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Rong Wen, Shuliang Jiao, Yiwen Li, Nusrat Yeasmin, Philip J Rosenfeld, Sander R Dubovy, Byron L Lam, ziqiang Guan; A2E distribution in lipofuscin granules isolated from human RPE cells. Invest. Ophthalmol. Vis. Sci. 2020;61(7):3097.
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© ARVO (1962-2015); The Authors (2016-present)
Lipofuscin accumulation is a hallmark of retinal pigment epithelium (RPE) aging and implicated in retinal diseases. To examine the distribution of A2E in RPE cells, we fractionated RPE granules from human eyes and characterized the dry weight, morphology, A2E content, and fluorescence emission of each fraction.
RPE cells were collected from 37 donor human eyes. Granules were fractionated by discontinuous sucrose gradient centrifugation (1.0, 1.2, 1.4, and 2M sucrose). The dry weight of each fraction was measured, which served as a reference for the characterization of A2E content and autofluorescence. The absolute quantification of A2E content was measured by liquid chromatography/mass spectrometry (LC/MS) using an A2E homolog as a spike-in standard. The autofluorescence of RPE granules was measured with a custom-built spectro-fluorometer.
Five fractions (F1-F5 in ascending order of density) of granules were collected. A low-density fraction (F1, density <1M sucrose) is found to be the major population of RPE granules. This fraction accounts for half of the dry weight of all RPE granules, 67% of A2E content, and 75% of projected autofluorescence emission. The previously reported RPE lipofuscin (F3 in this study) is surprisingly the least abundant population of RPE granules. A significant amount of A2E was detected in every fraction, even in the heavily pigmented granules (F5) regarded as the melanosomes. Autofluorescence measurement showed a progressive decrease in autofluorescence in granules from F2 to F4, which is seemly inversely correlated with pigmentation. No detectable autofluorescence emission was found in F5, despite its significant A2E content.
Three significant findings from our systemic analysis of human RPE granules could change our current view of A2E and RPE lipofuscin. First, we discovered a major population of lipofuscin granules (F1). Granules in F1, not described before, bear all the marks of RPE lipofuscin and thus F1 should be regarded as a major RPE lipofuscin population. In addition, a significant amount of A2E in every fraction of RPE granules, including melanosomes (F5), raising the question as to how A2E is integrated into non-lipofuscin granules such as melanosomes. Furthermore, our results highlight the signal attenuation of autofluorescence by RPE melanin, which should be taken into consideration for quantitative fundus autofluorescence imaging.
This is a 2020 ARVO Annual Meeting abstract.
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