An investigation of amyloid (Aβ) induced pathology in age-related macular degeneration

Abstract

Age-related Macular Degeneration (AMD) is the leading cause of irreversible vision loss in the western world, accounting for 8.7% of global blindness. However, current therapeutics are only applicable to 50% of patients and are ineffective in long-term treatment. This is due to an incomplete understanding of the aetiology underpinning Retinal Pigment Epithelium (RPE) and photoreceptor atrophy in AMD. Lysosomal malfunction within the RPE is believed to result in failed photoreceptor outer segment (POS) phagocytosis and the increased accumulation of protein/lipid aggregates in early AMD. Amyloid beta (Aβ), a highly toxic and penetrative peptide associated with Alzheimer’s disease, has also been shown to deposit in the ageing retina and associated with key stages of AMD. The purpose of this work was to elucidate the association of Aβ with AMD pathology and test the hypothesis that Aβ accumulates within RPE lysosomes and perturbs normal organelle function. An in vitro model of the outer retina was successfully established and exploited to investigate the subcellular localisation of Aβ1-42, as well as its effects on lysosomal function. Colocalisation analysis showed that 40.7 ± 8.6% of Aβ resided within RPE lysosomes, whilst live cell imaging and QPCR determined temporal changes in Cathepsin B activity and expression. Elevated Cathepsin B activity was found at 0.5 (p<0.0001) and 3 hours (p=0.0095) post exposure but returned to baseline at 24 and 48 hours, however mRNA expression remained consistent. Increased Aβ colocalisation with lysosomal Cathepsin B continued up to 24 hours despite a decline in activity. A functional assay also assessed Aβ effects on RPE phagocytic function, which showed decreased POS colocalisation with LAMP-1 at 20 hours. Insights into the biological function of Aβ within the retina were gained through ELISA, which demonstrated increased secretion of Aβ from the basal RPE surface (p=0.0004). We also assessed the toxicity of Aβ in vivo using subretinal injections in C57BL6/J mice. Here, ERG and OCT revealed no significant effect on global retinal function or thickness of constituent layers, although Aβ appeared to induce retinal pathology akin to CNV. Finally, a case-control pilot study was initiated to determine the association of vitreous Aβ concentration with AMD. The work presented within this thesis demonstrates the presence of Aβ within RPE lysosomes for the first time, where it potentially modulates the function of RPE clearance mechanisms. Similarly, we associate Aβ with CNV in situ through the novel use of non-invasive imaging techniques. These studies provide mechanistic insights into the cytotoxic effects of Aβ within the retina that could ultimately drive the RPE atrophy and inflammatory processes associated with AMD pathology. In particular, the functional consequences of Aβ on the RPE endolysosomal system may identify Aβ as a novel therapeutic target for early AMD