The Protective Effect Of Neprilysin On Retinal Pigment Epithelial Cells Apoptosis Induced By Amyloid β_1-42 Peptide

Age-related macular degeration (AMD) is a leading cause of blindness and visual impairment in older adults. The incidence is about6.8%-7.7%among people age50and older in our country. There are "dry" and "wet" forms, dry AMD results from atrophy of the retinal pigment epithelial layer below the retina, which causes vision loss through loss of photoreceptors (rods and cones) in the central part of the eye, and the "wet" form of AMD, causes vision loss due to abnormal blood vessel growth (choroidal neovascularization) in the choriocapillaris, through Bruch’s membrane. Because of imperfect structure of these new blood vessels, bleeding, leaking, and scarring from these new blood vessels, and eventually cause irreversible damage to the photoreceptors and vision loss if left untreated. Drusen and the changement of pigment are the same characters of these two forms of AMD. Drusen, a accumulations of extracellular material that build up between Bruch’s membrane and the retinal pigment epithelium of the eye, is a common cause and also the result of AMD. Beta-amyloid is an important concentration of drusen. Alzheimer’s disease is a chronic neurodegenerative disease, extracellular amyloid beta protein deposition is the main pathological features of Alzheimer’s disease, which is a key factor that initiates the onset of Alzheimer’s disease. The function of amyloid-β in AMD may share some same pathogenesis with Alzheimer’s disease.As a very important factor involved in nerve damage and cell apoptosis, amyloid β is a degradation production of amyloid-precursor protein. Amyloid-precursor protein can be degradated by α-secretase, β-secretase and γ-secretase, generating cytotoxic products Aβ1-40, Aβ1-42and Aβ1-43. The cytotoxic products further lead to cell apoptosis. As a result, the removal of Aβ provides potential treatment to Alzheimer’s disease. A peptidase named neprilysin is a major Aβ nature degradate enzyme. However, the relationship between Aβ and the development of age-related macular degeration has not been widely studied. In this study, it is hypothesized the role of Aβ in the process of age-related macular degeration is the same as in the central nervous system. In addition, we examined the influence of Aβ1-42on the growth of retinal pigment epithelial cells. On the other hand, the UF11vector expressing HA tagged with NEP was constructed, and the Aβ-degradation activity of the vector was assessed, we explored the the protective effect and the mechanism of neprilysin on retinal pigment epithelial cells apoptosis induced by amyloid β1-42peptide.1. The role of NEP in Aβ1-42-induced retinal pigment epithelial cell injuryCell viability was detected by MTT assay at different time points after the retinal pigment epithelial cells stimulated by different concentrations of Aβ1-42. The results showed that Aβ1-42affected the activity of retinal cells by modulating the growth of retinal pigment epithelial cells. Based on above experimental data, we determined the best treatment condition to induce the Aβ1-42injury was1μM Aβ1-42for6hours.2. The protective effect of neprilysin on retinal pigment epithelial cells apoptosis induced by amyloid β1-42peptideThe UF11vector which expresses HA tag and NEP was constructed and transfected into the retinal pigment epithelial cells. The results of SDS-PAGE and Western-blot showed that the target protein was correctly expressed. The purified target protein was incubated with fibrosis of Aβ1-42for a certain time in vitro to simulate the in vivo physiological conditions. The residual Aβ1-42was detected with Western-blot. An NEP inhibitor thirophan control was set up at the same time to confirm the role of NEP. The results showed that NEP indeed enhanced degradation ofAβ1-42.In order to further explore the role of NEP on Aβ1-42induced damage on the retinal pigment epithelial cell, we transfected the NEP expressing UF11vector into the Aβ1-42damaged retinal pigment epithelial model cells. Then we detected the relationship between Aβ1-42and NEP protein levels using Western blot. We also analyzed the effect of NEP gene on the cell growth (MTT method), the mRNA levels of BAX and NEP (RT-PCR). The results showed that the number of apoptosis cells in the group transfected with the NEP expressing UF11vector were significantly lower. The RT-PCR result showed that the BAX mRNA expression in NEP gene adding group was lower than non-adding group, The result from the Western blot experiment showed that the protein content of Aβ1.42decreased when the NEP expression was increased, and also indicated the effect of NEP on Aβ1-42protein. All These results showed that the overexpression of NEP reduced the cytotoxic effect of Aβ1-42.In this study, we preliminary examined the pathogenesis of the age-related macular degeration. We also successfully established an Aβ1-42-induced retinal pigment epithelial cells damage model. In addition, the role of NEP on Aβ1-42degradation was validated. In general, this study provides a potential therapeutic method for the age-related macular degeration.