Hall, Michael James;
(2020)
Lysosome Turnover in the Retinal Pigment Epithelium in Health, Ageing and Age- related Macular Degeneration.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
The Retinal Pigment Epithelium (RPE) phagocytoses and degrades spent Photoreceptor Outer Segments (POS) every day, placing an unparalleled burden on lysosomes in RPE cells, which require well-regulated turnover to maintain degradative capacity. With age and in Age-related Macular Degeneration (AMD), debris accumulates inside and outside RPE cells leading to the hypothesis that lysosome dysfunction could contribute to AMD pathology. This project aimed to identify the functional relevance of different subpopulations of lysosome within the heterogenous lysosomal compartment of RPE cells. Models of lysosome dysfunction were then generated in order to determine whether they recapitulate hallmarks of aging and AMD. Electron microscopy (EM) and electron tomography of primary porcine RPE (pRPE) revealed morphologically distinct subpopulations of lysosomes that form a complex network. Pulse-chase experiments showed that multilamellar Cathepsin D-rich lysosomes transition to electron dense lysosomes that stained poorly for Cathepsin D, suggesting that these subpopulations represent different stages of the lysosome cycle. Tracking of phagocytosed POS suggests that POS degradation occurs by both full fusion and kiss-and-run interactions between phagosomes and lysosomes. Correlative light and electron microscopy, and high content imaging, demonstrated that POS loading induces the gradual formation of lipofuscin-like autofluorescent granules. Loading cells with degradation- resistant UV-irradiated POS induced the formation of more and larger autofluorescent granules, thus recapitulating one of the hallmarks of aging and AMD. Treatment with the acidotropic compound chloroquine caused accumulation of undigested phagocytic, endocytic and autophagic cargo, consistent with lysosome dysfunction. This was accompanied by accumulation of lamellar lysosomes containing unprocessed lysosomal Cathepsin D, a feature 4 recapitulated by direct protease inhibition and prevented by inhibiting protein synthesis. Together, the findings in this PhD project shed new light on the identity, function and fate of lysosomes in the RPE and suggest that lysosome dysfunction could contribute to the pathology of AMD, as well as to chloroquine-induced retinopathy.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Lysosome Turnover in the Retinal Pigment Epithelium in Health, Ageing and Age- related Macular Degeneration |
| Event: | UCL (University College London) |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2020. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > Institute of Ophthalmology |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10102314 |
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