Hypothesis

Cycling NMN‑induced SIRT1 activation does not merely boost overall autophagy flux; it selectively reprograms the cargo‑receptor hierarchy to prioritize mitophagy over aggrephagy. This shift is mediated by SIRT1‑dependent deacetylation of the adaptor protein p62/SQSTM1, which alters its ubiquitin‑binding affinity and LC3 interaction, thereby changing the order in which damaged mitochondria versus protein aggregates are sequestered. Disrupting this SIRT1‑driven reordering—either by blocking p62 deacetylation or by forcing constitutive aggrephagy—will accelerate age‑related decline, whereas enhancing the mitophagy‑first hierarchy will extend healthspan even without changes in total autophagy levels.

Mechanistic Rationale

1. SIRT1 deacetylates p62 at lysine residues K420 and K440 (sites shown to affect ubiquitin binding in structural studies) [5]. Deacetylated p62 exhibits higher affinity for K63‑linked ubiquitin chains on depolarized mitochondria and reduced binding to K48‑linked aggregates, biasing autophagosome formation toward mitophagy.
2. FOXO3a transcriptional program downstream of SIRT1/AMPK upregulates BNIP3 and NIX, mitophagy‑specific receptors, while concurrently downregulating p62‑independent aggrephagy receptors such as NBR1 [3]. This creates a coordinated shift in receptor expression levels.
3. Ubiquitin landscape remodeling: SIRT1 activates the deubiquitinase OTUB1, which preferentially removes K48 linkages, further decreasing the ubiquitin signal that aggrephagy receptors rely on [6].

Testable Predictions

• Prediction 1: In wild‑type mice receiving intermittent NMN, p62 immunoprecipitation will show decreased acetylation and increased mitochondrially‑derived ubiquitin peptides compared with controls, while aggregate‑associated ubiquitin peptides remain unchanged.
• Prediction 2: CRISPR knock‑in of acetylation‑mimetic p62 (K420Q/K440Q) will abolish the NMN‑induced bias toward mitophagy, resulting in LC3‑II accumulation that colocalizes equally with mitochondrial (TOM20) and aggregate (p62) markers.
• Prediction 3: Mice expressing acetylation‑mimetic p62 will exhibit shortened median lifespan and accelerated frailty markers despite normal overall autophagy flux measured by lysosomal cathepsin activity.
• Prediction 4: Pharmacological enhancement of SIRT1 activity (e.g., with SRT2104) in acetylation‑deficient p62 knock‑in mice will rescue the mitophagy‑first hierarchy and improve grip strength and mitochondrial respiration.

Experimental Approach

1. Generate p62‑K420Q/K440Q knock‑in mice and littermate controls.
2. Treat cohorts with NMN cycling (400 mg/kg, 5 days on/2 days off) for 6 months.
3. Assess p62 acetylation status by IP‑Western, ubiquitin linkage profiling by tandem ubiquitin‑binding entity (TUBE) pull‑followed by mass spectrometry.
4. Measure organelle‑specific autophagic flux using mt‑Keima for mitochondria and GFP‑mCherry‑aggregates reporters in primary fibroblasts and brain slices.
5. Monitor longitudinal healthspan (grip strength, gait analysis, glucose tolerance) and survival.
6. Rescue experiments with SIRT1 activator or AAV‑mediated wild‑type p62 expression.

If the hypothesis holds, the data will show that NMN‑driven longevity depends not on how much autophagy occurs, but on what the cell chooses to eat first—a nuance that could redirect interventions toward selective autophagy modulation rather than global induction.