Hypothesis

Aging hepatocytes in zone 3 of the MASLD liver exhibit heightened ferritinophagy-mediated labile iron accumulation, which drives ferroptosis and sustains the Aging Hepatocyte Gene Signature (AHGS); inhibiting NCOA4‑dependent ferritinophagy specifically in zone 3 will reduce AHGS+ cells, attenuate fibrosis, and restore metabolic function even in advanced MASLD.

Mechanistic Rationale

Recent work shows that aging accelerates ferroptosis in MASLD hepatocytes and that AHGS correlates with disease severity et al.. Zone 3 hepatocytes are uniquely vulnerable to lipid overload and iron‑dependent death due to their high de novo lipogenesis activity Sanyal et al.. Ferritinophagy, the autophagic degradation of ferritin via NCOA4, releases stored iron and can exacerbate ferroptotic stress when lysosomal activity is altered with age Gao et al.. We hypothesize that in aged MASLD livers, zone 3 hepatocytes up‑regulate NCOA4, increasing labile iron pools that potentiate PUFA peroxidation and ferroptosis, thereby locking cells into the AHGS state. Blocking this pathway should lower iron‑catalyzed lipid peroxidation, allowing ferroptosis inhibitors like ferrostatin‑1 to work more efficiently and enabling epigenetic reprogramming that reverses senescence signatures.

Experimental Design

1. Model: Use aged (18‑month) C57BL/6 mice fed a methionine‑choline deficient (MCD) diet to induce MASLD with fibrosis; include young (3‑month) controls.
2. Intervention: Deliver an adeno‑associated virus (AAV8) carrying a CRISPRi construct targeting NCOA4 under a zone‑3‑specific promoter (e.g., CYP2E1) versus a control AAV‑scramble.
3. Groups (n=10 per group):
   • Young + MCD + scramble
   • Aged + MCD + scramble
   • Aged + MCD + NCOA4‑CRISPRi
   • Aged + MCD + NCOA4‑CRISPRi + low‑dose ferrostatin‑1
4. Readouts (at 8 weeks):
   • Histology: H&E, Sirius Red for fibrosis, Perl's iron staining.
   • Molecular: qPCR and Western blot for NCOA4, ferritin H/L, ACSL4, 4‑HNE; AHGS markers (p16, SASP factors, lamin B1 loss).
   • Functional: Serum ALT/AST, glucose tolerance test, insulin tolerance test.
   • Epigenetic: DNA methylation age (Horvath clock) from isolated hepatocytes.
   • Cell death: TUNEL and C11‑BODIPY flow cytometry for lipid peroxidation.
5. Analysis: Compare AHGS+ hepatocyte proportion, fibrosis stage, and metabolic parameters across groups using ANOVA with post‑hoc Tukey.

Predicted Outcomes

• NCOA4 knockdown in zone 3 will reduce labile iron (Perl's staining) and lipid peroxidation (C11‑BODIPY) in aged MASLD mice.
• AHGS+ hepatocyte frequency will drop toward young levels, accompanied by decreased p16 and SASP expression.
• Fibrosis area (Sirius Red) will be significantly lower in the NCOA4‑CRISPRi group versus scramble, with additive benefit when combined with ferrostatin‑1.
• Metabolic tests will show improved glucose tolerance and reduced serum transaminases.
• Epigenetic age will be reduced, indicating partial reversal of aging signatures. If these results are observed, the hypothesis is supported; lack of change in iron, AHGS, or fibrosis despite NCOA4 suppression would falsify the proposed mechanism.