Hypothesis

Aging selectively remodels chromatin accessibility at enhancers of selective autophagy receptors (SQSTM1, CALCOCO2, OPTN) without altering core autophagy gene promoters, thereby rewiring the substrate hierarchy of the autophagic 'cannibalism ritual.' Total autophagic flux remains unchanged, but mis‑directed cargo degradation leads to loss of quiescence‑promoting factors and stem‑cell dysfunction.

Rationale

• ATAC‑seq in aged stem cells shows erosion at metabolic/quiescence regulators, not at ATG5/ATG7/BECN1/LC3 promoters [1][2]
• Selective autophagy relies on receptors such as p62/SQSTM1, NDP52/CALCOCO2, OPTN to ubiquitinate mitochondria, ROS aggregates, and specific protein complexes [3] [4]
• The hierarchy of receptor usage determines which substrates are prioritized; altering receptor levels shifts autophagy from protective mitophagy to indiscriminate bulk degradation or to degradation of stemness factors [5]
• Therefore, age‑linked loss of accessibility at receptor enhancers could change receptor expression, rewiring the autophagy hierarchy while leaving core machinery intact.

Novel Mechanistic Insight

We propose that the autophagy machinery operates like a ranked‑choice system: receptors compete for limited ATG8‑LC3 pools, and their relative affinity (set by expression and post‑translational modifications) decides the order of substrate engagement. Chromatin erosion reduces enhancer activity for high‑priority receptors (e.g., SQSTM1 for damaged mitochondria), lowering their expression. Consequently, lower‑affinity receptors (e.g., OPTN) gain preferential access to LC3, redirecting autophagy toward non‑optimal cargos such as transcription factors that maintain quiescence (e.g., FOXO3, p57^Kip2^). This selective loss of quiescence factors triggers differentiation bias and senescence, even though LC3‑II turnover assays show normal flux.

Testable Predictions

1. Chromatin: ATAC‑seq and H3K27ac ChIP‑seq will reveal significant loss of accessibility and active enhancer marks at SQSTM1, CALCOCO2, and OPTN loci in aged hematopoietic and neural stem cells, whereas ATG5/ATG7/BECN1/LC3 promoters remain unchanged.
2. Expression: mRNA and protein levels of the selective receptors will be downregulated in aged cells, with a concomitant shift in the receptor‑LC3 interaction profile (measured by proximity ligation or BioID) favoring lower‑affinity receptors.
3. Substrate Specificity: Quantitative proteomics of autophagosome‑isolated cargo will show enrichment of quiescence‑maintaining proteins and depletion of damaged mitochondria in aged cells relative to young.
4. Functional Rescue: CRISPR‑a mediated upregulation of SQSTM1 in aged stem cells will restore mitochondrial clearance, reduce ROS, and improve quiescence‑exit capacity without altering overall LC3‑II flux.
5. Falsification: If receptor enhancer accessibility and expression are unchanged with age, or if restoring receptor levels fails to correct substrate selectivity and stem‑cell phenotypes, the hypothesis is refuted.

Experimental Approach (brief)

• Isolate young (3 mo) and aged (24 mo) HSCs and NSCs; perform ATAC‑seq, H3K27ac ChIP‑seq, RNA‑seq, and quantitative Western blot for autophagy components.
• Use LC3‑interacting protein pull‑down followed by mass spectrometry to map receptor‑LC3 dynamics.
• Conduct mito‑Keima assays and ROS measurements to assess mitophagy efficiency.
• Perform immunostaining and flow cytometry for quiescence markers (FOXO3, p57^Kip2^) and differentiation markers (CD11b, GFAP).
• Rescue experiments: lentiviral CRISPR‑a targeting SQSTM1 enhancer; assess functional readouts.

Implications

This reframes age‑related autophagy decline not as a loss of degradative capacity but as a qualitative rewiring of the autophagy hierarchy. Therapeutic strategies that selectively boost specific autophagy receptors—rather than globally inducing autophagy—may preserve stem cell function during aging.