RESUMO
Numerous studies have focused on the deteriorate role of amyloid-ß (Aß) on retina, implying the potential pathogenic mechanism underlying age-related macular degeneration (AMD). However, the mechanism underlying the Aß deposition in AMD patients remains unknown. Beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1), rate-limiting enzyme for Aß production, plays an important role in Aß deposition in the brain. In the current study, we aimed to clarify the regulation mechanism of BACE1 and explore potential drug targets using a lipofuscinfluorophore A2E-mediated photo-oxidation model. In this model, Aß1-40 and Aß1-42 levels increased simultaneously with the enhanced BACE1 expression. These changes were associated with the hypomethylation of specific loci within the BACE1 gene promoter and the decreased levels of DNA methyltransferase 1 (DNMT1). Furthermore, we noticed overlapping regions of differentially methylated CpG islands and specificity protein (Sp1) binding sites within the BACE1 promoter. We employed chromatin immunoprecipitation (ChIP) assay to verify that the decreased BACE1 promoter methylation by DNMT1 enabled increased binding between Sp1 and the BACE1 promoter, which further enhanced BACE1 transcription. The inhibition of Sp1 with mithramycin A (MTM) could down-regulate the expression of BACE1 as well as alleviate the RPE barrier morphology and function impairment. Our results for the first time show the competitive regulation of BACE1 by transcription factor Sp1 and DNMT1 after photo-oxidation and confirm the potential novel protective role of MTM on RPE cells.
Assuntos
Secretases da Proteína Precursora do Amiloide/biossíntese , Ácido Aspártico Endopeptidases/biossíntese , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Estimulação Luminosa/efeitos adversos , Epitélio Pigmentado da Retina/metabolismo , Retinoides/toxicidade , Fator de Transcrição Sp1/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/genética , Peptídeos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/genética , Sequência de Bases , Linhagem Celular , Relação Dose-Resposta a Droga , Expressão Gênica , Humanos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Plicamicina/análogos & derivados , Plicamicina/farmacologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/patologia , Pigmentos da Retina/toxicidadeRESUMO
We present the first evidence that purified rhodopsin can induce experimental autoimmune uveoretinitis (EAU) in monkeys. Injection of a highly purified lipid-free rhodopsin preparation provokes severe chorioretinitis with concomitant anterior uveitis. The onset of disease is earlier, its frequency is higher, and the inflammation is considerably more severe than in EAU induced under similar conditions by opsin. The first inflammatory cells are observed in the ciliary body and pars plana. Within a few days the inflammation extends into the anterior chamber, choroid, and retina. Retinitis predominates in the central area, while chorioretinitis is observed in the periphery, both accompanied by damage to and elimination of the photoreceptor cells. The monkeys develop high cellular and humoral immune responses against rhodopsin and opsin. The cellular response maximum just precedes the onset of EAU. This may indicate that cellular immunity has an important role in the pathogenesis of rhodopsin-induced EAU.