May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Downstream Consequences of A2E-Induced Cholesterol Accumulation in Retinal Pigment Epithelial Cells
Author Affiliations & Notes
  • A. Lakkaraju
    Dyson Vision Research Institute, Department of Ophthalmology, Weill Medical College of Cornell University, New York, New York
  • E. Rodriguez-Boulan
    Dyson Vision Research Institute, Department of Ophthalmology, Weill Medical College of Cornell University, New York, New York
  • Footnotes
    Commercial Relationships  A. Lakkaraju, None; E. Rodriguez-Boulan, None.
  • Footnotes
    Support  NIH EY08538, NIH F32 EY015363, Research to Prevent Blindness Foundation, Dyson Foundation
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 2436. doi:https://doi.org/
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    • Get Citation

      A. Lakkaraju, E. Rodriguez-Boulan; Downstream Consequences of A2E-Induced Cholesterol Accumulation in Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2436. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : We recently reported that the lipofuscin fluorophore A2E causes cholesterol accumulation in late endosomes of retinal pigment epithelial (RPE) cells by displacing membrane cholesterol (Lakkaraju, et al, PNAS 104:11026, 2007). Late endosomal cholesterol levels are critical for regulating organelle motility and intracellular trafficking. The Niemann-Pick C1 and C2 (NPC1 & NPC2) cholesterol transporters are responsible for effluxing cholesterol out of late endosomes. Here, we further characterize the biophysical basis of A2E-induced cholesterol accumulation and investigate how cholesterol overload in RPE cells affects late endosomal protein and lipid trafficking.

Methods: : We used accessibility of membrane cholesterol to cholesterol oxidase to measure cholesterol displacement from lipid bilayers (± A2E). We tested a range of A2E concentrations spanning that found in aging human eyes. Late endosomal internal membranes are rich in lyso-bisphospatidic acid (LBPA), which is a cone-shaped lipid like A2E and cholesterol. We used bilayers with A2E and LBPA to test if LBPA expedites cholesterol dissociation. We used immunofluorescence and confocal microscopy to study the subcellular distribution of cholesterol transporters in polarized ARPE-19 cells in the presence or absence of A2E. Intracellular cholesterol was visualized with filipin.

Results: : A2E caused a dose-dependent increase in cholesterol dissociation from the bilayer (r2 = 0.94). A2E is present in late endosomal internal membranes of ARPE-19 cells since it colocalized with LBPA and CD63. Cholesterol dissociation significantly increased in bilayers with A2E and LBPA compared to A2E alone. A2E caused the redistribution of NPC1 from a punctate cytoplasmic pattern to a perinuclear lysosomal pattern that colocalized with A2E and filipin.

Conclusions: : A2E can cause late endosomal cholesterol accumulation even at low cellular levels because A2E displaces membrane cholesterol over a wide concentration range. Late endosomes are major sorting stations from where proteins and lipids are either sent to lysosomes for degradation or recycled to other intracellular destinations. Since membranes with LBPA transport cholesterol out of late endosomes, accelerated displacement of cholesterol in the presence of LBPA and A2E likely prevents cholesterol efflux from late endosomes. This leads to the entrapment of NPC1, resulting in a feed-forward cycle where decreased cholesterol transport out of late endosomes further exacerbates the A2E-induced cholesterol accumulation.

Keywords: retinal pigment epithelium • lipids • retinal degenerations: cell biology 
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