Hypothesis

Circadian amplitude attenuation predicts mortality risk by disrupting the rhythmic autophagy‑lysosome flux that normally gates proteostatic repair during the rest phase. When circadian amplitude falls below a critical threshold, the phase‑locked activation of lysosomal hydrolases becomes arrhythmic, leading to chronic accumulation of damaged macromolecules and accelerated biological aging.

Mechanistic Insight

The core clock drives not only transcription of autophagy genes (e.g., LC3, ATG5) but also the rhythmic expression of lysosomal v‑ATPase subunits and cathepsins, creating a temporal gating mechanism that aligns autophagosome formation with lysosomal degradation capacity. This coupling ensures that cytosolic waste is cleared efficiently during the subjective night. Age‑related decline in BMAL1/CLOCK activity reduces the amplitude of both arms, uncoupling autophagosome formation from lysosomal readiness. The resulting "autophagic bottleneck" increases cytosolic p62/SQSTM1, elevates inflammasome activation, and drives systemic inflammation—a known mortality predictor.

Testable Predictions

1. In human peripheral blood mononuclear cells (PBMCs) collected every 4 h over 24 h, individuals with low circadian amplitude (measured by PER2::LUC bioluminescence or serum cortisol rhythm) will show a blunted LC3‑II/LLC‑I ratio peak and a concomitant rise in basal p62 levels compared with high‑amplitude counterparts.
2. Longitudinally, baseline circadian amplitude will inversely correlate with all‑cause mortality over 5 years, independent of age, sex, and comorbidities, and this relationship will be mediated by the autophagic flux index (LC3‑II turnover after bafilomycin A1 treatment).
3. Pharmacological enhancement of lysosomal acidity (e.g., low‑dose chloroquine withdrawal or TFEB activators) in low‑amplitude subjects will restore rhythmic LC3‑II degradation and reduce inflammatory cytokines (IL‑6, TNF‑α) in a crossover trial.

Experimental Approach

• Cohort: Recruit 200 adults aged 50‑80, stratify by actigraphy‑derived circadian amplitude (high vs low).
• Sampling: Collect PBMCs at 4‑hour intervals for 24 h; measure PER2 mRNA, LC3‑II, LC3‑I, p62, cathepsin D activity, and cytokine panel.
• Flux assay: Treat aliquots with bafilomycin A1 for 4 h to calculate autophagic flux.
• Follow‑up: Track mortality and morbidity for 5 years.
• Intervention sub‑study: Randomize low‑amplitude participants to TFEB activator or placebo for 12 weeks; repeat 24‑h sampling.

Potential Confounds & Controls

• Control for sleep disorders, shift work, and medication affecting autophagy.
• Adjust for NAD+ levels, given the Clock‑NAD+-Sirtuin axis.
• Use mixed‑effects modeling to account for intra‑individual variability.

If validated, this hypothesis would position circadian amplitude not merely as a biomarker but as a modifiable upstream regulator of lysosomal autophagy, offering a concrete target for geroprotective interventions.

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