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Kimberly Toops, Jin Xu, Aparna Lakkaraju; Isoform-specific interactions between human apolipoprotein E and amyloid-beta in the retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4582.
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To investigate how different apolipoprotein E (ApoE) isoforms (a) influence the expression of amyloid-beta (Aβ) in the retinal pigment epithelium (RPE) and (b) preferentially interact with Aβ under conditions of RPE stress. Human ApoE occurs in three isoforms (E2, E3, E4) that vary in only two amino acids but have different conformations, stabilities and interactions with lipids and proteins such as Aβ. The E4 isoform is strongly associated with increased risk of Alzheimers’ disease (AD), but has a protective effect in age-related macular degeneration (AMD). Drusen contain ApoE and Aβ and the RPE may be a source of both proteins. Increased intracellular cholesterol escalates neuronal Aβ production and ApoE participates in clearing Aβ. Since RPE cells with lipofuscin store cholesterol, we investigated how lipofuscin and cholesterol storage affects Aβ expression and ApoE-Aβ interactions.
Primary porcine RPE cells were transfected with GFP-tagged ApoE2, E3 or E4 expressed from either a CMV or Best1 promoter. Cells were treated with the lipofuscin component A2E or with U18666A to induce cholesterol storage directly. Expression of ApoE and Aβ was measured by immunoblot in total cell lysates or after immunoprecipitation with an ApoE antibody. ApoE and Aβ cellular localization was examined by immunocytochemistry. Cholesterol was measured biochemically and by filipin staining. Live imaging was performed using spinning disk confocal microscopy.
Expression of human ApoE isoforms in pig RPE induces Aβ production. The amount of Aβ varies depending on the ApoE isoform. Pull-down assays show that the Aβ-ApoE interaction is resistant to reducing agents. Our data also suggest that lipofuscin and intracellular cholesterol affect numerous steps of the ApoE-Aβ cascade including ApoE trafficking, the amount of Aβ generated in the cell and the binding of ApoE to Aβ.
ApoE trafficking and ApoE-Aβ interactions are modulated by intracellular cholesterol and lipofuscin content in the RPE. In the retina, it is likely that ApoE isoforms participate in clearing Aβ from the RPE and direct it into the choroidal circulation. Thus, while there may be common pathological processes in AD and AMD, our data point to key functional differences at the cellular level that could help explain how the same ApoE isoform can confer risk in one disease and protection in another.
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