Hypothesis

Aging myenteric neurons experience persistent oxidative stress that constitutively activates AMPK‑ULK1‑dependent autophagy initiation, yet lysosomal acidification fails due to ROS‑mediated inhibition of the V‑ATPase proton pump. This creates a futile autophagy loop where autophagosomes engulf damaged mitochondria but cannot fuse with or degrade them in acidic lysosomes, leading to incomplete mitophagy, ATP depletion, and selective loss of cholinergic and nitrergic neurons. The loop explains why interventions that merely boost autophagy initiation (e.g., rapamycin, caloric restriction) improve survival only when they also restore lysosomal function, and why chronic oxidative conditions convert autophagy from a protective housekeeping process into a maladaptive rationing system that exhausts neuronal reserves.

Mechanistic Reasoning

1. ROS‑driven AMPK activation → sustained ULK1 phosphorylation → constant phagophore formation.
2. ROS attacks V‑ATPase subunits (especially ATP6V0C) → lysosomal pH rises from ~4.5 to >6.0 → cathepsin activity drops, impairing autophagosome‑lysosome fusion and cargo degradation.
3. Accumulation of undigested autophagosomes sequesters LC3‑II and p62, detectable as elevated autophagy flux markers despite blocked degradation.
4. Energy crisis: futile cycling consumes ATP without yielding reusable metabolites, worsening mitochondrial dysfunction and triggering apoptosis preferentially in high‑firing cholinergic/nitrergic neurons.
5. Neuronal subtype vulnerability: these neurons have high basal mitochondrial turnover and limited glycolytic capacity, making them reliant on efficient mitophagy.

Testable Predictions

• Prediction 1: In aged rat myenteric plexus, lysosomal pH will be significantly higher than in young controls, and V‑ATPase activity will be inversely correlated with ROS levels (1).
• Prediction 2: Pharmacological re‑acidification of lysosomes (e.g., with vacuolar‑ATPase activators such as MLN4924 at low dose or overexpression of ATP6V0C) will restore LC3‑II turnover and reduce p62 accumulation, indicating completed autophagic flux.
• Prediction 3: Neuronal survival assays will show that combining an autophagy initiator (rapamycin) with a lysosomal acidulant (e.g., leucine‑ethylester) yields greater preservation of cholinergic/nitrergic neuron numbers than either treatment alone in aged colonic explants.
• Prediction 4: Genetic knockdown of ATP6V0C in young myenteric neurons will phenocopy the aged phenotype: increased autophagosome formation, mitochondrial loss, and slowed colonic transit in vivo.

Falsifiability

If lysosomal pH remains unchanged with age, or if restoring acidification fails to improve mitophagy flux and neuronal survival, the hypothesis would be refuted, suggesting that the bottleneck lies elsewhere (e.g., impaired autophagosome‑lysosome tethering or lysosomal enzyme inactivation independent of pH).

Conclusion

Reframing autophagy as a siege‑induced rationing system predicts that the key therapeutic target in age‑related constipation is not merely boosting autophagy initiation but repairing lysosomal acidification to break the futile cycle and allow genuine mitochondrial recycling.