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Open AccessArticle

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

1
Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, MP806, Tremona Road, Southampton SO16 6YD, UK
2
Biomedical Imaging Unit, University of Southampton, MP12, Tremona Road, Southampton SO16 6YD, UK
3
Biological Sciences, Faculty of Environmental and Life Sciences, Life Sciences Building 85, University of Southampton, Highfield Campus, Southampton SO17 1BJ, UK
4
Eye Unit, University Hospital Southampton NHS Foundation Trust, Southampton SO16 6YD, UK
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2020, 21(18), 6647; https://doi.org/10.3390/ijms21186647
Received: 12 July 2020 / Revised: 30 August 2020 / Accepted: 7 September 2020 / Published: 11 September 2020
(This article belongs to the Special Issue Molecular Biology of Age-Related Macular Degeneration (AMD) 2.0)
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. View Full-Text
Keywords: RPE; oxidized POS; proteolysis; lysosomes; autophagy; autofluorescence; retina; AMD; diet; aging RPE; oxidized POS; proteolysis; lysosomes; autophagy; autofluorescence; retina; AMD; diet; aging
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MDPI and ACS Style

Keeling, E.; Culling, A.J.; Johnston, D.A.; Chatelet, D.S.; Page, A.; Tumbarello, D.A.; Lotery, A.J.; Ratnayaka, J.A. An In-Vitro Cell Model of Intracellular Protein Aggregation Provides Insights into RPE Stress Associated with Retinopathy. Int. J. Mol. Sci. 2020, 21, 6647. https://doi.org/10.3390/ijms21186647

AMA Style

Keeling E, Culling AJ, Johnston DA, Chatelet DS, Page A, Tumbarello DA, Lotery AJ, Ratnayaka JA. An In-Vitro Cell Model of Intracellular Protein Aggregation Provides Insights into RPE Stress Associated with Retinopathy. International Journal of Molecular Sciences. 2020; 21(18):6647. https://doi.org/10.3390/ijms21186647

Chicago/Turabian Style

Keeling, Eloise; Culling, Annabelle J.; Johnston, David A.; Chatelet, David S.; Page, Anton; Tumbarello, David A.; Lotery, Andrew J.; Ratnayaka, J. A. 2020. "An In-Vitro Cell Model of Intracellular Protein Aggregation Provides Insights into RPE Stress Associated with Retinopathy" Int. J. Mol. Sci. 21, no. 18: 6647. https://doi.org/10.3390/ijms21186647

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