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Alyssa Cwanger, Ying Song, Joshua L Dunaief; Iron Overload in the RPE Causes Particulate Autofluorescence Associated with Exosome Marker, CD63. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4205.
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Age-related Macular Degeneration (AMD) is associated with the accumulation of autofluorescent deposits including lipofuscin in the retinal pigment epithelium (RPE). We investigate the pattern of particulate autofluorescence accumulation in the iron-overloaded RPE of Cp/Heph double knockout (DKO) mice as well as autofluorescence attributed to lipofuscin in human post-mortem retinas and determine if exosome marker CD63 is associated with this autofluorescence. We hypothesize that RPE lipofuscin may be captured within and transported by exosomes.
Using confocal microscopy, we examined RPE lipofuscin distribution in cryosections and flat mounts of 86-year-old human AMD and normal post-mortem retinas with lipofuscin and six to nine-month-old mice with iron-induced particulate autofluorescence. The spatial association of exosome marker CD63 with autofluorescence was assessed by immunofluorescence. Particulate autofluorescence resulted from RPE iron overload caused by knockout of the ceruloplasmin (Cp) gene and a hypomorphic mutation in the hephaestin (Heph) gene in mice known as DKO as we generated and published previously (Hahn et al, 2004).
Particulate autofluorescence in mouse RPE and lipofuscin in human RPE accumulated in a mosaic manner with higher levels in some cells than in neighboring cells. Bright autofluorescence extended beyond tight junction boundaries connecting the fluorescent cell with its neighbors. Specifically, a series of confocal images including extended focus and orthogonal views of a six-month-old DKO RPE flat mount illustrates that particulate autofluorescence (green) signal is located inside single RPE cells and extends beyond the tight junctions delineated by anti-ZO-1 immunolabeling (red, Cy3). In addition, exosome marker CD63 co-localized with the autofluorescence in both autofluorescence-laden cells and in the proximal edges of neighboring cells, corresponding to the location of autofluorescence.
This spatial arrangement of particulate autofluorescence in the RPE of DKO mice and lipofuscin in the RPE of human post-mortem retinas and its co-localization with the exosome marker CD63 is consistent with a hypothesis that the autofluorescent material is associated with and transported by exosomes. Further investigation of the spatial arrangement of this autofluorescent material and proposed exosome-mediated RPE lipofuscin transport is warranted.
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