LC3 and promotes mitophagy 1. In aging, NAD+ troughs blunt this signal, leading to accumulation of damaged mitochondria and mtDNA deletions 2. Exogenous NAD+ precursors (e.g., nicotinamide riboside) can raise cytosolic NAD+, but their efficacy depends on timing relative to the endogenous rhythm; administering them when NAD+ is lowest should amplify the amplitude of the oscillation and re‑engage SIRT3‑mediated mitophagy.

Experimental Design

• Animals: Young (3 mo) and aged (18 mo) C57BL/6J mice, plus liver‑specific Bmal1 knockout (L‑Bmal1KO) to test clock‑independence.
• Intervention: Oral nicotinamide riboside (NR) 300 mg/kg/day delivered via drinking water either at ZT6 (NAD+ peak) or ZT18 (NAD+ trough) for 12 weeks. Control groups receive water alone.
• Readouts:
  • Hepatic NAD+ levels (LC‑MS) at 4‑h intervals to confirm rhythm restoration.
  • SIRT3 activity (acetyl‑LC3 Western blot) and LC3‑II/I ratio (mitophagy flux) with and without bafilomycin A1.
  • Mitochondrial DNA copy number and deletion frequency (qPCR long‑range assay).
  • ROS production (MitoSOX fluorescence) and oxidative stress markers (4‑HNE).
  • Behavioral metrics (activity rhythms) and survival analysis.

Predicted Outcomes

• NR given at ZT18 will increase NAD+ amplitude >30 % versus ZT6 or controls, rescue SIRT3‑LC3 deacetylation, and raise mitophagic flux by ~2‑fold in aged WT mice.
• This will halve mtDNA deletion frequency and reduce ROS to levels seen in young mice.
• In L‑Bmal1KO mice, ZT18 NR will still improve mitophagy and lower deletions, indicating that timed NAD+ can bypass a broken transcriptional clock.
• Lifespan extension (median survival) of ~15 % in aged WT mice receiving ZT18 NR, but not in ZT6 NR or controls.

Potential Confounds and Controls

• NR metabolism may produce nicotinamide that inhibits sirtuins; we will measure NAMPT activity and include a nicotinamide‑only group to rule out inhibitory effects.
• Feeding behavior could shift with NR timing; we will monitor food intake and adjust pair‑feeding cohorts.
• Circadian leakage from peripheral tissues; we will sample muscle and brain to confirm liver‑specific effects.

If the hypothesis fails—i.e., ZT18 NR does not improve mitophagy or mtDNA integrity despite raising NAD+—then the premise that NAD+ timing alone can drive SIRT3‑dependent mitophagy would be falsified, pointing to additional clock‑controlled cofactors (e.g., acetyl‑CoA rhythms) as necessary.