Lysosomal pH–Dependent Reordering of Autophagic Substrate Priority Drives Age‑Related Clonal Expansion of Chromosomally Unstable Cells

The autophagy hierarchy described in ’s work removes damaged mitochondria first, then nuclear chromatin, and finally whole cells harboring copy number variations (CNVs). We hypothesize that this order is not fixed but is gated by lysosomal luminal pH, which shifts toward alkalinity with age. When lysosomes become less acidic, the activity of pH‑dependent hydrolases (e.g., cathepsins B, L, D) declines disproportionately for substrates that require rapid proteolysis, such as ubiquitinated mitochondrial outer‑membrane proteins. Consequently, the initial mitophagy step slows, while downstream chromatin‑fragment degradation—reliant on less pH‑sensitive proteases—continues relatively unchanged. This reordering allows ROS‑producing mitochondria to persist in CNV‑bearing cells, fostering a permissive environment for clonal expansion.

Mechanistic reasoning: Lysosomal pH regulates the conformational state of the V-ATPase and the recruitment of autophagosome‑lysosome tethering complexes (HOPS, STX17). Acidic lysosomes promote rapid fusion and activation of cathepsins that preferentially cleave K63‑linked ubiquitin chains on damaged mitochondria. Alkalinization impairs this ubiquitin‑sensing step, delaying mitophagy while leaving the recognition of histone H2AX‑γ (a marker of chromatin damage) intact because its ubiquitination relies on K48‑linked chains that are processed by a broader set of proteases. The resulting substrate‑selection skew means that autophagy no longer functions as a tumor‑suppressive triage but instead selectively degrades nuclear material, potentially increasing mutagenesis while failing to curb oxidative stress.

Testable predictions:

1. In young human hematopoietic stem cells (HSCs), lysosomal pH measured with LysoSensor Yellow/Blue will correlate inversely with mitophagy flux (mt‑Keima assay) and positively with clearance of γ‑H2AX foci.
2. Pharmacological lysosomal alkalinization (e.g., chloroquine low dose) in young HSCs will reproduce the aged autophagy hierarchy: delayed mitochondrial clearance, unchanged chromatin‑fragment degradation, and increased survival of CNV‑induced HSCs under oxidative stress.
3. Genetic restoration of lysosomal acidity in aged HSCs (overexpression of V‑ATPase subunit ATP6V0D2) will rescue mitophagy priority, reduce ROS, and diminish the expansion of CNV‑bearing clones in vivo.
4. Single‑cell multi‑omics of aged human blood will reveal a subpopulation with elevated lysosomal pH signatures, high mitochondrial mass, low ubiquitinated mitochondrial proteins, and persistent γ‑H2AX, correlating with clonal hematopoiesis driver mutations (DNMT3A, TET2, JAK2).

Falsifiability: If lysosomal pH modulation does not alter the sequential degradation of mitochondria versus chromatin fragments, or if rescuing acidity fails to mitigate CNV‑bearing clone expansion, the hypothesis would be refuted. This positions lysosomal pH as a tunable checkpoint that encodes the survival priorities of the autophagy "cannibalism ritual," offering a novel avenue to counteract age‑related clonal hematopoiesis and associated cancer risk.