Molecular mechanisms of phagosome maturation and degradation in the retinal pigment epithelium in health and disease

The retinal pigment epithelium (RPE) is a single cell layer in the eye that lies immediately beneath the photoreceptors, the cells that detect light. Photoreceptors must undergo a process of daily partial renewal in order to function properly. Everyday at about the onset of daylight, photoreceptors shed their tips, which are engulfed (phagocytosed) by the RPE and degraded. RPE cells do not normally divide in the adult so a single RPE cell phagocytoses and degrades an enormous amount of photoreceptor material during a 70 year lifespan. This prodigious activity takes its toll and as the RPE ages undigested products of phagocytosis accumulate within the cells and this is believed to contribute to the accumulation of debris with age, particularly in people suffering from age-related macular degeneration, the leading cause of blindness in the Western world. Whilst considerable progress has recently been made in identifying the molecules that regulate the initial engulfment process, much less is known about what regulates the subsequent degradation of the vacuole containing the phagocytosed material (the phagosome). In cells where phagosome degradation has been better characterised the phagosome has been shown to undergo a maturation process involving the sequential acquisition of specific proteins that are necessary for the phagosome to gain the ability to fuse with the lysosome. Within the lysosome enzymes degrade the phagosome. Using high resolution microscopic methods we will identify the transport steps required for phagocytosed photoreceptor outer segments to be delivered to the lysosome and degraded. We will then determine which molecules regulate phagosome maturation and degradation in RPE cells, focussing on Rab proteins and motor proteins, some of which we have already shown are present in RPE cells and regulate other transport steps within the cell. This study will identify mechanisms whereby a process that is essential to human vision takes place and will help us to understand how this process breaks down with age and in eye disease.

Phagocytosis of photoreceptor outer segments (POS) occurs in all mammalian retinal pigment epithelial (RPE) cells and a failure to efficiently degrade the products of phagocytosis leads to a gradual build up of undigested products in all aging retinae but is particularly marked in diseases such as age-related macular degeneration (AMD), the leading cause of blindness in the Western world. Whilst some of the molecules required for the initial engulfment of the phagosome have recently been identified, the subsequent degradation of the phagosome is a poorly understood process likely to involve interaction with endosomes, lysosomes and possibly melanosomes. The molecular mechanisms governing transport between these organelles are unclear, as is their contribution to the lipofuscin and melanolipofuscin granules that characterise the aging and diseased retina. Our aims are to characterise phagosome maturation and degradation in RPE cells, to identify molecules that regulate sequential steps in the maturation process and determine how phagosome maturation/degradation are modified in aging and disease. We will identify markers of the maturing phagosome in RPE cells and measure the kinetics of phagosome maturation, phagosome-lysosome fusion and degradation of phagosome content. We will then analyse the role of potential molecular regulators of phagosome maturation/degradation, focussing on rab proteins and unconventional myosins. Myosin VIIa has been shown to play an as yet uncharacterised role in phagocytosis in RPE cells and rabs 5, 7 and 27a and myosin Va have been shown to play a role in phagosome maturation in other systems. Phagosome maturation/degradation in vivo will be analysed by measuring phagosome numbers and distribution within the cell (by conventional EM) and acquisition of endosomal and lysosomal markers (by immuno-EM). In vitro studies will be performed in cultured primary RPE cells incubated with purified POS. Phagosome degradation will be measured by quantitative confocal microscopy and phagosome maturation by immunofluorescence and immunoEM. To determine the roles of specific myosins and rabs we will study retinae and isolated RPE cells from mice carrying mutations in the relevant proteins and by depleting wild type RPE cells of the relevant protein by virally introduced shRNAs. We will also compare phagosome maturation/degradation in young and in aging mice and determine how the level of pigmentation in the retina affects phagosome maturation/degradation. Together these studies will elucidate molecular mechanisms regulating a phagocytic process that is fundamental to vision and will hopefully provide new insights into retinal degenerative disease and age.