Cholesterol distribution & regulation of AB generation

描述(由申请人提供)：最近的研究表明，胆固醇水平改变了细胞和转基因动物中抗体的产生。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。其中两个细胞系过度产生胆固醇，一个(AC29)作为膜结合的游离胆固醇(4倍)，另一个(25RA)作为胆固醇酯(6倍)。第三个细胞系M19在调节胆固醇生物合成方面存在缺陷，其游离胆固醇含量比对照细胞低90%。我们用APP751或APP751/V7171稳定地转染了这三种胆固醇突变细胞，发现Af3的生成根据游离和酯化形式的胆固醇在细胞内的分布而受到不同的调控。研究发现，AB的生成与胆固醇酯水平特别相关，与游离胆固醇无关。具体地说，与对照CH0751细胞相比，AC29751和AC29751/V7171细胞(缺乏胆固醇酯)的抗体分泌都减少了~95%。相反，25RA细胞中高水平的胆固醇酯与显著增加的抗体生成有关。与这些数据一致的是，ACAT抑制剂减少了AF3的生成。我们还展示了在胆固醇酯减少的情况下，基于PSI加速降解而导致抗体产生减少的可能机制。我们还发现，鞘脂与胆固醇富集区(RAFT)中的胆固醇密切相关，是抗体生成的强大调节因子。这些发现表明，通过特定的ACAT抑制剂或膜胆固醇的调节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。这项建议的首要目标是确定细胞内胆固醇运输和细胞区隔如何影响抗体的产生。具体目标1旨在阐明降低的胆固醇酯水平对APP和BACE在神经细胞和非神经细胞以及ACAT‘小鼠中的加工、成熟和运输的影响。在具体目标2中，我们将进一步探讨在低水平胆固醇酯存在的情况下抗体水平的降低，特别是PSI周转的潜在作用。具体目标3将测试A13代是否需要胆固醇筏的结构完整性，如通过靶向破坏或过量生产富含胆固醇的结构域来评估。这些研究应该能更清楚地了解胆固醇依赖调节抗体生成的分子和生物化学事件。

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.