Autophagy functions as a metabolic gate that sets the temporal window for safe epigenetic reset during partial reprogramming. When OSKM factors are expressed transiently, autophagy peaks early, supplying acetyl‑CoA and α‑ketoglutarate while removing differentiation‑linked proteins Autophagy pulses during reprogramming. These metabolites fuel TET‑dependent DNA demethylation and histone acetylation, creating a permissive chromatin state that allows epigenetic age reversal without triggering pluripotency Beneficial effects require active DNA demethylation by TET1 and TET2. If autophagy persists beyond this early pulse, the continued catabolism depletes essential cofactors and accumulates toxic metabolites, pushing cells toward aberrant mesenchymal activation or senescence Some tissues show undesirable mesenchymal gene activation. Thus, the longevity benefits of cyclic OSKM depend not on autophagy activation per se, but on restricting its activity to a narrow, early phase that matches the metabolic demand of epigenetic rewiring.

We hypothesize that the amplitude and duration of the autophagy pulse directly determine the efficiency and safety of epigenetic reset. Specifically, a brief, high‑intensity autophagy flux maximizes metabolite supply for TET and histone acetyltransferases while minimizing collateral degradation of nuclear lamina and lineage‑specific transcription factors. Extending autophagy flux—either by genetic overexpression of ATG5/7 or by pharmacological inhibition of lysosomal repletion—will shift the balance toward either insufficient metabolite provision (failed reset) or excessive self‑cannibalism (identity loss, teratoma risk, mesenchymal shift).

To test this, we will use inducible ATG5 and ATG7 alleles in mice subjected to cyclic OSKM (2 days on/5 days off). Four groups will receive: (1) control OSKM only, (2) OSKM + transient ATG5 overexpression limited to the first 24 h of each cycle, (3) OSKM + sustained ATG5 overexpression throughout the on‑phase, and (4) OSKM + lysosomal blockade with chloroquine administered after the autophagy peak. We will measure: (a) epigenetic age via liver and kidney DNA methylation clocks, (b) transcriptomic signatures of pluripotency and mesenchymal genes, (c) circulating metabolites (acetyl‑CoA, α‑ketoglutarate, lactate), and (d) functional readouts such as fibrosis scores and tumor incidence over 12 months Cyclic OSKM induction in physiologically aged mice restores youthful DNA methylation, transcriptomic, and lipidomic profiles Short‑term systemic OSK via AAV in 124‑week‑old mice extends median remaining lifespan by 109%. A successful validation will show that group 2 exhibits the greatest epigenetic age reduction with minimal off‑target markers, whereas groups 3 and 4 display either incomplete reset or increased mesenchymal/tumor phenotypes.

This hypothesis links autophagy’s role as a siege‑rationing system to the precise metabolic timing required for epigenetic reprogramming, offering a falsifiable framework to refine cyclic OSKM protocols for therapeutic use.