title: Multiple A2E treatments lead to melanization of rod outer segment-challenged ARPE-19 cells.
abstract: OBJECTIVE
  Daily phagocytosis of outer segments (OSs) and retinoid recycling by the RPE lead to the accumulation of storage bodies in the RPE containing autofluorescent lipofuscin, which consists of lipids and bisretinoids such as A2E and its oxidation products. Accumulation of A2E and its oxidation products is implicated in the pathogenesis of several retinal degenerative diseases. However, A2E accumulates in the RPE during normal aging. In this study, we used a cell model to determine the homeostatic mechanisms of RPE cells in response to A2E accumulation.
  
  
  METHODS
  To distinguish between pathologic and normal responses of the RPE to A2E accumulation, we treated established ARPE-19 cells (cultured for 3 weeks after reaching confluence) with low micromolar amounts of A2E for several weeks. We compared the lysosomal function, lysosomal pH, degree of OS digestion, and melanization of the treated cells to untreated control cells in response to a challenge of purified rod OSs (ROSs). A2E was analyzed with high-performance liquid chromatography (HPLC); and A2E and melanin were identified with mass spectrometry.
  
  
  RESULTS
  We found that post-confluent ARPE-19 cells took up and accumulated A2E under dim light conditions. Spectral analysis of the HPLC separations and mass spectrometry showed that A2E-fed cells contained A2E and oxidized A2E (furan-A2E). A2E accumulation led to a modest increase (up to 0.25 unit) in lysosomal pH in these cells. The specific activity of cathepsin D and lysosomal acid phosphatase was reduced in the A2E-treated cells, but ROS degradation was not impaired. We found that, upon challenge with ROSs, melanin pigment was induced in the lysosomal fraction of the A2E-treated ARPE-19 cells. Thus, the ARPE-19 cells responded to the A2E treatment and ROS challenge by producing a melanin-containing lysosome fraction. We speculate that this prevents them from becoming impaired in OS processing.
  
  
  CONCLUSIONS
  We used a modified ARPE-19 cell model in which melanization was elicited as a response to chronic accumulation of A2E. We found that although A2E treatment led, as has been previously reported, to modest lysosomal alkalinization and lysosomal impairment of ARPE-19 cells, a potential homeostatic mechanism may involve production of a special type of lysosomes containing melanin.
authors.affiliation: Laboratory of Retinal Cell and Molecular Biology, NEI, NIH, Bethesda, MD.
authors.forename: Eugenia
authors.initials: E
authors.lastname: Poliakov
authors.affiliation: Ophthalmic Genetics and Visual Function Branch, NEI, NIH, Bethesda, MD.
authors.forename: Natalya V
authors.initials: NV
authors.lastname: Strunnikova
authors.affiliation: NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, NIH, Rockville, MD.
authors.forename: Jian-kang
authors.initials: JK
authors.lastname: Jiang
authors.affiliation: Laboratory of Retinal Cell and Molecular Biology, NEI, NIH, Bethesda, MD.
authors.forename: Bianca
authors.initials: B
authors.lastname: Martinez
authors.affiliation: Laboratory of Retinal Cell and Molecular Biology, NEI, NIH, Bethesda, MD.
authors.forename: Toral
authors.initials: T
authors.lastname: Parikh
authors.affiliation: Department of Ophthalmology and Visual Sciences, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI.
authors.forename: Aparna
authors.initials: A
authors.lastname: Lakkaraju
authors.affiliation: NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, NIH, Rockville, MD.
authors.forename: Craig
authors.initials: C
authors.lastname: Thomas
authors.affiliation: Ophthalmic Genetics and Visual Function Branch, NEI, NIH, Bethesda, MD.
authors.forename: Brian P
authors.initials: BP
authors.lastname: Brooks
authors.affiliation: Laboratory of Retinal Cell and Molecular Biology, NEI, NIH, Bethesda, MD.
authors.forename: T Michael
authors.initials: TM
authors.lastname: Redmond
mesh_terms: D000468:Alkalies / Q000378:metabolism; D000588:Amines / Q000378:metabolism; D000818:Animals; D055162:Biocatalysis / Q000187:drug effects; D002417:Cattle; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005453:Fluorescence; D006801:Humans; D006873:Hydroquinones / Q000633:toxicity; D008247:Lysosomes / Q000187:drug effects / Q000201:enzymology; D008543:Melanins / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects; D010857:Pigment Epithelium of Eye / Q000166:cytology*; D012176:Retinoids / Q000494:pharmacology*; D012374:Rod Cell Outer Segment / Q000187:drug effects / Q000378:metabolism*
chemical_list: C112040:A2-E (N-retinylidene-N-retinylethanolamine); D000468:Alkalies; D000588:Amines; D006873:Hydroquinones; D008543:Melanins; C493330:Red DND-99; D012176:Retinoids; C031927:hydroquinone