An In-Vitro Cell Model of Intracellular Protein Aggregation Provides Insights into RPE Stress Associated with Retinopathy

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.

3D-Reconstructed Retinal Pigment Epithelial Cells Provide Insights into the Anatomy of the Outer Retina

“…The phenomenon of processing photoreceptor outer segments as part of the daily photoreceptor renewal is unique to RPE, whereby the distal 10% of OS are internalised every 24 h by RPE cells and degraded in the phagosome or autophagy-dependent lysosomal pathways [ 33 ]. The high photo-oxidative environment of the retina as well as defects in cargo processing have been shown to increase the proteolytic burden of RPE cells and contribute to retinopathy [ 4 , 34 , 35 , 36 ]. The age-related accumulation of lipofuscin within RPE, which fills ~20% of the cytoplasm by the eighth decade of life, places a further burden on cellular stress [ 37 , 38 ].…”

Formation of Lipofuscin-Like Autofluorescent Granules in the Retinal Pigment Epithelium Requires Lysosome Dysfunction

“…These results confirmed that AFGs have resulted from the fusion of lysosomes to form phagolysosomes and further suggest that CTSD is consumed within AFGs while LAMP1 is retained at the surface of the granules. A recent study followed the fate of POS and oxidatively modified POS (oxPOS) in ARPE-19 cells for 72 h and found that POS were sequentially trafficking to LAMP1 and LAMP2 as reported here but in that study the use of fluorescently-labelled POS prevented the analysis of formation of AFGs 32,33,38 . In post-mortem RPE derived from AMD patients, enlarged and annular LAMP1-positive compartments were observed 39 , which could represent AFGs even though AF was not studied.…”

“…Several reports have also shown LC3 colocalization with internalized fluorescently-labelled POS 32,33,38 , as LC3-associated phagocytosis contributes . CC-BY-ND 4.0 International license perpetuity.…”

“…The above results predict that AFGs contain markers of phagolysosomes 32,33 , Therefore, we immunostained RPE cells with the established lysosome markers, LAMP1 and CTSD (Fig. 3A, B).…”

“…Several reports have also shown LC3 colocalization with internalized fluorescently-labelled POS 32, 33, 38 , as LC3-associated phagocytosis contributes to efficient POS degradation and recycling of bis-retinoids (38). However, we were unable to colocalize LC3 with AFGs either formed in hfRPE or in ARPE-19 cells (results not shown).…”