Redox‑ControlledPP2A Gates TFEB Dephosphorylation and Autophagy Reactivation in Aging

Hypothesis: Age‑associated oxidative stress inactivates the serine/threonine phosphatase PP2A through oxidation of a conserved cysteine in its B56δ regulatory subunit, which blocks dephosphorylation of TFEB at mTORC1‑targeted sites. Consequently, TFEB remains phosphorylated, bound to 14‑3‑3 proteins, and trapped in the cytoplasm even when mTORC1 activity fluctuates. This creates a dual‑lock mechanism: mTORC1 hyper‑phosphorylates TFEB, while oxidized PP2A fails to remove the phosphate, making autophagy suppression resistant to transient mTORC1 inhibition. During partial reprogramming, Sox2 not only lowers mTORC1 expression (as shown)[4] but also induces expression of thiol‑reductases (e.g., TXNDC5) that restore the reduced state of the PP2A B56δ cysteine, re‑activating PP2A phosphatase activity toward TFEB. Dephosphorylated TFEB dissociates from 14‑3‑3, translocates to the nucleus, and drives the transcriptional program required for autophagy induction, an early event in reprogramming[6]. Thus, the reversibility of autophagy decline with age depends on restoring PP2A redox state, not merely relieving mTORC1‑mediated phosphorylation.

Predictions:

1. In aged mouse liver and muscle, PP2A B56δ cysteine will show higher oxidation (e.g., sulfinylation) and PP2A activity toward phospho‑TFEB will be reduced compared with young tissue; this will correlate with cytoplasmic TFEB sequestration despite variable mTORC1 activity (see [1][2]).
2. Pharmacological activation of PP2A (e.g., low‑dose FTY720) or expression of a redox‑resistant B56δ mutant (Cys→Ser) in aged cells will increase TFEB nuclear localization and LC3‑II accumulation without rapamycin treatment; conversely, PP2A knockdown will blunt the autophagic response to mTORC1 inhibitors.
3. Sox2‑driven autophagy induction in aged fibroblasts will be lost when PP2A is silenced or when the B56δ cysteine is mutated to mimic oxidation (Cys→Asp), even though Sox2 still suppresses mTORC1 expression[4]; rescuing PP2A activity will restore autophagy.

These predictions are testable with immunoblotting for phospho‑TFEB, subcellular fractionation, autophagy flux assays (bafilomycin A1 chase), and redox‑sensitive probes for PP2A cysteine oxidation. Falsification would occur if PP2A oxidation status does not change with age, or if manipulating PP2A redox state fails to affect TFEB localization or autophagy flux under the conditions described.