Deciphering Underlying Mechanisms Of Photoreceptor Outer Segment Degradation; Involvement Of Intracellular Trafficking

Age related macular degeneration (AMD) is the leading cause of vision loss among adults 50 and older in the United States. AMD is characterized by the degeneration of photoreceptors and retinal pigment epithelium (RPE) cells in the macula region of the retina. RPE maintain the health of the retina by providing support to the photoreceptor cells and the retina through various tasks. One major task RPE cells are involved in, is daily uptake, trafficking and degradation of shed photoreceptor outer segment (POS). It has been shown that with age and as a result of cellular stress this process can become impaired. Impairment in this unique phagocytic process can lead to degeneration of the RPE cells as well as the adjacent photoreceptor cells. In order to understand the pathogenesis of AMD and other retinal degenerative diseases it is important to uncover the molecular factors that are involved in the phagocytic activity of RPE uptake and clearance of POS. Lack of molecular methods and assays have limited our knowledge of these factors. In this project I developed a novel method which is used to uncover new factors specifically involved in POS trafficking in the RPE. Foster Resonance Energy Transfer or FRET is an energy transfer mechanism used to monitor interactions between proteins. This method was utilized to monitor movement of phagocytosed POS within the RPE to the lysosome. Using FITC labeling of POS and Lysotracker labelling of lysosomes I developed a novel assay to monitor the energy transfer of FITC labelled POS to Lysotracker labelled lysosome. The FRET assay is validated using Chloroquine treatment of ARPE-19 cells and knockdown of Atg5. This unique assay gives specific information regarding the trafficking of POS to the lysosome within the RPE. The assay is useful for uncovering novel factors involved in the trafficking stage of POS phagocytosis. With the developed assay I show the role of vesicular protein sorting subunits 18 and 11 in POS trafficking. In VPS18 and VPS11 siRNA knockdown ARPE-19 cells, POS trafficking is impaired. The working model suggests that disruption of endocytic pathway complex, HOPS and COVERT leads to impaired trafficking in RPE cells. The developed trafficking assay uncovered the role of the endocytic pathway in POS phagocytosis in RPE.