Hypothesis

Aging drives a measurable breakdown in the topological hierarchy of autophagy substrate selection, detectable as the loss of persistent 1‑dimensional loops (representing ordered co‑expression of mitophagy → lipophagy → aggrephagy modules) in single‑cell transcriptomic manifolds. This topological decay precedes functional decline in autophagic flux and lifespan, and experimentally restoring the hierarchical order rescues both.

Mechanistic Basis

• Autophagy selectivity is governed by a temporal cascade of cargo receptors (NIX/BNIP3L → OPTN → SQSTM1/p62) whose expression is coordinated by successive transcription factor waves (FOXO3 → TFEB → ATF4).
• In young cells, this wave creates a reproducible loop in gene‑expression space: early mitophagy genes rise, then decline as lipophagy genes ascend, followed by aggrephagy genes—a trajectory captured by a persistent 1‑D hole in the persistence diagram.
• With age, epigenetic drift and stochastic noise desynchronize these waves, causing the loop to fragment (shorter persistence) or fill in (birth–death pairs converge), reflecting a loss of hierarchical ordering.
• The resulting ‘topological noise’ manifests as indiscriminate organelle consumption, driving the functional deficits attributed to generic autophagy decline.

Testable Predictions

1. Persistence Diagram Metric: The bottleneck distance between the persistence diagram of autophagy‑related genes in young stressed cells and that in aged stressed cells will increase with chronological age across species (yeast, worms, mice).
2. Correlation with Function: Greater bottleneck distance will predict lower mitophagy‑to‑lipophagy flux ratios (measured by mt‑Keima and BODIPY‑C12) and shorter median lifespan, independent of overall autophagy gene expression levels.
3. Causal Rescue: Forced re‑expression of the early‑phase receptor NIX (or optogenetic activation of FOXO3) in aged cells will restore the 1‑D loop persistence (reducing bottleneck distance) and re‑establish the mitophagy‑first consumption order, thereby improving flux and extending lifespan.
4. Pharmacological Perturbation: Treating young cells with rapamycin (which globally induces autophagy) should increase topological disorder (higher bottleneck distance) if it bypasses the natural hierarchy, providing a negative control.

Experimental Design

• Data Generation: Perform scRNA‑seq on isolated tissues (e.g., mouse liver, C. elegans intestine) from young (3 mo) and aged (24 mo) mice under acute oxidative stress (paraquat). Include conditions: wild‑type, NIX‑overexpressing aged, and rapamycin‑treated young.
• Analysis Pipeline:
  1. Extract expression of a curated autophagy selectivity gene set (mitophagy, lipophagy, aggrephagy, ubiquitin‑binding receptors).
  2. Build a Vietoris–Rips complex on the normalized expression matrix; compute persistence diagrams (dimensions 0 and 1).
  3. Summarize the 1‑D persistence landscape; calculate bottleneck distance to the young wild‑type reference diagram.
  4. Correlate distance with functional assays (flux, ROS, lifespan).
• Validation: Use multiplexed immunofluorescence to verify the temporal order of organelle clearance (MitoTracker, BODIPY, p62 aggregates) in the same samples.

Falsifiability

If aged cells show no significant increase in bottleneck distance compared to young cells, or if restoring NIX fails to recover the 1‑D loop and does not improve flux/lifespan, the hypothesis is refuted. Conversely, a consistent topological metric that predicts functional outcomes and is manipulable by hierarchy‑specific interventions would support the claim that autophagy decline is a failure of ordered selectivity, not merely reduced capacity.