Hypothesis

Premature activation of ER‑phagy through aberrant OPTN phosphorylation flips the normal autophagy substrate hierarchy, causing mitochondria to be spared while ER is over‑cleared. This shift leads to mitochondrial dysfunction, ROS accumulation, and accelerates age‑related decline.

Mechanistic Basis

• Autophagy substrate hierarchy is set by competitive binding of SARs (OPTN, NDP52, p62) to ubiquitinated cargos and their phosphorylation‑dependent recruitment of ULK1/TBK1 complexes [1][3]
• In inflammation‑induced muscle atrophy, TNF‑α upregulates SEC62 and OPTN, biasing the system toward ER‑phagy early in the atrophy process [2]
• We propose that a kinase (e.g., CK2) phosphorylates OPTN at serine residues that normally respond to ER stress, but chronic low‑grade inflammation drives constitutive phosphorylation, giving OPTN a competitive advantage over mitophagy receptors (NDP52, p62) even when mitochondrial damage signals are present
• Consequently, ER is phagocytosed preferentially, mitochondria escape timely removal, and damaged mitochondria accumulate, increasing ROS and triggering inflammasome activation—a feed‑forward loop that exacerbates ER stress and further OPTN phosphorylation

Testable Predictions

1. Cells expressing a phospho‑dead OPTN mutant (S→A) will retain normal mitophagy priority under TNF‑α exposure, showing reduced mitochondrial ROS compared with wild‑type OPTN
2. Phospho‑mimetic OPTN (S→D) will cause ER‑phagy dominance, mitochondrial accumulation, and shortened replicative lifespan in human fibroblasts
3. In vivo, muscle‑specific overexpression of phospho‑mimetic OPTN in mice will accelerate age‑related grip‑strength loss and increase markers of mitochondrial dysfunction, whereas phospho‑dead OPTN will extend healthspan

Experimental Approach

• Generate CRISPR‑edited C2C12 myoblasts with OPTN S→A or S→D mutations
• Treat with TNF‑α (10 ng/mL) for 6 h, then measure:
  • ER‑phagy flux (SEC62‑LC3 colocalization) vs mitophagy flux (mt‑Keima assay)
  • Mitochondrial ROS (MitoSOX) and membrane potential (TMRE)
  • Seahorse OCR/ECAR
• In vivo: create AAV9‑mediated muscle‑specific OPTN S→A or S→D expression in C57BL/6J mice; monitor grip strength, treadmill endurance, and muscle histology at 6, 12, and 18 months
• Statistical analysis: two‑way ANOVA with genotype and age as factors; n ≥ 8 per group for adequate power

If phospho‑dead OPTN preserves mitophagy and delays functional decline, the hypothesis is supported; if altering OPTN phosphorylation does not shift substrate hierarchy or affect aging phenotypes, the hypothesis is falsified.