The role of high-fat diet and oxidative stress in driving the structural and functional deficits associated with Age-related Macular Degeneration

Abstract

Age-related Macular Degeneration (AMD) is the leading cause of irreversible blindness in the developed world. Current treatments are only suitable for a proportion of patients and are usually ineffective in the long-term. This is partially due to an incomplete understanding of the underlying aetiologies that drive pathology in the Retinal Pigment Epithelium (RPE) and adjacent tissues of the outer retina. Many risk factors for AMD, including consumption of a high fat diet (HFD), increase oxidative stress in the retina. I studied the link between a HFD and structural changes to the tissues in the outer retina. Next, I investigated how these pathogenic changes could cause disease phenotypes in the RPE at a single cell resolution. To achieve these objectives, I used serial block-face scanning electron microscopy (SBSEM) to study healthy tissues in the mouse outer retina. An entire patch of RPE and overlying photoreceptors were reconstructed in 3D, and comparisons were made between mono-nucleate and bi-nucleate RPE cells, of which the latter is associated with early AMD. This novel approach provided new data on the structure and arrangement of RPE and photoreceptors, as well as their relationship to each other. 3D approaches have not been extensively used before, thus we have gained new types of structural information on these tissues. In mice fed a HFD, I used conventional TEM alongside confocal immunofluorescence imaging to study the pathogenic changes in tissues of the outer retina. Detailed studies of HFD mouse eyes have not been carried out to this extent before. My findings revealed HFD-induced changes to the structure of outer retinal tissues, showing the effects of an unhealthy diet and oxidative stress in living eyes. Next, my work delved into these pathogenic mechanisms at single-cell resolution. A HFD is associated with oxidative stress and impaired acidification of intracellular compartments in the lysosomalautophagy pathway. Using confocal-immunofluorescence microscopy and conventional TEM, I studied how these insults impaired the trafficking of photoreceptor outer segments (POS) in cultured RPE cells. My discoveries reveal the trafficking and breakdown of POS cargos in early and late endosomes/ phagosomes, and their eventual transport to lysosomes and autophagy bodies in healthy RPE cells. My data also provides novel mechanistic insights into how HFD associated oxidative stress and impaired acidification alters POS trafficking, potentially contributing to early stages of RPE pathology. Notably, it appears that damage may be confined to only a proportion of RPE lysosomes, suggesting that at least some compartments in the endo-lysosomal pathway remains functional, even in diseased cells. Collectively, my work has revealed novel structural and functional insights into pathogenic mechanism in the RPE and adjacent tissues of the outer retina, which could significantly influence the way in which disease processes in the aging retina are understood. Such insights are critical if effective treatments against AMD are to be devised in the future

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Identifiers

ORCID for Eloise Keeling: orcid.org/0000-0003-0399-359X

ORCID for Janaka Ratnayaka: orcid.org/0000-0002-1027-6938

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