Hypothesis

The autophagy substrate hierarchy is not fixed; it is tunable through phosphorylation of selective autophagy receptors (SARs). We hypothesize that chronic inflammatory signaling in aging skews TBK1‑dependent phosphorylation toward ubiquitin‑binding SARs (p62, OPTN) at the expense of mitophagy receptors (BNIP3/NIX), causing a bottleneck that spares damaged mitochondria while allowing lipofuscin‑forming aggregates to accumulate. Restoring a balanced phosphorylation state—by enhancing TBK1 activity on mitophagy SARs or inhibiting it on p62—should reset the hierarchy, re‑prioritize mitochondrial clearance, and reduce lipofuscin burden in aged cells.

Mechanistic Rationale

• SARs compete for limited LC3/GABARAP (ATG8) and FIP200 binding sites; phosphorylation by TBK1 or ULK1 increases affinity and thus priority in the hierarchy [1].
• In young cells, oxidative stress preferentially activates ULK1‑driven mitophagy (BNIP3/NIX) and TBK1‑driven clearance of ubiquitinated proteins, creating a dynamic equilibrium.
• Aging is associated with low‑grade inflammasome activation and sustained TBK1 signaling that hyper‑phosphorylates p62/OPTN, sequestering LC3 and outcompeting mitophagy receptors [2]. This shifts the hierarchy toward protein aggregate clearance, leaving mitochondria under‑degraded and contributing to ROS‑driven lipofuscin formation.
• Lipofuscin resists autophagy because its crosslinking occurs after mitochondrial damage; if mitochondria are not removed first, aggregates become entrenched.

Testable Predictions

1. Phospho‑specific SAR profiling: Aged human fibroblasts will show increased p62‑S403/OPTN‑S177 phosphorylation and decreased BNIP3‑S17/NIX‑S95 phosphorylation relative to young cells (Western blot with phospho‑specific antibodies).
2. Hierarchy rescue: Expressing phospho‑deficient (S→A) p62/OPTN or phospho‑mimetic (S→D/E) BNIP3/NIX in aged cells will restore LC3 binding to mitophagy receptors (measured by LC3‑pull‑down) and increase mitochondrial turnover (mt‑Keima assay) without affecting total autophagic flux (LC3‑II turnover with bafilomycin).
3. Lipofuscin reduction: The SAR re‑balancing interventions will decrease lipofuscin autofluorescence (excitation 360 nm, emission 460 nm) by ≥30 % after 72 h, whereas scavenging ROS alone will not.
4. In vivo relevance: Mice treated with a TBK1‑biased inhibitor that spares ULK1 activity will exhibit improved mitophagy (COX‑IV loss) and lower lipofuscin in brain and muscle versus wild‑type aged controls.

Falsifiability

If manipulating SAR phosphorylation does not alter the relative LC3 binding of mitophagy versus ubiquitin receptors, or if lipofuscin levels remain unchanged despite restored mitophagy, the hypothesis is refuted.

[1] https://pmc.ncbi.nlm.nih.gov/articles/PMC12669772/ [2] https://pmc.ncbi.nlm.nih.gov/articles/PMC8486182/