Hypothesis

Aging hepatocytes accumulate lysosomal iron that is released into the cytosol during metabolic stress, triggering ferroptosis preferentially in zone 3 where hypoxia and lipogenesis already sensitize cells to lipid‑peroxide damage. Enhancing lysosomal biogenesis via TFEB activation sequesters excess iron, lowers the labile iron pool, and attenuates ferroptotic death, thereby reversing the Aging Hepatocyte Gene Signature (AHGS) and preventing fibrosis.

Mechanistic Rationale

• Lysosomal iron storage: With age, autophagic flux declines and iron‑rich ferritin accumulates in lysosomes (see lysosomal iron overload in aging models) [[https://pmc.ncbi.nlm.nih.gov/articles/PMC12810195/]]. In zone 3 hepatocytes, high glycolytic flux and low oxygen limit ferritin degradation, expanding a labile iron pool that can catalyze Fenton reactions.
• Ferroptosis trigger: The released Fe²⁺ reacts with polyunsaturated fatty acids (PUFAs) generated by SREBP‑1c/ChREBP‑driven de novo lipogenesis, amplifying lipid peroxidation—a core ferroptosis signal [[https://pmc.ncbi.nlm.nih.gov/articles/PMC6105174/]].
• TFEB as a counter‑regulator: TFEB drives lysosomal biogenesis and autophagy, promoting ferritin sequestration and iron export via ferroportin. Pharmacologic TFEB activators (e.g., trehalose) or genetic overexpression reduce cytosolic iron and lipid ROS in aged liver cells.

Testable Predictions

1. Biomarker prediction: In human MASLD biopsies, the co‑localization of lysosomal marker LAMP1 with ferritin heavy chain will correlate with AHGS score and ferroptosis marker 4‑HNE, specifically in zone 3.
2. Intervention prediction: Treating aged MASH mice with a TFEB activator (trehalose or a small‑molecule agonist) will decrease hepatic labile iron (measured by Calcein‑AM quenching), lower AHGS expression, and reduce perisinusoidal fibrosis compared with vehicle, even without direct ferroptosis inhibitors.
3. Falsification prediction: If TFEB activation fails to reduce cytosolic iron or AHGS despite increasing lysosomal mass, the hypothesis that lysosomal iron release drives zone‑3 ferroptosis is weakened.

Experimental Approach

• Human tissue: Multiplex immunofluorescence on MASLD liver sections (mild, moderate, severe) to quantify zone‑3 lysosomal ferritin, AHGS (p16, p21), and ferroptosis (ACSLO4, 4‑HNE). Correlate with clinical fibrosis stage.
• Mouse models: Use aged (>18 mo) western‑diet‑induced MASH mice. Treat with trehalose (2 % in drinking water) or TFEB‑AAV8 for 8 weeks. Measure labile iron (FeRhoNox‑1), AHGS qPCR, fibrosis (hydroxyproline, Sirius Red), and serum ALT.
• Rescue experiments: Co‑treat with ferrostatin‑1 to confirm that any benefit of TFEB activation is ferroptosis‑dependent.

Potential Impact

Confirming that lysosomal iron release couples aging to zone‑3 ferroptosis would reposition lysosomal biogenesis as a upstream, druggable node that complements ferroptosis inhibitors and de‑novo lipogenesis blockers. It would also provide a non‑invasive biomarker (lysosomal ferritin signal) for patient stratification in trials targeting ferroptosis or senescence.

Keywords: lysosomal iron, TFEB, ferroptosis, zone 3 hepatocytes, MASLD, aging hepatocyte gene signature.

References

• Aging drives MASLD progression via ferroptosis and reversible AHGS [[https://pmc.ncbi.nlm.nih.gov/articles/PMC12810195/]]
• Epigenetic clocks confirm accelerated biological aging in MASLD [[https://pmc.ncbi.nlm.nih.gov/articles/PMC12933295/]]
• Zone 3 vulnerability due to hypoxic, lipogenic microenvironment [[https://onlinelibrary.wiley.com/doi/10.1111/liv.15025]]
• Steatosis severity and zone 3 fibrosis risk [[https://pmc.ncbi.nlm.nih.gov/articles/PMC2346454/]]
• De novo lipogenesis, VLDL export, PNPLA3 I148M amplify zone 3 lipotoxicity [[https://pmc.ncbi.nlm.nih.gov/articles/PMC6105174/]]
• Ferroptosis‑targeting drug reverses metabolic liver disease [[https://medicalxpress.com/news/2026-01-drug-candidate-reverses-metabolic-liver.html]]