Reordering the autophagic cannibalism hierarchy via NAD+-dependent adaptor phosphorylation selectively primes senescent cells for senolysis

Hypothesis

We propose that the order of substrate degradation within the autophagic cannibalism hierarchy of senescent cells is dynamically regulated by the phosphorylation state of selective autophagy adaptor proteins (e.g., p62/SQSTM1, NBR1, OPTN) in response to intracellular NAD+ levels. When NAD+ is high, adaptor phosphorylation favors clearance of proteotoxic agents (aggrephagy, ER‑phagy) first, preserving mitochondrial function and sustaining the SASP. A drop in NAD+ re‑phosphorylates these adaptors to prioritize mitophagy, shifting the hierarchy and causing accumulation of protein aggregates and ER stress that pushes senescent cells past a proteotoxic threshold into senolysis.

Mechanistic Rationale

1. NAD+‑sensing kinases such as SIRT1 and AMPK are known to modulate autophagy activity [3] and can phosphorylate adaptor proteins, altering their affinity for ubiquitin‑cargo complexes [1].
2. In senescent cells, basal autophagy sustains mTORC1 signaling by recycling amino acids [3], creating a feedback loop that couples energy status to substrate selection.
3. Recent work shows that disrupting the hierarchy—e.g., by ATG3 knockout—leads to mitochondrial backlog, ROS‑driven SASP, and eventual senolysis [4]. We argue that the trigger for this backlog is not loss of autophagy per se, but a NAD+‑dependent re‑ranking of cargo preference that stalls aggrephagy/ER‑phagy while mitophagy proceeds.

Testable Predictions

• Prediction 1: Pharmacological elevation of NAD+ (e.g., nicotinamide riboside) will increase the phosphorylation of p62 at serine residues linked to aggrephagy/ER‑phagy cargo binding, measurable by phospho‑specific immunoblotting, and will shift the autophagic flux order toward protein aggregate clearance before mitophagy in senescent fibroblasts.
• Prediction 2: Conversely, NAD+ depletion (via FK866 or CD38 overexpression) will reverse adaptor phosphorylation, causing a relative increase in mitophagy flux (mt‑Keima assay) concomitant with accumulation of p62‑positive aggresomes and ER‑stress markers (CHOP, BiP).
• Prediction 3: Inducing the NAD+‑low shift in senescent cells will sensitize them to sub‑lethal doses of senolytics (e.g., low‑dose dasatinib) because the proteotoxic overload lowers the threshold for autophagic cell death, whereas proliferating controls remain unaffected.
• Prediction 4: In aged mice, intermittent NAD+ boosting will delay the emergence of the hierarchical shift, preserving autophagy‑mediated SASP suppression without inducing senolysis, thereby extending healthspan; NAD+ blockade will accelerate senescent cell clearance but also increase tissue inflammation if dosing is not timed.

Experimental Design

• In vitro: Use human IMR‑90 fibroblasts rendered senescent by irradiation. Treat with NR (1 g/kg equivalent) or FK866 (10 nM) for 48 h. Measure:
  • Phospho‑p62 (Ser403/Ser407) by Western blot.
  • Mitochondrial flux (mt‑Keima) vs. aggrephagy flux (GFP‑LC3‑RFP‑p62 reporter).
  • ROS (MitoSOX), ER‑stress (CHOP), SASP cytokines (IL‑6, IL‑8 ELISA).
  • Viability after low‑dose dasatinib (50 nM) or quercetin (5 µM).
• In vivo: Aged (20‑month) C57BL/6 mice receive NR (400 mg/kg chow) or FK866 (0.5 mg/kg i.p. twice weekly) for 8 weeks. Assess:
  • Tissue‑specific senescence (p16^Ink4a^‑GFP reporters).
  • Autophagy hierarchy via tandem reporters for mitophagy and aggrephagy in isolated macrophages.
  • SASP serum levels, frailty index, and histological damage.

Falsifiability

If NAD+ manipulation does not alter adaptor phosphorylation or the relative order of mitophagy versus aggrephagy/ER‑phagy, or if these changes fail to affect senescent cell sensitivity to senolytics, the hypothesis would be falsified. Likewise, if NAD+ elevation in aged mice does not delay senescence‑associated functional decline despite altering autophagy flux, the proposed mechanistic link between NAD+‑dependent hierarchy remodeling and senolysis would be refuted.

By targeting the sequence rather than the capacity of autophagy, this hypothesis converts a descriptive cannibalism hierarchy into a precise, druggable checkpoint for selective senescent cell removal.