Hypothesis

Berberine’s longevity benefits stem from AMPK‑driven selective mitophagy that purges mitochondria carrying high levels of pathogenic mtDNA heteroplasmy, thereby lowering the mutational load below a tissue‑specific threshold and breaking the vicious cycle of deletion‑mutant amplification.

Mechanistic Rationale

1. AMPK activation by berberine phosphorylates the mitophagy receptors FUNDC1 and BNIP3, increasing their affinity for LC3 on depolarized mitochondria.
2. Depolarized mitochondria are enriched for mtDNA lesions and high heteroplasmy because damaged genomes impair oxidative phosphorylation and membrane potential.
3. Selective removal of these organelles reduces the fraction of mutant mtDNA copies without triggering compensatory biogenesis, as PGC‑1α activation is coupled to a quality‑control checkpoint that senses reduced ROS and restored NAD⁺/NADH ratio.
4. Consequently, the net mtDNA copy number may stay stable or even decline in diseased cells, while healthy repopulation occurs from the remaining low‑mutagen load.

Testable Predictions

• Prediction 1: In mtDNA mutator (PolG⁻/⁻) mice, chronic berberine treatment will decrease heteroplasmy at common deletion sites (e.g., mtDNA4977) in skeletal muscle and brain by ≥30 % compared with vehicle, measured by droplet digital PCR.
• Prediction 2: The heteroplasmy reduction will correlate with improved respiratory control ratio (RCR) and decreased ROS emission, whereas total mtDNA copy number remains unchanged or slightly reduced.
• Prediction 3: Muscle‑specific AMPKα1/α2 double knockout will abolish berberine‑induced heteroplasmy shift and fail to extend survival, despite normal drug exposure.
• Prediction 4: Pharmacological inhibition of mitophagy (e.g., using the ULK1 inhibitor SBI‑0206965) or genetic ablation of Atg5 in muscle will block berberine’s effect on heteroplasmy, confirming mitophagy dependence.
• Prediction 5: Single‑mitochondrion sequencing will show a shift in the distribution of mutant load toward lower heteroplasmy percentages, indicating purging rather than uniform dilution.

Falsifiability

If berberine treatment does not lower pathogenic heteroplasmy in any tissue, or if the longevity benefit persists in AMPK‑ or mitophagy‑deficient models, the hypothesis is refuted. Conversely, demonstration of selective mutant depletion would support the claim that targeting mtDNA quality control, rather than generic AMPK signaling, is a key mediator of berberine’s anti‑aging action.