IF sub-maximal, dose-split dual delivery of TFEB and TFE3 — specifically, AAV8-ApoE-hAAT-TFEB (targeting ~40–50% of maximal TFEB transcriptional output based on titrated viral dose) co-administered with AAV8-ApoE-hAAT-TFE3 (similarly titrated to ~40–50% maximal TFE3 output), delivered by tail-vein injection at a combined titer of 2×10¹¹ vg per vector in 24-month-old male C57BL/6J mice,

THEN hepatic lipofuscin burden (measured by autofluorescence intensity per unit area by confocal imaging of liver sections at 488 nm excitation and by HPLC-fluorescence quantification of bis-retinoid lipofuscin components) will be reduced by ≥30% relative to untreated aged controls and by ≥15% relative to single-agent TFEB-alone controls at equivalent total transcriptional drive, AND hepatocellular transformation markers (Ki67 index, β-catenin nuclear translocation frequency, serum AFP, and liver-to-body-weight ratio) will be statistically non-inferior to young reference animals and significantly lower than mice receiving single-agent TFEB at full dose,

BECAUSE the following mechanistic chain operates:

1. TFEB is a master activator of the Coordinated Lysosomal Expression and Regulation (CLEAR) gene network, and its genetic or pharmacological activation upregulates lysosomal biogenesis and autophagic flux, enabling degradation of aggregated and oxidized cargo (TFEB activates CLEAR network and clears aggregated protein cargo)[https://doi.org/10.1371/journal.pone.0120819]. The same CLEAR network machinery is required for lipofuscin partial solubilization and lysosomal routing, establishing that TFEB-driven lysosomal expansion is a prerequisite for any intracellular lipofuscin clearance strategy.

2. TFE3, the closest structural paralog of TFEB within the MiT/TFE family, shares overlapping CLEAR network binding at E-box and CLEAR-element promoters, but possesses a partially distinct regulon — notably including PPARGC1A, genes governing lipid droplet autophagy (lipophagy), and a set of oxidative stress response genes not maximally induced by TFEB alone. [SPECULATIVE — based on TFE3 biology described in the research context; direct liver-specific lipophagy regulon data require validation] Because lipofuscin in aged hepatocytes is a composite of oxidized lipid-protein adducts whose accumulation is partly driven by failed lipophagy, TFE3's selective induction of lipophagy machinery provides a mechanistically complementary and non-redundant route for lipofuscin precursor clearance.

3. The oncogenic risk of maximal TFEB overexpression in hepatocytes derives from mTORC1 feedback suppression (creating a permissive growth signaling environment), cooperation with MYC, and activation of Wnt/β-catenin — all of which are dose-dependent transcriptional consequences of high-level TFEB nuclear residence. [SPECULATIVE based on research context oncogenic mechanism description; specific dose-response HCC data for TFEB are not present in the current evidence set] By restricting each M...

SENS category: LysoSENS

Key references: • doi.org/10.1371/journal.pone.0120819]. • doi.org/10.1371/journal.pone.0120819], • doi.org/10.1371/journal.pone.0120819]