Mechanism of Lysosome Functional Impairment in Cholesterol-engorged Foam Cells

描述（由申请人提供）：溶酶体功能的进行性下降与许多疾病有关。功能下降的一个原因是溶酶体内不可消化物质的积累，这可以产生功能障碍。这种联系在遗传性溶酶体贮积性疾病中最为明显，但在其他疾病中也很明显。溶酶体功能障碍的发生随着年龄的增长而增加，并且可能是造成细胞内稳态机制和代谢失衡的继发性效应的结果。先天性和获得性溶酶体贮积障碍几乎总是有病理后果，往往是致命的。近年来，人们越来越认识到溶酶体在胆固醇代谢疾病中的重要性，如Niemann-Pick C型、阿尔茨海默氏症和最显著的动脉粥样硬化。巨噬细胞泡沫细胞中的溶酶体固醇积聚是动脉粥样硬化的一个普遍存在但尚未得到充分了解的方面。与致动脉粥样硬化颗粒一起培养的巨噬细胞也表现出溶酶体中游离胆固醇和酯化胆固醇的积累。我们的研究表明，最初，游离胆固醇作为脂蛋白衍生的胆固醇酯的正常溶酶体水解的结果而积累，但产生的游离胆固醇仍然被困在溶酶体中。游离胆固醇积累后，胆固醇酯水解受到抑制，因此未降解的脂蛋白胆固醇酯也在溶酶体中积累。我们的初步数据表明，水解抑制至少部分是通过诱导溶酶体无法维持酸性pH来介导的。提出的研究系统地调查了甾醇积累抑制溶酶体维持低pH的能力的机制，并测试了游离胆固醇至少部分介导这种抑制的假设。在目的1中，细胞和分离的溶酶体被用来确定特定脂质的能力：1)抑制质子泵入溶酶体，2)改变溶酶体质子泵的合成、稳定性和运输，3)增加溶酶体膜的漏性，4)是否影响其他底物的水解。溶酶体的脂质含量会通过与脂蛋白或其他类似于动脉粥样硬化病变中发现的含脂颗粒的孵育而改变。Aim 2直接调节体外溶酶体的固醇、甘油三酯和鞘脂含量，以确定是否有任何或所有这些影响溶酶体维持酸度的能力。目的3探讨溶酶体无法维持活性pH是否与胆固醇介导的膜序改变有关。最后，目的4研究溶酶体内脂质氧化是否解释了游离胆固醇在溶酶体内的捕获和/或随后对CE水解的抑制。公共卫生相关性：建立泡沫细胞中溶酶体固醇积聚的机制和后果将增加我们对动脉粥样硬化病变发展的理解。这项研究对其他胆固醇调节疾病，如尼曼-匹克症和阿尔茨海默病也有更广泛的意义。

DESCRIPTION (provided by applicant): Progressive decreases in lysosomal function have been linked to a number of diseases. One cause of decreased function is build-up of indigestible material within lysosomes, which can produce dysfunction. This link is clearest in genetic lysosomal storage disorders but is also apparent with other diseases. The occurrence of lysosomal malfunction increases with age and can be the result of secondary effects creating imbalances in cell homeostatic mechanisms and metabolism. Both innate and acquired lysosomal storage disorders almost always have pathologic consequences and are often fatal. Recently, there has been a growing understanding of the importance of lysosomes in diseases of cholesterol metabolism, such as Niemann-Pick type C, Alzheimer's and most notably atherosclerosis. Lysosomal sterol accumulation in macrophage foam cells is a ubiquitous but not well-understood aspect of atherosclerosis. Macrophages in culture incubated with atherogenic particles also exhibit an accumulation of both free and esterified cholesterol in lysosomes. Our studies show that, initially, free cholesterol accumulates as a result of normal lysosomal hydrolysis of lipoprotein-derived cholesteryl ester but the generated free cholesterol remains trapped in the lysosomes. Subsequent to the free cholesterol accumulation, cholesteryl ester hydrolysis is inhibited and so undegraded lipoprotein cholesteryl ester also accumulates in the lysosomes. Our preliminary data suggests that the inhibition of hydrolysis is mediated, at least in part, through the induction of an inability of lysosomes to maintain an acidic pH. The proposed studies systematically investigate the mechanism by which sterol accumulation inhibits the lysosome's ability to maintain a low pH and tests the hypothesis that free cholesterol, at least partially, mediates the inhibition. In aim 1, cells and isolated lysosomes are used to determine the ability of specific lipids to 1) inhibit pumping of protons into lysosomes, 2) alter the synthesis, stability and trafficking of lysosome proton pumps, 3) increase leakiness of lysosome membranes and 4) whether hydrolysis of other substrates is affected. The lipid content of lysosomes will be altered by incubation with lipoprotein or other lipid-containing particles similar to those found in atherosclerotic lesions. Aim 2 directly modulates sterol, triglyceride, and sphingolipid content of lysosomes ex-vivo to determine if any or all of these affect the lysosomes ability to maintain acidity. Aim 3 explores whether the inability of lysosomes to maintain an active pH is related to cholesterol-mediated alterations in membrane order. Finally, aim 4 investigates whether intra-lysosomal oxidation of lipid explains the trapping of free cholesterol within lysosomes and/or the subsequent inhibition of CE hydrolysis. PUBLIC HEALTH RELEVANCE: Establishing the mechanism and consequences of lysosomal sterol accumulation in foam cells will increase our understanding of atherosclerosis lesion development. The research also has broader implications for other sterol-modulated diseases such as Niemann-Pick and Alzheimer's.