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Intraluminal acidity plays a key role in the function of lysosomes in RPE cells

Intraluminal acidity plays a key role in the function of lysosomes in RPE cells
Intraluminal acidity plays a key role in the function of lysosomes in RPE cells
Purpose : impaired lysosomal function in retinal pigment epithelial (RPE) cells is linked with incomplete photoreceptor outer segment (POS) degradation and their accumulation as lipofuscin; a well-defined pathway of RPE atrophy in Age-Related Macular Degeneration (AMD). Here, we tested the hypothesis that intraluminal lysosomal acidity (pH) is a key determinant in POS degradation that becomes impaired in AMD.

Methods : the molecular probe Lyso-pHluorin, which increases in fluorescence with diminishing acidification, was expressed in RPE (ARPE-19) cells and exposed to oxidative stress (10mM H2O2, 24Hrs) or Aβ (1µM human oligomeric Aβ1-42, 3Hrs) which is elevated in aged/AMD retinas. Cells were then synchronously fed POS (4µg/cm2). Parallel cultures were fed OxPOS, which were produced by UV cross-linked POS that becomes sequestered in lysosomes. RPE cells without insults acted as controls. Bafilomycin A1 was used to obtain maximal Lyso-pHluorin response (positive control). Whilst these studies were carried out after fixation, the use of CypHer5E conjugated POS provided insights into dynamic changes to lysosomal pH in living RPE. Co-labelling with LysoTracker Green DND-26 provided readouts of lysosomal size.

Results : POS (p=0.0077) as well as OxPOS (p=0.0025) co-localisation to lysosomes significantly diminished their intraluminal acidity compared to lysosomes without cargos. Interestingly, there was no appreciable difference in the fluorescence intensity of lysosomes with POS vs. OxPOS cargos. Lysosomal acidity also diminished after exposure to oxidative stress and Aβ, whilst CypHer5E showed dynamic alterations to lysosomal acidity.

Conclusions: our studies revealed that intraluminal lysosomal acidity becomes significantly diminished following POS and OxPOS accumulation. However, POS is rapidly degraded in healthy RPE, whilst OxPOS is known to be sequestered in RPE lysosomes for prolonged periods (akin to lipofuscin). Exposure to AMD-linked disease pathways also impaired lysosomal acidity. Our findings support a key role for intraluminal lysosomal pH in the ability to effectively degrade POS cargos, revealing novel mechanistic insights into the pathogenesis of AMD.
lysosome, retina, RPE
0146-0404
Miller, Rebecca Denise
2fdf8af7-fc30-4254-a301-ba32ca1fa1d9
Lynn, Savannah Amy
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Keeling, Eloise
3207bbdb-d391-44af-8abc-a60c08dce45b
Ellis, Charles
72e80fc3-f499-442c-85d4-00e2ea489a42
Chatelet, David
6aea1ff6-ad01-4927-82da-b4a1874f0010
Johnston, David A.
87eb13df-1613-4b49-894b-7304bb6ca89a
Tumbarello, David
75c6932e-fdbf-4d3c-bb4f-48fbbdba93a2
Lotery, Andrew J.
5ecc2d2d-d0b4-468f-ad2c-df7156f8e514
Ratnayaka, Arjuna
002499b8-1a9f-45b6-9539-5ac145799dfd
Miller, Rebecca Denise
2fdf8af7-fc30-4254-a301-ba32ca1fa1d9
Lynn, Savannah Amy
949c1189-1dd6-4ec1-b80e-97517a1f7b4b
Keeling, Eloise
3207bbdb-d391-44af-8abc-a60c08dce45b
Ellis, Charles
72e80fc3-f499-442c-85d4-00e2ea489a42
Chatelet, David
6aea1ff6-ad01-4927-82da-b4a1874f0010
Johnston, David A.
87eb13df-1613-4b49-894b-7304bb6ca89a
Tumbarello, David
75c6932e-fdbf-4d3c-bb4f-48fbbdba93a2
Lotery, Andrew J.
5ecc2d2d-d0b4-468f-ad2c-df7156f8e514
Ratnayaka, Arjuna
002499b8-1a9f-45b6-9539-5ac145799dfd

Miller, Rebecca Denise, Lynn, Savannah Amy, Keeling, Eloise, Ellis, Charles, Chatelet, David, Johnston, David A., Tumbarello, David, Lotery, Andrew J. and Ratnayaka, Arjuna (2023) Intraluminal acidity plays a key role in the function of lysosomes in RPE cells. Investigative Ophthalmology & Visual Science, 64 (8), [3891].

