Hypothesis

Aging spiral ganglion neurons (SGNs) exhibit a blockade at the lysosomal degradation step of autophagy, marked by LC3‑II/SQSTM1 accumulation and TFEB cytoplasmic sequestration. While mTORC1 hyperactivation is implicated, we propose that primary lysosomal membrane damage—driven by iron‑mediated lipid peroxidation—prevents TFEB translocation even when mTORC1 activity is normalized. Repairing lysosomal membranes with exogenous phosphatidylethanolamine (PE)‑rich liposomes will restore TFEB nuclear entry, re‑establish autophagic flux, and reduce lipofuscin without directly inhibiting mTORC1.

Mechanistic Rationale

1. Lysosomal permeabilization as a sensor – Oxidative stress in the cochlea generates labile iron that catalyzes peroxidation of lysosomal phospholipids, compromising membrane integrity. Damaged lysosomes fail to recruit the Ragulator‑Rag complex properly, keeping TFEB phosphorylated and cytoplasmic despite low mTORC1 activity.
2. TFEB regulation is dual‑controlled – TFEB nuclear translocation requires both mTORC1‑dependent phosphorylation status and lysosomal‑derived calcium/phospholipid signals (via calcineurin). Membrane damage uncouples the latter, creating a "false fed" signal.
3. Rescue via membrane replenishment – Supplementing PE‑rich liposomes fuses with damaged lysosomal membranes, restoring lipid composition, sealing leaks, and permitting proper Ragulator‑Rag recruitment. This should allow calcineurin‑mediated dephosphorylation of TFEB and its nuclear translocation even if mTORC1 remains active.

Testable Predictions

• Prediction 1: In aged mouse SGNs, lysosomal membranes will show increased 4‑HNE adducts and reduced PE-to-phosphatidylcholine ratio compared with young controls.
• Prediction 2: Acute treatment with PE‑liposomes (non‑toxic, size <100 nm) will increase lysosomal membrane integrity (measured by galectin‑3 puncta reduction) within 6 h.
• Prediction 3: PE‑liposome treatment will increase nuclear TFEB immunoreactivity in SGNs by ≥2‑fold without altering phospho‑S6K levels (mTORC1 activity readout).
• Prediction 4: Concomitant LC3‑II turnover (using bafilomycin A1 chase) will show restored flux (decreased LC3‑II accumulation) and reduced SQSTM1/p62 aggregates.
• Prediction 5: Lipofuscin autofluorescence in the organ of Corti will decline by ≥30 % after 2 weeks of weekly PE‑liposome intracochlear injections.
• Prediction 6: Auditory brainstem response (ABR) thresholds will improve by ≥10 dB SPL at 16–32 kHz relative to vehicle‑treated aged mice.

Experimental Approach

1. Animal model: C57BL/6J mice, 12‑month‑old (presbycusis phenotype).
2. Intervention: PE‑liposomes (1,2‑dioleoyl‑sn‑glycero‑3‑phosphoethanolamine) formulated with PEG for stability; administered via round‑window membrane (0.5 µL, 2 mg/mL) weekly for 4 weeks.
3. Controls: (a) vehicle (PBS), (b) empty PEG‑liposomes, (c) rapamycin (mTORC1 inhibitor) as positive control for TFEB nuclear shift.
4. Readouts:
   • Lysosomal lipidomics (LC‑MS) for PE/PC ratio and 4‑HNE adducts.
   • Immunofluorescence for TFEB (nuclear/cytoplasmic ratio), LAMP1, galectin‑3.
   • Autophagic flux assay: LC3‑II with/without bafilomycin A1 in microdissected SGNs.
   • Lipofuscin quantification (autofluorescence imaging).
   • ABR thresholds before and after treatment.
5. Statistical analysis: ANOVA with post‑hoc Tukey; n ≥ 6 per group for adequate power.

Falsifiability

If PE‑liposome treatment fails to increase nuclear TFEB or autophagic flux despite verified lysosomal membrane repair, or if mTORC1 inhibition (rapamycin) does not further enhance TFEB nuclear localization beyond liposome treatment, the hypothesis would be falsified. Conversely, demonstration that membrane rescue alone restores flux and improves hearing would support the notion that lysosomal integrity, not merely mTORC1 signaling, is the critical checkpoint governing autophagic competence in aging SGNs.

Potential Impact

Confirming this mechanism would shift therapeutic focus from broad mTOR inhibition—which risks impairing protein synthesis in high‑activity cells like the stria vascularis—to targeted lysosomal membrane stabilization. Such precision could alleviate the energetic siege in SGNs without compromising the metabolic demands of adjacent nonsensory cells, offering a viable strategy to mitigate age‑related hearing loss.