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Kimberly A Toops, Li Xuan Tan, Aparna Lakkaraju; Retinal pigment epithelium organelle biogenesis and trafficking in models of macular degeneration. Invest. Ophthalmol. Vis. Sci. 2016;57(12):No Pagination Specified.
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© ARVO (1962-2015); The Authors (2016-present)
The endo-lysosomal pathway is responsible for directing the recycling or degradation of proteins and lipids. Sequential maturation of endosomes is critical for maintaining cellular homeostasis. In the retinal pigment epithelium (RPE) the terminal organelle of this pathway, the lysosome, is compromised in both inherited and age-related macular degenerations by the accumulation of complex vitamin A dimers and cholesterol. Here we investigated how endosome maturation occurs in the RPE and whether this is affected in models of macular degeneration.
Adult primary RPE were transfected or transduced with fluorescent organelle markers and imaged live using high-speed spinning disk microscopy. Organelle numbers, morphology and trafficking were quantified using 4D image analysis. Immunoblotting and immunohistochemistry of RPE monolayers and wild type and Stargardt disease (Abca4-/-) mouse RPE were used to confirm live imaging data. Primary RPE and mice were treated with the LXRa agonist TO901317 to increase cholesterol efflux.
Compared to wild types, RPE from Abca4-/- mice had swollen early endosomes and significantly fewer late endosomes, indicating a defect in endosome maturation. Identical data were obtained from polarized primary RPE exposed to the vitamin A dimer A2E. Live-cell imaging revealed fewer late endosomes that associated poorly with microtubules. A switch from Rab5 to Rab7 on the early endosomal membrane is essential for endosome maturation. In RPE with vitamin A dimers, Rab7 is sequestered in lysosomes, decreasing the pool available for driving late endosome maturation. This is because excess lysosomal cholesterol in these cells recruits the cholesterol sensor ORP1L, which in turn binds Rab7 and its effector RILP, thus preventing the biogenesis of late endosomes. Removal of cholesterol restores RPE organelle numbers, volume and traffic in vitro and in Abca4-/- mice.
Our data indicate that vitamin A dimers in the RPE lead to widespread organelle defects that originate in the lysosome and proceed in a retrograde manner (lysosome sequestration of Rab 7 leads to defective late endosome maturation, which results in swollen early endosomes). These studies also show that the cholesterol storage caused by vitamin A dimers is more insidious than the vitamin A dimers themselves, because cholesterol removal is sufficient to correct all upstream endosomal defects.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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