Central to the retinal pigment epithelial cells'(RPE)1 role as professional phagocytes is the regulated biogenesis and maturation of lysosomes which serves as the major degrading compartment, containing over 50 acid-dependent hydrolytic enzymes (1). In RPE cells the daily phagocytic challenge produces not only a high degradative load but since these cells are post-mitotic, over the long-term, virtually continuous phagocytosis produces additional metabolic stress on the RPE. Incomplete digestion of internalized OSs leads to the accumulation of autofluorescent aggregates called lipofuscin within the membrane bound lysosomes. We have identified a novel putative regulator of lysosome function, called melanoregulin (MREG). In the Mreg- /- mouse, loss of MREG results in diminished lysosomal hydrolase activity and deregulation of Cathepsin-D processing. This has deleterious consequences to the RPE resulting in delayed phagosome degradation, accumulation of opsin positive phagolysosomes and in aging animals, enhanced production of toxic photoproducts. Our studies are focused on understanding the molecular mechanism by which MREG regulates lysosomogenesis. We will test the hypothesis that MREG is necessary in the dynamic regulation of lysosome function. We plan to use an integrated approach involving in vivo studies using Mreg-/- and age-matched control Mreg+/+ mice in combination with in vitro Mregdsu gene silencing techniques to assess the consequences of abnormal lysosome maturation on human RPE health. In specific aim 1, we will delineate how MREG contributes to lysosome maturation and how defects in maturation lead to diminished lysosomal hydrolase function. The cumulative, long-term effect of lysosomal dysfunction will be addressed by quantifying the levels of A2E in the RPE. In the second specific aim we will focus on the effect of lysosomal dysfunction on the processing of Cat-D and establish the relationship between Cat-D sorting and, missorting due to loss of MREG, and phagocytosis. We predict that loss of MREG contributes to RPE dysfunction leading to long-term pathologic changes in RPE, Bruch's membrane (BM), and choroid. This proposal expands previous RPE intracellular trafficking studies in a new direction and represents a unique approach to advance our understanding of how lysosomal enzymes are trafficked and what effect the collective loss of several hydrolases has on RPE health. These studies provide the underpinnings for future studies directed at the development of MREG as therapeutic agent. PUBLIC HEALTH RELEVANCE: The accumulation of debris in the retina contributes to pathologic changes associated with retinal degenerative disease as well as normal human aging. Essential for the efficient degradation of ingested material are lysosomes. They play a particularly crucial role in the degradation of ingested photoreceptor material by the retinal pigment epithelia (RPE). Defective maturation of essential lysosomal enzymes and missorting of lysosomal proteases are hallmarks of the RPE's response to oxidative stress, as well as age- associated changes in the RPE. In these studies we will evaluate a novel regulator of lysosome function called melanoregulin. Understanding how melanoregulin contributed to lysosome function will allow us to develop therapeutic approaches to enhancing lysosome function during normal human aging.