Hypothesis

The hierarchy of selective autophagy is not static but is rewired each day by a circadian phospho‑switch on the receptor p62/SQSTM1. During the light phase, CK1ε phosphorylates p62 at Ser403, increasing its affinity for K63‑linked ubiquitin chains while reducing LC3 interaction. This biases the receptor‑driven, cargo‑first pathway toward removal of damaged mitochondria and protein aggregates. In the dark phase, dephosphorylation by PP1 restores balanced p62‑LC3 binding, allowing bulk, starvation‑induced autophagy to dominate and sustain amino‑acid recycling. Disruption of this phospho‑cycle—through chronic light‑at‑night, aging‑related kinase decline, or phospho‑dead mutants—locks p62 in a state that preferentially engages bulk autophagy, depriving the cell of timely selective clearance and leading to accumulation of toxic cargo, mitochondrial dysfunction, and inflammaging.

Testable Predictions

1. Phospho‑specific antibodies will show peak p62‑Ser403 phosphorylation in mouse liver at ZT6 (mid‑light) and nadir at ZT18 (mid‑dark).
2. Liver‑specific expression of a phospho‑dead p62‑S403A mutant will flatten the circadian rhythm of autophagosome abundance, increase basal LC3‑II levels, and reduce mitochondrial protein turnover measured by pulse‑chase labeling of COXIV.
3. Conversely, a phospho‑mimetic p62‑S403D will enhance mitochondrial clearance during the light phase, increase mitophagy flux (mt‑Keima), and improve glucose tolerance in aged mice.
4. Chronic exposure to dim light at night will dampen p62‑Ser403 phosphorylation, shift autophagy toward bulk degradation, and accelerate age‑related decline in grip strength and cognitive performance; rescuing CK1ε activity with a small‑molecule agonist will restore the phospho‑rhythm and extend healthspan.
5. In human peripheral blood monocytes, serum melatonin inversely correlates with p62‑Ser403 phosphorylation, linking the light‑dark cycle to receptor state.

Mechanistic Insight

The canonical model places cargo receptors downstream of isolation membrane formation for selective autophagy. Our hypothesis adds a temporal layer: circadian kinases modify the receptor’s binding hierarchy, converting the same molecular machinery from a cargo‑first, selective mode to a bulk, starvation‑driven mode. This dual‑use strategy lets the cell prioritize quality control when metabolic activity is high (light phase) and switch to nutrient scavenging when intake is low (dark phase). When the switch fails, the cell either over‑digests essential components or under‑clears damage, both hallmarks of aging. Targeting the phospho‑switch offers a precise way to repair the autophagy hierarchy without globally inhibiting or activating the pathway, addressing the core idea that it is the sequence of cargo consumption—not the process itself—that encodes survival priorities.

Experimental Approach

• Generate CRISPR‑knockin mice expressing p62‑S403A or p62‑S403D.
• Monitor autophagic flux with mCherry‑GFP‑LC3 reporters in liver and brain across circadian time.
• Quantify organelle turnover using organelle‑specific reporters (mt‑Keima for mitochondria, DQ‑BSA for lysosomes).
• Assess lifespan and healthspan under ad lib feeding and time‑restricted feeding regimens.
• Parallel human studies: isolate PBMCs from shift workers vs day workers, measure phospho‑p62 by flow cytometry, correlate with circadian markers and clinical aging scores.

If the phospho‑switch dictates the selective‑bulk autophagy hierarchy, restoring its circadian rhythm should selectively improve clearance of damaged cargo while preserving bulk recycling, thereby delaying age‑related decline. Falsification would come from showing that manipulating p62‑Ser403 phosphorylation does not alter the temporal pattern of substrate preference or health outcomes.