Abstract
Impaired cargo trafficking and the aggregation of intracellular macromolecules are key features of neurodegeneration, and a hallmark of aged as well as diseased retinal pigment epithelial (RPE) cells in the eye. Here, photoreceptor outer segments (POS), which are internalized daily by RPE cells, were modified by UV-irradiation to create oxidatively modified POS (OxPOS). Oxidative modification was quantified by a protein carbonyl content assay. Human ARPE-19 cells were synchronously pulsed with POS or OxPOS to study whether oxidatively modified cargos can recapitulate features of RPE pathology associated with blinding diseases. Confocal immunofluorescence microscopy analysis showed that OxPOS was trafficked to LAMP1, LAMP2 lysosomes and to LC3b autophagy vacuoles. Whilst POS were eventually degraded, OxPOS cargos were sequestered in late compartments. Co-localization of OxPOS was also associated with swollen autolysosomes. Ultrastructural analysis revealed the presence of electron-dense OxPOS aggregates in RPE cells, which appeared to be largely resistant to degradation. Measurement of cellular autofluorescence, using parameters used to assess fundus autofluorescence (FAF) in age-related macular disease (AMD) patients, revealed that OxPOS contributed significantly to a key feature of aged and diseased RPE. This in vitro cell model therefore represents a versatile tool to study disease pathways linked with RPE damage and sight-loss.
Highlights
The retinal pigment epithelium (RPE) plays a critical role in vision and is intimately associated with photoreceptors of the neuroretina
Since autofluorescence is a characteristic feature of RPE cells which is thought to originate from accumulated intracellular material [30], we studied whether this feature of the aged and diseased retina could be recapitulated in our model
Elevated proteolytic stress of RPE cells in the aging retina is an important mechanism of early damage which leads to eventual cellular atrophy, a key feature in several blinding conditions
Summary
The retinal pigment epithelium (RPE) plays a critical role in vision and is intimately associated with photoreceptors of the neuroretina. Intense light levels in the retina, in the lower wavelengths [14] alongside high metabolic activity coupled with hypoxic conditions, create a unique environment, where proteins and lipids become susceptible to modification by photo-oxidation This results in the formation of a variety of intracellular compounds, such as pyridinium bis-retinoid A2E, malondialdehyde (MDA) and 4-hydroxynonenal (HNE), amongst others, which are known to have different pathogenic effects in the RPE [15,16,17,18,19,20,21]. Such compounds target activities of the proteolytic pathway. A nuanced understanding of these mechanisms could result in elucidating the etiology of retinopathy and its progression, so that effective new treatments can be developed for diseases that currently result in irreversible sight-loss
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