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An in-vitro cell model of intracellular protein aggregation provides insights into RPE stress associated with retinopathy

An in-vitro cell model of intracellular protein aggregation provides insights into RPE stress associated with retinopathy
An in-vitro cell model of intracellular protein aggregation provides insights into RPE stress associated with retinopathy
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.
AMD, Aging, RPE, autofluorescence, autophagy, diet, lysosomes, oxidized POS, proteolysis, retina
1422-0067
1-23
Ratnayaka, J. Arjuna
002499b8-1a9f-45b6-9539-5ac145799dfd
Lotery, Andrew
5ecc2d2d-d0b4-468f-ad2c-df7156f8e514
Tumbarello, David
75c6932e-fdbf-4d3c-bb4f-48fbbdba93a2
Ratnayaka, J. Arjuna
002499b8-1a9f-45b6-9539-5ac145799dfd
Lotery, Andrew
5ecc2d2d-d0b4-468f-ad2c-df7156f8e514
Tumbarello, David
75c6932e-fdbf-4d3c-bb4f-48fbbdba93a2

Ratnayaka, J. Arjuna, Lotery, Andrew and Tumbarello, David (2020) An in-vitro cell model of intracellular protein aggregation provides insights into RPE stress associated with retinopathy. International Journal of Molecular Sciences, 21 (18), 1-23, [6647]. (doi:10.3390/ijms21186647).

Record type: Article

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.

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More information

Accepted/In Press date: 7 September 2020
e-pub ahead of print date: 11 September 2020
Published date: September 2020
Additional Information: This article belongs to the Special Issue: Molecular Biology of Age-Related Macular Degeneration (AMD) 2.0)
Keywords: AMD, Aging, RPE, autofluorescence, autophagy, diet, lysosomes, oxidized POS, proteolysis, retina

Identifiers

Local EPrints ID: 443956
URI: http://eprints.soton.ac.uk/id/eprint/443956
ISSN: 1422-0067
PURE UUID: b19ea042-ad3f-448a-8d19-23febc4de620
ORCID for J. Arjuna Ratnayaka: ORCID iD orcid.org/0000-0002-1027-6938
ORCID for Andrew Lotery: ORCID iD orcid.org/0000-0001-5541-4305
ORCID for David Tumbarello: ORCID iD orcid.org/0000-0002-5169-0561

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Date deposited: 17 Sep 2020 16:42
Last modified: 22 Nov 2021 03:06

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