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L. Yu, G. Malek, U. Kelly, B. E. Mace, P. M. Sullivan, C. Bowes Rickman; Cholesterol Modulation of Amyloid Precursor Protein (APP) Processing and Amyloid-ß (Aß) Peptide Generation in RPE Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2190.
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Cholesterol, its transporter apolipoprotein E (apoE), and ß-amyloid peptide (Aß) are found in basal deposits and drusen in age-related macular degeneration (AMD). We have established a murine model of AMD that exhibits pathologic changes found in human AMD including basal and drusenoid deposits. To test a molecular mechanism for development of basal deposits and drusen, we investigated amyloid deposition in our model as well as the effects of cholesterol on APP and Aß generation in cultured human RPE cells.
ApoE4 targeted-replacement mice aged over 60 weeks were fed a high fat cholesterol-rich diet (apoE4-HFC) or normal mouse chow (apoE4-ND) for 8 weeks. Total plasma cholesterol levels were measured. Aß deposition in the eye was examined by immunohistochemistry. In vitro, cultured ARPE19 cells were exposed to 60, 120, 240 or 480 µM cholesterol for 17 hrs ±10 µM lovastatin (a cholesterol-depleting agent). Processing of APP was revealed by Western blots. Levels of secreted Aß40 and Aß42 were measured by Aß40- and Aß42-specific ELISA. Protein levels of ß-secretase-1 (BACE1), a major proteolytic enzyme in the amyloidogenic process, were analyzed by Western blots and activities of BACE1 were determined using a ß-secretase activity kit.
In apoE4-HFC mice plasma cholesterol levels were 2.5-fold higher than in controls. Aß immunoreactivity was detected in basal deposits in the apoE4-HFC mice and was absent in controls. Immunoblots of lysates of ARPE19 cells exposed to cholesterol revealed significant loss of mature (glycosylated) full-length APP with a concomitant significant increase of immature APP and significantly increased secretion of Aß40, but not of Aß42. As expected, there was a decrease in secreted Aß40 in the presence of lovastatin, but there was no change in secretion of Aß42. BACE1 activities and protein levels did not change significantly.
We have demonstrated that Aß peptide is present in the basal deposits of a murine model of AMD with a higher plasma cholesterol level and that Aß generation in human cultured RPE cells was induced by cholesterol. Both findings implicate cholesterol in APP metabolism and amyloid deposition and suggest these interactions contribute to the pathogenesis of AMD. There was no difference in activity and protein expression of BACE1, while mature APP was decreased in RPE cells exposed to cholesterol. This suggests that cholesterol-stimulated Aß generation is regulated by abnormal APP processing.
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