Epigenetic Mechanism Of Increased Expression Of Amyloid-beta In RPE Cells In AMPO Model

Objective Age-related macular degeneration(AMD) is the important reason for the elderly center vision loss. Most of them are with dry subtype and there is still no effective treatment. Drusen is the "Clinical hallmark" of this disease. Amyloid beta(Aβ) is an important composition of drusen and the mechanism of metabolic abnormalities in AMD remains unknown. Our previous meta-analysis showed that AMD patients had elevated blood levels of homocysteine, implicating abnormal DNA methylation circulation in the development of AMD. In this study, we explored the epigenetic mechanisms of increased expression of β-amyloid of retinal pigment epithelium(RPE) cells in the A2 E mediated oxidative stress(A2E-meadiated photooxidation, AMPO) model.Materials and methods ARPE-19 cell viability were detected by MTS with different concentrations of A2 E and different time points in AMPO model. Flow cytometry was used to detect the ROS levels in RPE cells between AMPO model and controls(DHE). Elisa kits were used to detect supernatant content of Aβ1-40 and Aβ1-42 in ARPE-19 cells between AMPO model and normal controls. Real-time PCR and Western blot technique were adopted to detect Aβ-associated protein amyloid precursor protein(APP), β-site APP-cleaving enzyme 1(BACE1), presenilin 1(PS1) m RNA expression and protein content between control and AMPO groups. Real-time PCR and Western blot were employed to detect the m RNA expression and protein content of methyltransferase DNMT1, DNMT3 a, methylation binding protein(MECP2) and histone deacetylase(HDAC1). Maldi-Tof MS technology was used for methylation sequencing of BACE1 gene promoter region 397 bp length(-388-2) loci.Results In AMPO model, MTS results implied a reduction of cell vitality with the extension of time and increased concentration of A2E(6.25 μM, 12.5 μM, 25 μM, 50 μM)(P<0.05). DHE probe fluorescence intensity within the threshold value increased with the increased concentration of A2 E. Significantly higher intracellular ROS levels were observed compared with the control group(P<0.05). Elisa results showed that supernatant of Aβ1-40 and Aβ1-42 were obviously higher than those of the controls(P< 0.05). Antioxidant NAC(20 m M) can inhibit the increased secretion of Abeta 1-40 and Abeta 1-42(P<0.05). BACE1 m RNA and protein expression level were obviously higher than those in the control group(P<0.05) and the APP and PS1 m RNA expression levels remained unchanged(P>0.05). Methyltransferase DNMT1, DNMT3 a content decreased in AMPO model(P<0.05). Maldi-Tof results showed that methylation of BACE1 gene promoter specific site 13, 14-15 and 16-18 decreased(P<0.05) among the 397 bp in length(-388-2), while the rest remained unchanged.Conclusion In AMPO model, photooxidation induced reduced methyltransferase expression. Specific loci of BACE1 gene promoter hypomethylation led to enhanced transcription. Elevated β-secretase production increased the formation of Amyloid β. The results implied the epigenetic regulation of amyloid β in the AMPO model.