Cholesterol distribution & regulation of AB generation

胆固醇分布

• 批准号: 6544694
• 负责人: DORA M KOVACS
• 金额: $ 32.87万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-23 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6544694
• 关键词: Alzheimer's disease; CHO cells; amyloid proteins; cell membrane; cholesterol; cholesterol esters; confocal scanning microscopy; endopeptidases; genetically modified animals; intracellular transport; laboratory mouse; lipid metabolism; plasma; protein localization; tissue /cell culture; transfection

DESCRIPTION (provided by applicant): Recent studies indicate that cholesterol levels alter Ab generation in cells and transgenic animals. To define the role of cholesterol in APP processing, we chose a genetic approach and took advantage of cholesterol-mutant CHO cell lines. Two of the cell lines overproduce cholesterol, one (AC29) as membrane-bound free cholesterol (4-fold), the other (25RA) as cholesteryl-esters (6-fold). The third cell line, M19, is defective in the regulation of cholesterol biosynthesis and contains 90% less free cholesterol than control cells. We stably transfected these three cholesterol-mutant cells with APP751 or APP751/V7171 and found that Af3 generation is differentially regulated in accord with the intracellular distribution of free and esterified forms of cholesterol. Ab generation was found to specifically correlate with cholesteryl-ester levels, independently of free cholesterol. Specifically, Ab secretion was reduced by ~95% in both AC29751 and AC29751/V7171 cells (lacking cholesteryl-esters) as compared to control CH0751 cells. In contrast, high levels of cholesteryl-esters in 25RA cells were associated with significantly increased Ab generation. In accord with these data, ACAT inhibitors reduced Af3 generation. We also show a possible mechanism for decreased Ab production in presence of reduced cholesteryl-esters based on accelerated PSi degradation. We also found that sphingolipids, which are tightly associated with cholesterol in cholesterol rich-domains (rafts), are strong regulators of Ab generation. These findings suggest that down regulation of cholesteryl-ester formation e.g. by specific ACAT inhibitors or modulation of membrane cholesterol may serve as a novel therapeutic strategy for treating or preventing AD. The overarching goal of this proposal is to determine how intracellular cholesterol trafficking and cellular compartmentation affect Ab generation. Specific Aim 1 is targeted at elucidating the effect of reduced cholesteryl ester levels on the processing, maturation and trafficking of both APP and BACE in neuronal and non-neuronal cells and ACAT' mice. In Specific Aim 2, we will further explore the reduction of Ab levels in the presence of low levels of cholesteryl-esters, and particularly the potential role of PSi turnover. Specific Aim 3 will test whether A13 generation requires the structural integrity of cholesterol rafts, as assessed by targeted disruption or overproduction of cholesterol-rich domains. These studies should provide a clearer understanding of the molecular and biochemicalevents involved in cholesterol-dependent regulation of Ab generation.

描述（由申请方提供）：最近的研究表明，胆固醇水平改变细胞和转基因动物中的Ab生成。为了确定胆固醇在APP加工中的作用，我们选择了遗传方法并利用胆固醇突变CHO细胞系。两个细胞系过量产生胆固醇，一个（AC 29）作为膜结合游离胆固醇（4倍），另一个（25 RA）作为胆固醇酯（6倍）。第三个细胞系M19在胆固醇生物合成的调节中有缺陷，并且含有比对照细胞少90%的游离胆固醇。我们用APP 751或APP 751/V7171稳定转染这三种胆固醇突变细胞，发现Af 3的产生与游离和酯化形式胆固醇的细胞内分布雅阁，受到差异调节。抗体生成被发现与胆固醇酯水平，独立于游离胆固醇。具体而言，与对照CH 0751细胞相比，AC 29751和AC 29751/V7171细胞（缺乏胆固醇酯）的Ab分泌减少约95%。相反，高水平的胆固醇酯在25 RA细胞与显着增加抗体生成。与这些数据雅阁的是，ACAT抑制剂减少了Af 3的产生。我们还显示了一个可能的机制，减少抗体生产的存在下，减少胆固醇酯的基础上加速PSi降解。我们还发现，鞘脂，这是密切相关的胆固醇富含结构域（筏），是抗体生成的强调节剂。这些发现表明，下调胆固醇酯形成，例如通过特异性ACAT抑制剂或调节膜胆固醇，可作为治疗或预防AD的新治疗策略。该提案的首要目标是确定细胞内胆固醇运输和细胞区室化如何影响Ab生成。具体目标1旨在阐明胆固醇酯水平降低对神经元和非神经元细胞以及ACAT小鼠中APP和BACE加工、成熟和运输的影响。在具体目标2中，我们将进一步探索在低水平胆固醇酯存在下Ab水平的降低，特别是PSi转换的潜在作用。具体目标3将测试A13产生是否需要胆固醇筏的结构完整性，如通过富胆固醇结构域的靶向破坏或过度产生所评估的。这些研究应该提供一个更清楚的了解分子和生化事件参与胆固醇依赖性调节抗体的产生。