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Aparna Lakkaraju, Kimberly Toops, Jin Xu; Endo-lysosome trafficking protects retinal pigment epithelial cells from complement-mediated attack. Invest. Ophthalmol. Vis. Sci. 2013;54(15):363.
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In the retinal pigment epithelium (RPE), endo-lysosome function is crucial for digesting photoreceptor outer segment disks and for recycling disk components. We have shown that the lipofuscin component A2E causes cholesterol accumulation in RPE late endosomes and lysosomes. Late endosomal cholesterol levels regulate organelle motility and protein-lipid sorting. Endo-lysosome function is critical for protecting the cell from the terminal complement complex (C5b-9) by recycling complement-regulatory proteins, secretion of exosomes and rapidly resealing pores via lysosome exocytosis. Here, we first investigated whether lipofuscin and cholesterol accumulation in the RPE cause widespread dysregulation of intracellular trafficking pathways or whether this is localized to late endosomes and lysosomes; next we examined the roles of RPE late endosome-lysosomes in protecting the cell from complement-mediated attack in the presence or absence of A2E.
Primary pig RPE cells (±A2E) were transfected with GFP-tagged markers for recycling endosomes (rab11 and transferrin receptor (TfR)), late endosomes (CD63 and the cholesterol transporter Niemann-Pick C1, (NPC1)) and lysosomes (LAMP2) and imaged live by spinning disk microscopy. Exosomes were purified by ultracentrifugation and analyzed by immunoblotting. Release of lysosomal hydrolases after lysosome exocytosis was used as a measure of membrane repair after exposure to pore-forming toxins or C5b-9. Polarity of complement-regulatory proteins CD55 and CD59 was assessed by immunofluorescence and in-cell westerns.
In the RPE, endosomes and lysosomes undergo rapid, bidirectional, long-range tubulo-vesicular trafficking. A2E significantly inhibits long-range movement of late endosomes and lysosomes, whereas trafficking of recycling endosomes is unaffected. Inhibition of NPC1 motility further exacerbates cholesterol storage in the RPE. A2E also interferes with the release of exosomes carrying complement-regulatory proteins and inhibits membrane repair after C5b-9 attack.
Our data show that in the RPE, late endosomes and lysosomes are critical hubs for protein-lipid sorting and intracellular communication. Lipofuscin and cholesterol interfere specifically with late endosome/lysosome traffic, which impairs the ability of the RPE to deal with complement attack and contributes to a chronic inflammatory environment in the outer retina.
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