IF combined treatment with trehalose (100 mM, continuous supplementation in culture medium) and SkQ1 (100 nM, plastoquinonyl-decyl-triphenylphosphonium) is administered for 72 hours to human ARPE-19 cells (and validated in primary human RPE) that have been pre-loaded with A2E via the established 20 μM × 4-hour pulse followed by a 7-day lysosomal chase period to establish mature lysosomal damage resembling aged RPE,

THEN intracellular A2E concentration, measured by HPLC-fluorescence (λex=430 nm, λem=600 nm) normalized to total protein, will be reduced by ≥60% relative to vehicle control and will exceed either monotherapy by ≥20 percentage points, with a statistically significant interaction term (p<0.05, two-way ANOVA), accompanied by: (a) increased TFEB nuclear/cytoplasmic ratio (>2-fold vs. vehicle by immunofluorescence), (b) elevated LC3-II/I ratio and LAMP1 protein levels by Western blot, (c) ≥40% reduction in mitochondrial superoxide by MitoSOX flow cytometry, and (d) measurable reduction in lipofuscin-like autofluorescence by confocal microscopy (488 nm excitation, 500–650 nm emission),

BECAUSE the following mechanistic chain operates through two complementary and mutually enabling arms that are neither redundant nor merely additive, but genuinely synergistic at a critical biochemical bottleneck:

1. Lysosomal A2E accumulation impairs the autophagy-lysosomal pathway in RPE cells — A2E, once trafficked to lysosomes following the 7-day chase, inhibits lysosomal acidification, reduces cathepsin activity, and blocks autophagic flux, establishing a state of lysosomal insufficiency that cannot be overcome by autophagy induction alone (autophagy induction without functional lysosomes delivers cargo to a non-degradative compartment). (A2E acutely induces autophagy and its functional impairment causes cytotoxicity)[https://doi.org/10.1016/j.fob.2014.11.003]

2. Trehalose induces TFEB nuclear translocation via mTOR-independent AKT inhibition, upregulating the entire CLEAR (Coordinated Lysosomal Expression and Regulation) gene network, expanding lysosomal number and biosynthetic capacity, and driving de novo autophagic flux. Critically, this mechanism bypasses the growth-factor-suppressing effects of mTOR inhibitors, preserving anabolic homeostasis in post-mitotic RPE cells. (Trehalose promotes TFEB nuclear translocation independently of mTOR via an AKT-dependent mechanism)[https://doi.org/10.1038/ncomms15750] (Trehalose is a potent mTOR-independent activator of functional autophagosomes)[https://doi.org/10.1016/j.reprotox.2019.02.005]

3. However, without mitochondrial ROS control, trehalose-driven autophagy is self-limiting and potentially counterproductive at high A2E loads [SPECULATIVE — mechanistically grounded but not yet directly demonstrated in RPE]. Mitochondrial superoxide, elevated by A2E-mediated disruption of the electron transport chain, drives photo-oxidation and chemical oxidation of intracellular A2E into far more cytotox...

SENS category: GlycoSENS

Key references: • doi.org/10.1038/ncomms15750] • doi.org/10.1016/j.fob.2014.11.003] • doi.org/10.1016/j.reprotox.2019.02.005] • doi.org/10.1101/2025.01.14.632938]. • doi.org/10.1091/mbc.e14-05-1028].