Hypothesis

When nutrients are scarce, cells execute a triage‑like selective autophagy program in which mitochondria are preferentially degraded (mitophagy) prior to the clearance of cytosolic protein aggregates (aggrephagy) and lipid droplets (lipophagy). This ordered hierarchy is not a by‑product of bulk autophagy but a regulated signaling cascade that prioritizes removal of the most deleterious damage first. We hypothesize that perturbing the temporal sequence — specifically, delaying mitophagy relative to aggrephagy — causes accumulation of dysfunctional mitochondria, increased ROS, and secondary proteotoxic stress, thereby driving the molecular hallmarks of aging. Conversely, preserving or enhancing the mitophagy‑first order should extend healthspan independent of overall autophagic flux.

Mechanistic Rationale

1. Hierarchy is encoded by differential ULK1 phosphorylation – AMPK‑dependent ULK1‑Ser317/Ser777 promotes mitophagy, whereas mTORC1‑sensitive ULK1‑Ser757 favors aggrephagy under prolonged stress. Nutrient fluctuations create a biphasic ULK1 activity profile that times mitophagy early and aggrephagy later.
2. Mitochondrial ROS acts as a secondary signal – Early mitophagy limits ROS‑mediated oxidation of cytosolic proteins, reducing the load on aggrephagy. If mitophagy is lagging, ROS‑damaged proteins overwhelm aggrephagy, leading to aggregate formation.
3. Feedback via TFEB – Successful mitophagy restores lysosomal competence, enabling TFEB nuclear translocation and upregulation of aggrephagy genes. Disrupted mitophagy keeps TFEB cytosolic, blunting aggrephagy capacity.

Thus, the hierarchy creates a self‑reinforcing quality‑control loop; breaking the loop uncouples mitophagy from aggrephagy and precipitates age‑associated decline.

Testable Predictions

• Prediction 1: In cultured human fibroblasts subjected to intermittent fasting, live‑cell reporters for mitophagy (mt‑Keima) will show a peak 2–4 h after glucose withdrawal, followed by a delayed aggrephagy reporter (GFP‑LC3‑p62) peak at 6–8 h. Pharmacological inhibition of AMPK (Compound C) will delay the mitophagy peak without changing overall LC3‑II conversion, resulting in higher mitochondrial ROS and increased p62‑positive aggregates at 8 h.
• Prediction 2: Expressing a phospho‑dead ULK1‑Ser317A mutant (which impairs AMPK‑driven mitophagy) in C. elegans will shift the autophagy hierarchy toward aggrephagy, measurable as earlier accumulation of LGG‑1::GFP in protein‑aggregate reporters (QC::YFP) and later mito‑ROS sensing (mito‑roGFP). These worms will exhibit shortened lifespan and accelerated motility decline despite normal basal autophagy flux.
• Prediction 3: Pulselow‑dose IGF‑1 combined with BPC‑157 (known to activate AKT‑mTOR) will bias ULK1 phosphorylation toward Ser757, postponing mitophagy. In aged mice, this treatment will increase mitochondrial DNA deletions and protein carbonyls in muscle, while aggrephagy markers (p62, NBR1) remain unchanged, leading to greater frailty scores compared with IGF‑1 alone.

Experimental Approach

1. Live‑cell imaging of dual‑fluorescence reporters (mt‑Keima and GFP‑LC3‑p62) in U2OS cells under serum starvation, with time‑lapse quantification of peak moments.
2. Genetic manipulation: CRISPR‑knock‑in of ULK1 phospho‑mutants (S317A, S757A) in HEK293 and C. elegans; assess mitochondrial Membrane Potential (TMRM), ROS (MitoSOX), and aggregate load (filter‑trap assay).
3. In vivo validation: Treat 18‑month‑old C57BL/6 mice with IGF‑1 (0.1 mg/kg, twice weekly) ± BPC‑157 (10 µg/kg) for 3 months; harvest gastrocnemius for mitophagy (LC3‑II/COXIV colocalization), aggrephagy (p62/SQSTM1), 8‑OH‑dG, and grip strength.

Falsifiability

If altering the order of selective autophagy (e.g., delaying mitophagy) does not lead to increased mitochondrial dysfunction, protein aggregation, or accelerated aging phenotypes across these models, the hypothesis would be falsified. Likewise, if rescuing mitophagy timing (via AMPK activators) fails to improve healthspan despite corrected hierarchy, the proposed causal link would be refuted.

Implications

Confirming that the selective autophagy hierarchy is a determinant of cellular longevity would shift focus from merely boosting autophagic flux to orchestrating the sequence of organelle and protein clearance. It would also provide a mechanistic framework for evaluating peptides like BPC‑157, TB‑500, and IGF‑1: their therapeutic value may hinge on whether they preserve or disrupt this triage‑like order rather than on their ability to stimulate bulk autophagy alone.