IF a single intravitreal co-injection of hydroxypropyl-β-cyclodextrin (HPβCD; 10–15 mM, 1.5 µL) combined with the mTOR-independent TFEB activator trehalose (100 mM, 1.5 µL in the same bolus, total volume 1.5 µL mixed formulation) is administered to 18-month-old male Abca4⁻/⁻ mice with pre-existing, peak-level lipofuscin deposits in the RPE,

THEN contralateral vehicle-controlled eyes will show ≥50% reduction in A2E and iso-A2E concentration by HPLC-MS/MS, ≥40% decrease in SW-AF intensity, and ≥35% reduction in TEM-quantified lipofuscin granule density at 8 weeks post-injection, significantly exceeding the effect of either agent alone (predicted: ~30–35% for HPβCD alone, ~20% for trehalose alone),

BECAUSE of the following orthogonal, synergistic mechanistic chain:

1. HPβCD directly solubilizes A2E from lysosomal membranes via hydrophobic inclusion-complex formation; β-cyclodextrins have been shown to extract A2E from ARPE-19 cells and reduce lipofuscin bisretinoid burden in RPE in vivo in 9-month-old Abca4⁻/⁻ mice via intravitreal injection (β-CDs extract A2E and reduce lipofuscin burden in vivo)[https://doi.org/10.1073/pnas.1400530111].

2. HPβCD mobilizes excess cholesterol from lysosomal membranes, attenuating LDLR/SREBP1 expression and restoring lysosomal membrane fluidity; critically, this cholesterol redistribution mechanism operates independently of TFEB-mediated lysosomal biogenesis, establishing the two pathways as non-redundant (HPβCD clears existing LF through cholesterol-associated pathway without TFEB involvement)[https://doi.org/10.1038/s41598-017-02387-8].

3. Cholesterol overload in A2E-laden lysosomes impairs lysosomal enzymatic function and autophagy; the compromised lysosomal environment reduces photoreceptor outer segment (POS) degradation, worsening lipofuscinogenesis in a self-reinforcing cycle (A2E impairs cholesterol and phospholipid metabolism and lysosomal function)[https://doi.org/10.1007/978-1-4614-3209-8_14]. HPβCD breaks this cycle at the lipid level.

4. HPβCD reshapes the retinal proteome toward vesicular trafficking, upregulating ATP10A (a flippase facilitating membrane phospholipid asymmetry) and downregulating LRP1 and DMXL2 (the latter being a critical regulator of V-ATPase assembly and lysosomal acidification), suggesting that cholesterol removal by HPβCD directly restores V-ATPase-dependent lysosomal acidification—a precondition for effective autophagic flux (HPβCD modulates endocytosis, vesicular trafficking, and lysosomal proteins in retina)[https://doi.org/10.1111/bph.15209].

5. Trehalose activates TFEB by an mTOR-independent mechanism distinct from HPβCD's cholesterol-extraction mechanism; pharmacological TFEB activation recapitulates TFEB overexpression by inducing nuclear translocation and upregulating the CLEAR network, including cathepsins and LAMP proteins required for lysosomal exocytosis (HPβCD and trehalose both activate TFEB nuclear translocation and CLEAR network up...

SENS category: LysoSENS

Key references: • doi.org/10.1073/pnas.1400530111]. • doi.org/10.1038/s41598-017-02387-8]. • doi.org/10.1007/978-1-4614-3209-8_14]. • doi.org/10.1111/bph.15209]. • doi.org/10.1371/journal.pone.0120819].