A Role for Melanoregulin in RPE-mediated Phagocytosis

描述(申请人提供)：视网膜色素上皮细胞(RPE)1作为专业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室，含有50多种依赖酸的水解酶(1)。在RPE细胞中，每天的吞噬挑战不仅产生高降解负荷，而且由于这些细胞是有丝分裂后的，长期的、几乎连续的吞噬作用在RPE上产生额外的代谢压力。不完全消化内化的OSS会导致自体荧光聚集体在膜结合的溶酶体内积累，称为脂褐素。我们已经确定了一种新的溶酶体功能调节因子，称为黑素调节蛋白(MREG)。在mreg-/-小鼠中，MREG的丢失会导致溶酶体水解酶活性降低和组织蛋白酶-D加工的放松调控。这会对RPE造成有害后果，导致吞噬小体降解延迟，视蛋白阳性吞噬小体积累，并在衰老的动物中增加有毒光产物的产生。我们的研究集中在了解MREG调节溶酶体发生的分子机制。我们将验证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的方法，包括使用Mreg-/-和年龄匹配的对照组Mreg+/+小鼠进行体内研究，并结合体外Mregdsu基因沉默技术来评估溶酶体异常成熟对人类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、长期影响将通过量化RPE中的A2E水平来解决。在第二个特定目标中，我们将重点研究溶酶体功能障碍对Cat-D加工的影响，并建立Cat-D分选与MREG丢失导致的分选错误和吞噬作用之间的关系。我们预测MREG的缺失会导致RPE功能障碍，导致RPE、Bruch膜(BM)和脉络膜的长期病理改变。这一建议将以前的RPE细胞内转运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共同丧失对RPE健康的影响的理解。这些研究为今后开发MREG作为治疗剂提供了基础。 公共卫生相关性：视网膜中碎片的积累导致与视网膜退行性疾病相关的病理变化以及正常的人类衰老。有效降解摄入物质的关键是溶酶体。它们在视网膜色素上皮(RPE)对摄取的光感受器物质的降解中起着特别关键的作用。RPE对氧化应激的反应以及RPE中与年龄相关的变化是RPE必需的溶酶体酶成熟缺陷和溶酶体蛋白酶分选错误的标志。在这些研究中，我们将评估一种新的溶酶体功能调节剂，称为黑素调节蛋白。了解黑素调节蛋白是如何促进溶酶体功能的，将使我们能够开发出在正常人类衰老期间增强溶酶体功能的治疗方法。

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.