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.
Autophagy/physiology, Cells, Cultured, Humans, Lysosomes/pathology, Macular Degeneration/pathology, Oxidation-Reduction, Oxidative Stress/physiology, Phagocytosis/physiology, Protein Aggregates/physiology, Retinal Diseases/pathology, Retinal Photoreceptor Cell Outer Segment/pathology, Retinal Pigment Epithelium/pathology
Keeling, Eloise
3207bbdb-d391-44af-8abc-a60c08dce45b
Culling, Annabelle J
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Johnston, David A
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Chatelet, David S
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Page, Anton
76ebbfb8-4fe3-495c-afff-1f2f34977fee
Tumbarello, David A
75c6932e-fdbf-4d3c-bb4f-48fbbdba93a2
Lotery, Andrew J
5ecc2d2d-d0b4-468f-ad2c-df7156f8e514
Ratnayaka, J Arjuna
002499b8-1a9f-45b6-9539-5ac145799dfd
11 September 2020
Keeling, Eloise
3207bbdb-d391-44af-8abc-a60c08dce45b
Culling, Annabelle J
a974bfef-6ac7-4345-a261-cddb14f6b919
Johnston, David A
b41163c9-b9d2-425c-af99-2a357204014e
Chatelet, David S
6371fd7a-e274-4738-9ccb-3dd4dab32928
Page, Anton
76ebbfb8-4fe3-495c-afff-1f2f34977fee
Tumbarello, David A
75c6932e-fdbf-4d3c-bb4f-48fbbdba93a2
Lotery, Andrew J
5ecc2d2d-d0b4-468f-ad2c-df7156f8e514
Ratnayaka, J Arjuna
002499b8-1a9f-45b6-9539-5ac145799dfd
Keeling, Eloise, Culling, Annabelle J, Johnston, David A, Chatelet, David S, Page, Anton, Tumbarello, David A, Lotery, Andrew J and Ratnayaka, J Arjuna
(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), [6647].
(doi:10.3390/ijms21186647).
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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ijms-21-06647 (2)
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Accepted/In Press date: 7 September 2020
e-pub ahead of print date: 11 September 2020
Published date: 11 September 2020
Additional Information:
Funding Information:
Funding: This research was funded by awards to J.A.R. from the UK Macular Society and the Gift of Sight Appeal.
Publisher Copyright:
© 2020 by the authors. Licensee MDPI, Basel, Switzerland.
Keywords:
Autophagy/physiology, Cells, Cultured, Humans, Lysosomes/pathology, Macular Degeneration/pathology, Oxidation-Reduction, Oxidative Stress/physiology, Phagocytosis/physiology, Protein Aggregates/physiology, Retinal Diseases/pathology, Retinal Photoreceptor Cell Outer Segment/pathology, Retinal Pigment Epithelium/pathology
Identifiers
Local EPrints ID: 443956
URI: http://eprints.soton.ac.uk/id/eprint/443956
ISSN: 1422-0067
PURE UUID: b19ea042-ad3f-448a-8d19-23febc4de620
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Date deposited: 17 Sep 2020 16:42
Last modified: 17 Mar 2024 03:56
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Contributors
Author:
Eloise Keeling
Author:
Annabelle J Culling
Author:
David A Johnston
Author:
David S Chatelet
Author:
Anton Page
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