A Role for Melanoregulin in RPE-mediated Phagocytosis

描述（由申请方提供）：视网膜色素上皮细胞（RPE）1作为专职吞噬细胞的核心作用是调节溶酶体的生物发生和成熟，溶酶体是主要的降解区室，含有超过50种酸依赖性水解酶（1）。在RPE细胞中，每天的吞噬攻击不仅产生高降解负荷，而且由于这些细胞是有丝分裂后的，因此长期而言，几乎连续的吞噬作用对RPE产生额外的代谢应激。内化的OS的不完全消化导致称为脂褐素的自发荧光聚集体在膜结合的溶酶体内积累。我们已经确定了一种新的假定的调节溶酶体功能，称为黑素调节蛋白（MREG）。在Mreg- /-小鼠中，MREG的丧失导致溶酶体水解酶活性降低和组织蛋白酶-D加工的失调。这对RPE具有有害的后果，导致延迟的吞噬体降解、视蛋白阳性吞噬溶酶体的积累，并且在衰老的动物中，毒性光产物的产生增加。我们的研究重点是了解MREG调节溶酶体生成的分子机制。我们将检验MREG在溶酶体功能的动态调节中是必要的这一假设。我们计划使用一种综合方法，包括使用Mreg-/-和年龄匹配的对照Mreg+/+小鼠结合体外Mregdsu基因沉默技术进行体内研究，以评估异常溶酶体成熟对人类RPE健康的影响。在具体目标1中，我们将描述MREG如何促进溶酶体成熟以及成熟缺陷如何导致溶酶体水解酶功能减弱。溶酶体功能障碍的累积长期效应将通过定量RPE中A2 E的水平来解决。在第二个具体目标中，我们将重点关注溶酶体功能障碍对Cat-D加工的影响，并建立Cat-D分选和由于MREG丢失导致的误分选与吞噬作用之间的关系。我们预测，MREG的损失有助于RPE功能障碍，导致RPE、布鲁赫膜（BM）和脉络膜的长期病理变化。该提案将以前的RPE细胞内运输研究扩展到一个新的方向，并代表了一种独特的方法，以促进我们对溶酶体酶如何运输以及几种水解酶的集体损失对RPE健康的影响的理解。这些研究为今后开发MREG作为治疗剂的研究提供了基础。 公共卫生相关性：碎片在视网膜中的积累有助于与视网膜变性疾病以及正常人类衰老相关的病理变化。溶酶体对于有效降解摄入物质至关重要。它们在视网膜色素上皮细胞（RPE）降解摄取的光感受器材料中发挥特别关键的作用。必需溶酶体酶的缺陷性成熟和溶酶体蛋白酶的错误分选是RPE对氧化应激的响应以及RPE中与年龄相关的变化的标志。在这些研究中，我们将评估一种新的调节溶酶体功能的称为黑素调节素。了解黑素调节蛋白如何促进溶酶体功能将使我们能够开发治疗方法来增强正常人类衰老过程中的溶酶体功能。

DESCRIPTION (provided by applicant): 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.