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Laura S. Frost, Janet R. Sparrow, Frank P. Stefano, Kathleen Boesze-Battaglia; Putative Role for Melanoregulin (Mreg) in Bisretinoid Lipofuscin Degradation in the Retinal Pigment Epithelium (RPE). Invest. Ophthalmol. Vis. Sci. 2012;53(14):6565.
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Lipofuscin build-up in aging RPE is linked to incomplete digestion of bisretinoid precursors which accumulate in lysosomal compartments. In addition to age related accumulation, A2E, a bisretinoid fluorophore found in lipofuscin is elevated in retinal degenerative diseases. This bisretinoid contributes to diminished photoreceptor outer segment (POS) degradation, lysosomal alkalinization and acts as a photosensitizer generating reactive oxygen species. The relationship between A2E accumulation and lysosome requiring process, specifically autophagy, is not well defined. Our laboratory has identified a 28 kDa peripheral membrane protein Melanoregulin (Mreg) which is required for autophagosome maturation. These studies focus will determine if A2E accumulation alters autophagic processes.
Using ARPE19 cells challenged with UV-oxidized POS, as a model of aged RPE, the effect of Mreg on autophagosome maturation was followed in Mreg knockdown (shRNA) ARPE19 cells. Using a well developed cell culture model that allows RPE cells to accumulate A2E we followed Mreg expression and autophagosome formation by qRT-PCR and western blotting. A2E accumulation was monitored by visualizing autofluorecence detectable by epifluorescence microscopy. Autophagosome-lysosome fusion was analyzed by live cell colocalization of RFP-LC3 and a self quenched dye conjugate of BSA (DQ Green BSA).
OxPOS challenge of RPE cells results in enhanced Mreg expression and increased LC3BII levels suggestive of an autophagic response. Mreg knockdown RPE cells challenged with oxPOS show a further increase in LC3BII suggesting an increase in autophagosome number. Consistent with this we see reduced autophagosome-lysosome fusion in Mreg knockdown cells. In A2E accumulating ARPE19 cells Mreg expression is increased approximately 2-3 fold compared to cells containing no A2E. We also see an increase in LC3BII and LAMP1 levels indicating an increase in the number of autophagosomes and lysosomes respectively. Higher levels of pro-CatD are seen in A2E cells though overall cathepsin D levels appear to be elevated possibly to compensate for the degradative defect associated with A2E.
Lipofuscin accumulation may induce an autophagic response in RPE cells. We see an increase in LC3BII in RPE cells containing A2E or challenged with oxPOS. Mreg expression is elevated in these cells indicating a role for Mreg in this process. Mreg expression and autophagosome/lysosome number may be elevated in order to compensate for defective phagolysosome function in RPE cells containing lipofuscin suggesting that Mreg may be a critical link between lysosome dysfunction and RPE pathology.
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