Sub-lethal digoxin exploits Na⁺/K⁺-ATPase inhibition to activate the TFEB→GPNMB lysosomal axis in apoptosis-resistant...

IF sub-lethal digoxin (0.25 mg/kg IP every 48 hours for 14 days) is administered to aged male C57BL/6J mice (22–24 months) prior to a GPNMB peptide-adjuvant vaccine,

THEN flow cytometric mean fluorescence intensity (MFI) of surface GPNMB on circulating and tissue-resident p16⁺/SA-β-gal⁺ senescent cells will increase ≥1.8-fold above vehicle-treated aged controls by day 14, and subsequent vaccination will reduce p16⁺ senescent cell burden by ≥40% more than vaccination alone, measured by p16-3MR reporter luminescence and tissue immunofluorescence at day 42,

BECAUSE the following causal chain operates:

1. Digoxin inhibits the Na⁺/K⁺-ATPase α1 subunit on senescent cells, elevating intracellular Na⁺ and triggering reversal-mode Na⁺/Ca²⁺ exchange, producing a sustained intracellular calcium transient (cardiac glycosides canonically inhibit the Na⁺/K⁺-ATPase)[https://doi.org/10.1038/s42255-019-0122-z].
2. Elevated intracellular Ca²⁺ activates the phosphatase calcineurin, which dephosphorylates TFEB at Ser211, promoting its nuclear translocation and activation of the CLEAR (Coordinated Lysosomal Expression and Regulation) gene network [SPECULATIVE: direct calcineurin→TFEB→GPNMB axis not yet demonstrated for cardiac glycosides; inferred from established calcineurin-TFEB literature].
3. Ouabain's senolytic mechanism in BRAF-V600E senescent cells operates through Na,K-ATPase–dependent signal transduction engaging the autophagy-lysosomal axis, and cardiac glycosides restore autophagy flux in cellular stress models, confirming lysosomal pathway engagement (ouabain engages Na,K-ATPase–dependent signal transduction rather than ion transport in senescent cells)[https://doi.org/10.1111/acel.13447] (cardiac glycosides restore autophagy flux)[https://doi.org/10.1101/2023.09.13.856416].
4. TFEB nuclear activity drives transcription of GPNMB, a lysosomal membrane glycoprotein regulated by the MiT/TFE family; senescent cells, which harbor massively expanded lysosomal compartments, are uniquely poised to accumulate and traffic newly synthesized GPNMB to the plasma membrane [SPECULATIVE: the senescence-specific lysosomal amplification of TFEB→GPNMB at sub-lethal CG doses is inferred, not directly demonstrated].
5. At sub-lethal doses, digoxin fails to overcome apoptosis resistance inherent in many senescent cell populations—apoptosis resistance of senescent cells is a demonstrated barrier to cardiac glycoside senolysis across multiple origins (apoptosis resistance is an intrinsic barrier for senolysis by cardiac glycosides)[https://doi.org/10.1007/s00018-021-03980-x]—meaning cells survive long enough to surface-display amplified GPNMB.
6. Elevated surface GPNMB density on surviving senescent cells provides a higher-valency antigenic target, allowing GPNMB peptide vaccine-elicited cytotoxic T lymphocytes and antibodies to more efficiently recognize, engage, and eliminate the primed senescent population; GPNMB already emerges as a top transcriptomic driver of mortalit...

SENS category: GlycoSENS

Key references: • doi.org/10.1038/s42255-019-0122-z], • doi.org/10.1038/s42255-019-0122-z]. • doi.org/10.1111/acel.13447] • doi.org/10.1101/2023.09.13.856416]. • doi.org/10.1007/s00018-021-03980-x]—meaning