Retinal Pigment Epithelium In Health And Disease PDF Download

The retinal pigment epithelium (RPE) is a key site of damage in macular degeneration, which causes irreversible blindness in 30-50 million people globally. Our work aims to elucidate disease mechanisms and identify promising therapeutic targets. Using live-cell imaging, biochemical approaches and a mouse model of disease, this thesis examined the role of organelle trafficking in autophagy and complement regulation, two pathways implicated in macular degeneration. Autophagy is a major cellular clearance pathway essential for maintaining homeostasis in post-mitotic tissues such as the RPE. However, little is known regarding how autophagy is regulated in the RPE. Here, we showed that pathological accumulation of vitamin A metabolites in macular degeneration traps cholesterol in the RPE and inhibits both autophagosome biogenesis and autophagic clearance. Mechanistically, we determined that cholesterol activates acid sphingomyelinase (ASMase), causing increased generation of ceramide. This in turn promotes aberrant stabilization of microtubules and constraints autophagosome trafficking, preventing effective clearance of autophagic substrates. The work in this thesis also establishes a central role for organelle trafficking in regulating protective mechanisms against the abnormal activation of the complement system, which has been implicated in macular degeneration. These novel protective mechanisms in the RPE include accelerated recycling of the complement regulatory protein CD59 to the cell surface, and lysosome exocytosis-mediated membrane repair. Both protective mechanisms are impaired in diseased RPE due to alteration in organelle trafficking, leading to mitochondrial damage and oxidative stress upon exposure to complement. A key RPE function indispensable for vision is the phagocytosis and degradation of photoreceptor outer segments. The thesis characterized sequential interactions of outer segment phagosomes with autophagic machinery as the phagosomes traffic from the cell surface into the RPE. Moreover, outer segment phagocytosis activates transcription factor EB (TFEB), which regulates a vast arsenal of autophagy and lysosomal genes. Thus, the RPE may employ autophagy machineries for efficient clearance of phagocytosed outer segments. Altogether, these studies highlight the importance of organelle trafficking in RPE health and function. Importantly, this research revealed that removing excess cholesterol or inhibiting ASMase using FDA-approved drugs corrects organelle trafficking, thereby restoring autophagy and complement regulation in models of macular degeneration.