Hypothesis

Engineering AAV6 to deliver TFEB under a stress‑inducible promoter (GRP78 or ATF4) will improve healthspan only when skeletal muscle experiences periodic physiological stress (e.g., intermittent fasting or exercise), because TFEB‑driven lysosomal biogenesis will be activated synchronously with autophagy flux, thereby converting a transient siege into a coordinated survival response. Constitutive TFEB expression will fail to extend lifespan and may exacerbate lysosomal overload.

Rationale

The seed idea frames autophagy as a rationing system invoked during chronic nutrient siege, not a constitutive housekeeping program. Supporting this, Beclin‑1‑dependent autophagy is stress‑responsive and dispensable for basal turnover【1】(https://pmc.ncbi.nlm.nih.gov/articles/PMC3242613/), while TFEB overexpression rescues age‑related autophagy‑lysosome deficits【2】(https://www.aging-us.com/article/101144/text). AAV serotype tropism shows AAV6 efficiently transduces skeletal muscle【3】(https://pmc.ncbi.nlm.nih.gov/articles/PMC10000783/). Pathological stress states such as myocardial infarction increase AAV transduction in affected tissue【4】(https://doi.org/10.1101/2024.06.10.597311), suggesting that delivery during stress enhances nuclear entry and expression. High AAV doses exhaust DNA repair, imposing a metabolic burden that can negate benefits【5】(https://doi.org/10.1101/2025.04.22.649920). Longevity effects of autophagy modulation are highly condition‑dependent【6】(https://elifesciences.org/reviewed-preprints/110620v1). Together, these data argue that TFEB should be expressed only when the cell is already activating autophagy, thereby avoiding unnecessary lysosomal biogenesis in rested fibers.

Experimental Design

1. Vector construction – Produce two AAV6 plasmids: (a) CAG‑TFEB (constitutive) and (b) GRP78‑TFEB (stress‑inducible). Include a fluorescent reporter for quantification.
2. Animal cohorts – Male and female C57BL/6J mice (n=20 per group) receive intravenous AAV6 at a low, sub‑saturating dose (1×10^11 vg) to avoid toxicity. Groups: (i) saline control, (ii) CAG‑TFEB, (iii) GRP78‑TFEB.
3. Stress regimen – Starting at 4 months, half of each vector cohort undergoes a 24‑hour fast twice weekly; the other half remains ad libitum. A parallel sub‑cohort performs voluntary wheel running to model mechanical stress.
4. Readouts – At 6, 12, and 18 months assess: (a) autophagy flux (LC3‑II/I, p62) by western blot in gastrocnemius; (b) lysosomal capacity (LAMP1, cathepsin activity); (c) TFEB nuclear localization (immunofluorescence); (d) circulating klotho and other myokines; (e) frailty index, grip strength, treadmill endurance; (f) lifespan.
5. Controls – Verify promoter specificity by qPCR of TFEB in liver, heart, and brain to confirm muscle‑restricted induction.

Predictions

• GRP78‑TFEB + intermittent stress will show heightened autophagy flux and lysosomal biogenesis specifically during fasting/exercise windows, leading to improved muscle function, elevated circulating klotho, and a significant extension of median lifespan (~15%) compared with controls.
• CAG‑TFEB will exhibit constitutive TFEB nuclear localization and lysosomal markers even in rested muscle, resulting in no functional benefit or a modest decline due to lysosomal overload and impaired proteostasis.
• Stress‑only groups (no vector) will show transient autophagy activation but lacking sustained lysosomal expansion, yielding only short‑term improvements.

Potential Pitfalls & Mitigations

• Promoter leakiness – Use a minimal GRP78 enhancer with multiple upstream stress‑response elements to reduce basal activity; validate with luciferase assays in C2C12 myotubes.
• AAV6 off‑target transduction – Quantify vector genomes in non‑muscle tissues by ddPCR; if liver or CNS expression exceeds 5% of muscle dose, switch to AAV6‑mut variants with enhanced muscle specificity.
• Compensatory mTOR activation – Monitor p‑S6K levels; if chronic mTOR repression occurs, incorporate a low‑dose rapamycin arm to test synergy.

This hypothesis directly tests whether matching TFEB expression to the cell’s innate stress‑induced autophagy "siege" converts a temporary survival tactic into a durable healthspan intervention, offering a falsifiable route beyond constitutive overexpression strategies.