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F. Mazzoni, J. Camakaris, E. Rodriguez-Boulan; Copper Homeostasis in the Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2010;51(13):492.
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© ARVO (1962-2015); The Authors (2016-present)
Recent work suggests that Cu2+ deficiency may play a role in Age Related Macular Degeneration (AMD). We examined the polarized expression in Retinal Pigment Epithelium (RPE) of three Cu2+ transporters, Menkes, Wilson and CRT1, which are poorly characterized in RPE.
Polarized filter-grown ARPE-19 cells were exposed to a Cu2+ chelator, BCS (300 µM) or to excess Cu2+ (100 µM CuCl2) for 2 h. Localization of endogenous and overexpressed transporters was analyzed by quantitative microscopy. Experiments in young and aged mice were conducted in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Enucleated eyes were fixed in 4% PFA and analyzed identically to cultured cells.
RPE expresses both Menkes and Wilson proteins and in low Cu2+ these transporters are stored in the Golgi compartment and move to the plasma membrane (PM) in high Cu2+. We found that in high Cu2+ both transporters were expressed at the basolateral PM of ARPE19 cells (i.e., facing the choroid circulation). In contrast, CTR1, the channel mediating the entrance of Cu2+ displayed a non-polar punctate pattern in the cytoplasm, in agreement with the concept that this transporter cycles between surface and intracellular pools. In aged mouse eyes the localization of the 3 endogenous transporters was comparable to that observed in cultures.
The basolateral localization of Menkes and Wilson in RPE differs from that reported in other tissues, e.g. placental trophoblasts, in which they are polarized to opposite PM domains. Our observations suggest that Menkes and Wilson play overlapping roles in RPE. The presence of CTR1 suggests an important role of this transporter in Cu2+ entry. We speculate that age may decrease the ability of these 3 transporters to maintain Cu2+ homeostasis, resulting in the Cu2+ deficiency recently reported in AMD donor eyes. We are currently characterizing the sorting of these three transporters in RPE and the participation of clathrin and clathrin adaptors in their polarized localization and function.
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