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Lysosomal impairment in the Retinal Pigment Epithelium (RPE) - a pathway of damage in the ageing retina

Lysosomal impairment in the Retinal Pigment Epithelium (RPE) - a pathway of damage in the ageing retina
Lysosomal impairment in the Retinal Pigment Epithelium (RPE) - a pathway of damage in the ageing retina
Purpose : A key function of the RPE is the phagocytosis/degradation of photoreceptor outer segments (POS). In age and disease, this pathway becomes impaired and lipofuscin accumulates within RPE lysosomes. Here, we used cultured RPE cells to identify the molecular pathways through which damage to the endosomal-lysosomal system occurs.
Methods : We developed an ex-vivo model of the RPE monolayer in which cells structurally and functionally resemble native RPE1. ARPE-19 cells were fed 4mg/cm2 POS-FITC. To optimise POS binding whilst synchronising internalisation2, cultures were incubated with POS for 30min at 17oC after which cells were returned to a 37oC incubator before fixation with 4% PFA at 2, 4, 6, 12, 24 and 48hrs. In order to mimic oxidative stress, cultures were pre-treated with 200mM Hydrogen Peroxide (H2O2) for 24hrs. Parallel cultures were pre-treated with 10nM Bafilomycin (Baf) for 48Hrs, which blocks vATPase mimicking effects of the lipofuscin-derivative A2E3. DMSO was fed to sister cultures as a control. Cells on transwell inserts were stained with endosomal (Rab5, Rab7), lysosomal (Lamp1, Lamp2), and autophagy marker (LC3B) for each time point. Data from n>6 cells/time point/treatment. Images collected on a Leica SP8 confocal microscope and analysed using Volocity (PerkinElmer).
Results : Our results show that trafficking of POS-FITC cargo occurs through endosomes, lysosomes and autophagosomes in a 2-48hr timescale. Pre-treatment with Baf increased accumulation in Rab5 and Rab7 (p<0.05) compartments at late time points (6hr-24hr), indicative of impaired maturation in late endosomes. Exposure to elevated oxidative stress (H2O2) increased cargo trafficking to lamp1/2 positive compartments between 2-4hrs (p<0.01). Exposure to Baf or H2O2 also increased the formation of autophagy bodies between 2-24hrs (p<0.05).
Conclusions : Exposure to an A2E mimic impaired maturation of late endosomes, whilst oxidative stress caused rapid trafficking of cargos to late compartments; revealing divergent mechanisms through which the RPE endosomal-lysosomal pathway becomes disrupted in old age, and how these perturbations increase autophagy. Such insights may be informative when designing future treatments.
0146-0404
4487
Keeling, Eloise
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Johnston, David
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Chatelet, David
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Tumbarello, David
75c6932e-fdbf-4d3c-bb4f-48fbbdba93a2
Lotery, Andrew
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Ratnayaka, J. Arjuna
002499b8-1a9f-45b6-9539-5ac145799dfd
Keeling, Eloise
abc30700-b1ed-49ed-ba1c-078856e01b7c
Johnston, David
b41163c9-b9d2-425c-af99-2a357204014e
Chatelet, David
6371fd7a-e274-4738-9ccb-3dd4dab32928
Tumbarello, David
75c6932e-fdbf-4d3c-bb4f-48fbbdba93a2
Lotery, Andrew
5ecc2d2d-d0b4-468f-ad2c-df7156f8e514
Ratnayaka, J. Arjuna
002499b8-1a9f-45b6-9539-5ac145799dfd

Keeling, Eloise, Johnston, David, Chatelet, David, Tumbarello, David, Lotery, Andrew and Ratnayaka, J. Arjuna (2018) Lysosomal impairment in the Retinal Pigment Epithelium (RPE) - a pathway of damage in the ageing retina. Investigative Ophthalmology & Visual Science, 59, 4487.

Record type: Meeting abstract

Abstract

Purpose : A key function of the RPE is the phagocytosis/degradation of photoreceptor outer segments (POS). In age and disease, this pathway becomes impaired and lipofuscin accumulates within RPE lysosomes. Here, we used cultured RPE cells to identify the molecular pathways through which damage to the endosomal-lysosomal system occurs.
Methods : We developed an ex-vivo model of the RPE monolayer in which cells structurally and functionally resemble native RPE1. ARPE-19 cells were fed 4mg/cm2 POS-FITC. To optimise POS binding whilst synchronising internalisation2, cultures were incubated with POS for 30min at 17oC after which cells were returned to a 37oC incubator before fixation with 4% PFA at 2, 4, 6, 12, 24 and 48hrs. In order to mimic oxidative stress, cultures were pre-treated with 200mM Hydrogen Peroxide (H2O2) for 24hrs. Parallel cultures were pre-treated with 10nM Bafilomycin (Baf) for 48Hrs, which blocks vATPase mimicking effects of the lipofuscin-derivative A2E3. DMSO was fed to sister cultures as a control. Cells on transwell inserts were stained with endosomal (Rab5, Rab7), lysosomal (Lamp1, Lamp2), and autophagy marker (LC3B) for each time point. Data from n>6 cells/time point/treatment. Images collected on a Leica SP8 confocal microscope and analysed using Volocity (PerkinElmer).
Results : Our results show that trafficking of POS-FITC cargo occurs through endosomes, lysosomes and autophagosomes in a 2-48hr timescale. Pre-treatment with Baf increased accumulation in Rab5 and Rab7 (p<0.05) compartments at late time points (6hr-24hr), indicative of impaired maturation in late endosomes. Exposure to elevated oxidative stress (H2O2) increased cargo trafficking to lamp1/2 positive compartments between 2-4hrs (p<0.01). Exposure to Baf or H2O2 also increased the formation of autophagy bodies between 2-24hrs (p<0.05).
Conclusions : Exposure to an A2E mimic impaired maturation of late endosomes, whilst oxidative stress caused rapid trafficking of cargos to late compartments; revealing divergent mechanisms through which the RPE endosomal-lysosomal pathway becomes disrupted in old age, and how these perturbations increase autophagy. Such insights may be informative when designing future treatments.

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ARVO abstract_2018
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More information

Published date: 1 July 2018
Additional Information: 2018 ARVO Annual Meeting Abstract

Identifiers

Local EPrints ID: 423103
URI: http://eprints.soton.ac.uk/id/eprint/423103
ISSN: 0146-0404
PURE UUID: d552f641-c631-45fb-ba7b-37fe4e9801bc
ORCID for David Johnston: ORCID iD orcid.org/0000-0001-6703-6014
ORCID for David Tumbarello: ORCID iD orcid.org/0000-0002-5169-0561
ORCID for Andrew Lotery: ORCID iD orcid.org/0000-0001-5541-4305
ORCID for J. Arjuna Ratnayaka: ORCID iD orcid.org/0000-0002-1027-6938

Catalogue record

Date deposited: 14 Aug 2018 16:30
Last modified: 22 Nov 2021 03:06

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Contributors

Author: Eloise Keeling
Author: David Johnston ORCID iD
Author: David Chatelet
Author: Andrew Lotery ORCID iD

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