June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Intraluminal acidity plays a key role in the function of lysosomes in RPE cells
Author Affiliations & Notes
  • Rebecca Denise Miller
    Clinical and Experimental sciences, University of Southampton, Southampton, Hampshire, United Kingdom
  • Savannah Amy Lynn
    Clinical and Experimental sciences, University of Southampton, Southampton, Hampshire, United Kingdom
  • Eloise Keeling
    Clinical and Experimental sciences, University of Southampton, Southampton, Hampshire, United Kingdom
  • Charles Ellis
    Clinical and Experimental sciences, University of Southampton, Southampton, Hampshire, United Kingdom
  • David Chatelet
    Biomedical Imaging Unit, University of Southampton, Southampton, Hampshire, United Kingdom
  • David A Johnston
    Biomedical Imaging Unit, University of Southampton, Southampton, Hampshire, United Kingdom
  • David A Tumbarello
    Biological Sciences, University of Southampton, Southampton, Hampshire, United Kingdom
  • Andrew J Lotery
    Clinical and Experimental sciences, University of Southampton, Southampton, Hampshire, United Kingdom
    Eye Unit, University Hospital Southampton NHS Foundation Trust, Southampton, United Kingdom
  • Arjuna Ratnayaka
    Clinical and Experimental sciences, University of Southampton, Southampton, Hampshire, United Kingdom
  • Footnotes
    Commercial Relationships   Rebecca Miller None; Savannah Lynn None; Eloise Keeling None; Charles Ellis None; David Chatelet None; David Johnston None; David Tumbarello None; Andrew Lotery None; Arjuna Ratnayaka None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 3891. doi:
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      Rebecca Denise Miller, Savannah Amy Lynn, Eloise Keeling, Charles Ellis, David Chatelet, David A Johnston, David A Tumbarello, Andrew J Lotery, Arjuna Ratnayaka; Intraluminal acidity plays a key role in the function of lysosomes in RPE cells. Invest. Ophthalmol. Vis. Sci. 2023;64(8):3891.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

Lyso-pHluorin response in RPE cells fed with: A: POS (n=3, p=0.0077) B: OxPOS (n=3, p=0.0025). C: Confocal images of Lyso-pHluorin (green) transfected RPE cells in each experimental group. Arrows indicate co-localisation between cargo (pink) and lysosome (red).

Lyso-pHluorin response in RPE cells fed with: A: POS (n=3, p=0.0077) B: OxPOS (n=3, p=0.0025). C: Confocal images of Lyso-pHluorin (green) transfected RPE cells in each experimental group. Arrows indicate co-localisation between cargo (pink) and lysosome (red).

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