Expression and regulation of key lysosomal effectors in RPE cells : implications for Age-related Macular Degeneration (AMD)

The retinal pigmented epithelium (RPE) is a monolayer of highly specialised polarised post-mitotic cells that help maintain retinal homeostasis. Accumulation of advanced glycation end products (AGEs), a phenomenon of the ageing process, in RPE and Bruch’s membrane (BrM) contribute to the development of age-related macular degeneration (AMD), the leading cause of blindness in the western world. AGEs induced oxidative insult which causes proteins to misfold and become dysfunctional. Removal of misfolded dysfunctional proteins by the lysosome is critical for cell survival. Key effectors of lysosomal enzymatic activity are cathepsins. In addition to lysosomal function, cathepsins also exert extracellular functions. Cathepsins activity is regulated by endogenous inhibitors and the balance between proteases and their inhibitors regulate several crucial cellular processes such as autophagy, apoptosis and extracellular remodelling. The most abundantly expressed protease inhibitor from the RPE is cystatin C. In addition, cystatin C, an inhibitor of cathepsins B, L and S, is susceptible to age-related alterations. Furthermore, the polymorphism rs1063049 in the cystatin C gene (CST3) is genetically associated with susceptibility to exudative AMD. The understanding of the effects of ageing on the balance between cathepsins and their inhibitors and how dysregulation could contribute to pathological changes associated with AMD is of crucial importance. Therefore, the overall aim of this present study is: 1) to investigate the endogenous expression and secretion of cathepsins B, L and S in RPE cells; 2) to assess the impact of age-related stresses on expression/processing and activity of cathepsins and 3) to further investigate the genetic association of CST3 with exudative AMD. Immunoblot and immunohistochemical (IHC) analysis confirmed the presence cathepsins B, L and S in RPE cells. In addition, immunoblot analysis of conditioned media detected the presence of cathepsins B, L and S, indicative of their secretion. RPE cells exposed to AGEs exhibited alterations in cathepsin L, which showed decreased protein and activity levels. Finally, the genetic analysis study showed that the rs1063049 was associated with exudative AMD in a recessive manner. The results presented in this thesis showed that cathepsins are susceptible to age-related changes in RPE cells. Alterations of cathepsins may impair important lysosomal processes such as autophagy, which may influence the age-related dysfunction of RPE. In addition, the genetic association of CST3 gene with exudative AMD further supports that proteolytic dysregulation involving cathepsins and cystatin C is a crucial event that contributes to RPE dysfunction and subsequent development of disease states such as AMD.