Partial reprogramming with transient OSK factors resets epigenetic clocks via TET1/2-dependent DNA demethylation, yet the role of autophagy in this process remains untested. We hypothesize that autophagy is not merely a by‑product of metabolic stress but a necessary supplier of acetyl‑CoA and α‑ketoglutarate (α‑KG) that sustains TET enzyme activity during OSK‑driven demethylation. In this view, autophagy functions as a rationing system that channels intracellular nutrients toward epigenetic remodeling, allowing the cell to survive the siege of reprogramming stress while achieving a youthful methylome.

Testable predictions

1. Genetic blockade of autophagy will impair OSK-induced demethylation. In vivo, inducible OSK expression in aged mice (124 weeks) will be combined with tissue‑specific knockout of essential autophagy genes (Atg5 or Atg7) using Cre‑loxP systems in liver, retina, and kidney. If autophagy is required for TET activity, Atg5/Atg7 loss will significantly blunt the reduction in Horvath multi‑tissue and LUC epigenetic age scores compared with OSK‑only controls, despite comparable OSK expression levels.
2. Metabolite supplementation will rescue the autophagy defect. Providing exogenous α‑KG or acetate (a precursor for acetyl‑CoA) in the drinking water of autophagy‑deficient, OSK‑expressing mice should restore TET activity and partially rescue epigenetic age reversal. This would demonstrate that the key function of autophagy in reprogramming is metabolic support rather than bulk cargo degradation.
3. Pharmacological inhibition of autophagy phenocopies genetic loss. Treatment with chloroquine or SAR405 (PI3K‑III inhibitor) during cyclic OSK administration will lead to reduced α‑KG and acetyl‑CoA levels (measured by LC‑MS in target tissues) and decreased 5‑hmC accumulation, a direct readout of TET activity. Correspondingly, frailty indices and functional readouts (e.g., optokinetic tracking for retina) will show limited improvement.
4. Autophagy flux correlates with TET occupancy. Chromatin immunoprecipitation sequencing (ChIP‑seq) for TET2 in OSK‑induced cells with intact versus blocked autophagy will reveal reduced TET2 binding at PRC2/H3K27me3‑marked loci when autophagy is inhibited, linking metabolite availability to enzyme recruitment.

Falsifiability If autophagy inhibition fails to attenuate OSK‑driven epigenetic clock reversal, metabolite levels, TET occupancy, or functional rejuvenation outcomes, the hypothesis would be falsified. Conversely, a consistent blockage of rejuvenation across genetic and pharmacological autophagy impairments, rescued by metabolite supplementation, would strongly support the model that autophagy acts as a metabolic rationing system enabling TET‑mediated DNA demethylation during partial reprogramming.