Record type: Meeting abstract

Abstract

Purpose : impaired lysosomal function in retinal pigment epithelial (RPE) cells is linked with incomplete photoreceptor outer segment (POS) degradation and their accumulation as lipofuscin; a well-defined pathway of RPE atrophy in Age-Related Macular Degeneration (AMD). Here, we tested the hypothesis that intraluminal lysosomal acidity (pH) is a key determinant in POS degradation that becomes impaired in AMD.

Methods : the molecular probe Lyso-pHluorin, which increases in fluorescence with diminishing acidification, was expressed in RPE (ARPE-19) cells and exposed to oxidative stress (10mM H2O2, 24Hrs) or Aβ (1µM human oligomeric Aβ1-42, 3Hrs) which is elevated in aged/AMD retinas. Cells were then synchronously fed POS (4µg/cm2). Parallel cultures were fed OxPOS, which were produced by UV cross-linked POS that becomes sequestered in lysosomes. RPE cells without insults acted as controls. Bafilomycin A1 was used to obtain maximal Lyso-pHluorin response (positive control). Whilst these studies were carried out after fixation, the use of CypHer5E conjugated POS provided insights into dynamic changes to lysosomal pH in living RPE. Co-labelling with LysoTracker Green DND-26 provided readouts of lysosomal size.

Results : POS (p=0.0077) as well as OxPOS (p=0.0025) co-localisation to lysosomes significantly diminished their intraluminal acidity compared to lysosomes without cargos. Interestingly, there was no appreciable difference in the fluorescence intensity of lysosomes with POS vs. OxPOS cargos. Lysosomal acidity also diminished after exposure to oxidative stress and Aβ, whilst CypHer5E showed dynamic alterations to lysosomal acidity.

Conclusions: our studies revealed that intraluminal lysosomal acidity becomes significantly diminished following POS and OxPOS accumulation. However, POS is rapidly degraded in healthy RPE, whilst OxPOS is known to be sequestered in RPE lysosomes for prolonged periods (akin to lipofuscin). Exposure to AMD-linked disease pathways also impaired lysosomal acidity. Our findings support a key role for intraluminal lysosomal pH in the ability to effectively degrade POS cargos, revealing novel mechanistic insights into the pathogenesis of AMD.

Text
Intraluminal acidity plays a key role in the function of lysosomes in RPE cells _ IOVS _ ARVO Journals - Accepted Manuscript
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More information

e-pub ahead of print date: 1 June 2023
Published date: 1 June 2023
Venue - Dates: Association for Research in Vision and Ophthalmology Annual Conference, Convention Centre , New Orleans, United States, 2023-04-23 - 2023-04-27
Keywords: lysosome, retina, RPE

Identifiers

Local EPrints ID: 479502
URI: http://eprints.soton.ac.uk/id/eprint/479502
ISSN: 0146-0404
PURE UUID: 5299e4da-80aa-4e69-b788-1ef8f659d1e1
ORCID for Eloise Keeling: ORCID iD orcid.org/0000-0003-0399-359X
ORCID for David Tumbarello: ORCID iD orcid.org/0000-0002-5169-0561
ORCID for Andrew J. Lotery: ORCID iD orcid.org/0000-0001-5541-4305
ORCID for Arjuna Ratnayaka: ORCID iD orcid.org/0000-0002-1027-6938

Catalogue record

Date deposited: 25 Jul 2023 16:48
Last modified: 06 Jun 2024 02:05

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Contributors

Author: Rebecca Denise Miller
Author: Savannah Amy Lynn
Author: Eloise Keeling ORCID iD
Author: Charles Ellis
Author: David Chatelet
Author: David A. Johnston

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