Amyloid Beta (1-42) Alters Structure and Function of Retinal Pigmented Epithelium

Abstract

Purpose: : Age-related macular degeneration (AMD) is characterized by the formation of drusen, extracellular deposits associated with atrophy of the retinal pigmented epithelium (RPE), disturbance of the transepithelial barrier and photoreceptor death. Amyloid-β (Aβ) is present in drusen but its role during AMD remains unknown. This study was conducted to investigate the involvement of Aβ in RPE alterations and dysfunctions leading to retinal degeneration.

Methods: : ARPE-19 cells were exposed to the oligomeric form of Aβ(1-42) – OAβ(1-42) . Mitochondrial redox potential was assessed by MTT assay, cell viability was determined by LDH release, trypan blue-counting, flow cytometry and TUNEL, and production of reactive oxygen species was quantified by flow cytometry using H2DCFDA. Disorganization of actin cytoskeleton and tight junction proteins was determined by immunohistochemistry, and alterations in transepithelial flux were measured by inward permeability of a 40kDa FITC-dextran probe. C57BL/6J mice were subretinally injected with OAβ(1-42). Morphometric analysis was performed on toluidine blue-stained retinal sections, and the effects of OAβ(1-42) were analysed either on RPE/choroid flat mounts and retinal sections by immunohistochemical staining of tight junction and visual cycle proteins.

Results: : Exposure of RPE cells to OAβ(1-42) reduced mitochondrial redox potential and increased the production of reactive oxygen species, but did not induce apoptosis in RPE cell cultures. It also disorganized the actin cytoskeleton and halved occludin expression, markedly decreasing attachment capacity and abolishing the selectivity of RPE cell transepithelial permeability. Antioxidant pretreatment partially reversed the effects of OAβ(1-42) on mitochondrial redox potential and transepithelial permeability. Subretinal injection of OAβ(1-42) induced pigmentation loss and RPE hypertrophy but not RPE cell apoptosis in C57BL/6J mice. Rapid OAβ(1-42)-induced disorganization of cytoskeletal actin filaments was accompanied by decreased RPE expression of the tight junction proteins Occludin and ZO-1 and of the visual cycle proteins CRALBP and RPE65. The number of photoreceptors decreased by half within a few days.

Conclusions: : Our study pinpoints the role of Aβ in RPE alterations and dysfunctions leading to retinal degeneration and suggests that targeting Aβ may help develop selective methods for treating diseases involving retinal degeneration, such as AMD.

Keywords: aging • oxidation/oxidative or free radical damage • retinal pigment epithelium