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Rina Sato, Tsutomu Yasukawa, Ayae Kubota, Aki Kato, Akiko Nishiwaki, Yuichiro Ogura; Role Of Amyloid Precursor Protein In Lipoprotein Deposition From Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5297.
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Amyloid ß is detectable in drusen in aging eyes and a biomarker in age-related macular degeneration as well as Alzheimer disease. We have previously developed the spheroid culture system of retinal pigment ephthelial (RPE) cells, which might be useful to elucidate unknown basal functions of RPE. The purpose of this study is to clarify the relationship of amyloid ß with lipoprotein deposition from RPE by use of this culture system.
Human RPE cells were cultured in medium with methylcellulose on a 96-well round-bottomed culture plate. RPE cells in each well aggregated to form a spheroid, on the surface of which a monolayer of RPE was constructed. The inhibitors of ß-secretase and γ-secretase, which cleave amyloid precursor protein (APP) to amyloid ß and other fragments, were added in the culture medium. A light microscope was used to measure the diameter of spheroids and observe the appearance periodically. Expression of amyloid ß, apoB-100 (a component of RPE-derived lipoproteins), and elastin (a major element of Bruch’s membrane) was evaluated by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA).
Diameters of spheroids treated with each secretase inhibitor were bigger than those in the control group throughout the observation period. In the groups with secretase inhibition, spheroids had smooth surface and reduced outward deposition of lipoproteins, as compared with the control group. Immunohistochemistry and ELISA showed that expression of apoB-100 and elastin on spheroids as well as amyloid ß was suppressed by each secretase inhibitor.
The deposition of RPE-lipoproteins and the turnover of Bruch’s membrane as well as the production of amyloid ß were reduced by secretase inhibitors. These results suggested that the cleavage of APP by secretases might play a crucial role in lipoprotein deposition and subsequent subclinical turnover of Bruch’s membrane.
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