Cholesterol distribution & regulation of AB generation

描述（由申请人提供）：最近的研究表明，胆固醇水平改变细胞和转基因动物中Ab的生成。为了确定胆固醇在APP加工中的作用，我们选择了一种遗传方法，并利用了胆固醇突变的CHO细胞系。两种细胞系过量产生胆固醇，一种（AC29）作为膜结合游离胆固醇（4倍），另一种（25RA）作为胆固醇酯（6倍）。第三种细胞系M19在胆固醇生物合成的调节方面存在缺陷，其游离胆固醇含量比对照细胞少90%。我们用APP751或APP751/V7171稳定转染了这三种胆固醇突变细胞，发现Af3的产生受到与游离胆固醇和酯化胆固醇在细胞内分布一致的差异调节。发现Ab生成与胆固醇-酯水平特异性相关，独立于游离胆固醇。具体而言，与对照CH0751细胞相比，AC29751和AC29751/V7171细胞（缺乏胆固醇酯）的Ab分泌减少了约95%。相反，25RA细胞中高水平的胆固醇酯与Ab生成显著增加相关。与这些数据一致，ACAT抑制剂减少了Af3的产生。我们还展示了一种可能的机制，即在降低胆固醇酯的情况下，基于加速PSi降解而减少Ab的产生。我们还发现，在胆固醇富集区域（筏）中与胆固醇密切相关的鞘脂是Ab生成的强调节因子。这些发现表明，通过特异性ACAT抑制剂或调节膜胆固醇来下调胆固醇酯的形成可能是治疗或预防AD的一种新的治疗策略。本提案的总体目标是确定细胞内胆固醇运输和细胞区隔如何影响Ab的产生。特异性目的1旨在阐明降低胆固醇酯水平对神经元和非神经元细胞以及ACAT小鼠中APP和BACE的加工、成熟和运输的影响。在Specific Aim 2中，我们将进一步探讨低水平胆固醇酯存在时Ab水平的降低，特别是PSi转换的潜在作用。Specific Aim 3将测试A13的生成是否需要胆固醇筏的结构完整性，通过靶向破坏或富含胆固醇的结构域的过度生产来评估。这些研究应该提供一个更清晰的分子和生物化学事件参与胆固醇依赖性调节的Ab生成。

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.