Study on cholesterol-dependent regulatory mechanisms of amyloid precursor protein processing in lipid microdomains

脂筏是膜微区，胆固醇是其中的主要成分之一。体外和体内研究表明，细胞内胆固醇水平对淀粉样前体蛋白（APP）向淀粉样β肽（Aβ）的加工有很大影响，A β是老年斑的主要成分。由于APP和淀粉样蛋白生成APP裂解酶（β-和γ-分泌酶）均存在于脂筏中，微区是胆固醇依赖性调节Aβ生成的关键位点，我们推测APP进入脂筏的分配受APP和脂筏蛋白之间的相互作用控制，并调节淀粉样蛋白生成分泌酶的进入。为了寻找筏中的候选蛋白，我们从小鼠脑或原代培养的皮层神经元中分离脂筏，然后对含有APP的筏进行免疫分离。免疫隔离筏的蛋白质组分的表征表明含APP的筏在神经元中用于细胞内囊泡运输的功能专业化。在含APP的脂筏和含γ-分泌酶的脂筏中发现了一些富含trans-Golgi网络/内体的蛋白质，提示脂筏可能参与了胆固醇依赖的Aβ生成调控，对免疫分离脂筏及其蛋白质组分的进一步分析将为研究脂筏相关的神经元APP加工调控机制提供新的思路。

Lipid rafts are membrane microdomains in which cholesterol is one of the major components. In vitro and in vivo studies have shown that intracellular cholesterol levels have a great impact on the processing of amyloid precursor protein (APP) to amyloid-β peptide (Aβ), the major constituent of senile plaques. Since APP and amyloidogenic APP-cleaving enzymes (β- and γ-secretases) have all been found in lipid rafts, the microdomain will be a critical site for cholesterol-dependent regulation of Aβ production.We hypothesize that partitioning of APP into the lipid rafts is controlled by interaction between APP and a raft protein(s) and that it regulates access to amyloidogenic secretases. To search for candidate proteins in rafts, we isolated lipid rafts from mouse brains or primary cultured cortical neurons and then performed immuno-isolation of APP-containing rafts. Characterization of protein components of the immuno-isolated rafts indicated functional specialization of APP-containing rafts for intracellular vesicular transport in neurons. Several proteins rich in the trans-Golgi network/endosomes are found in APP-containing rafts as well as in γ-secretase-containing ones, suggesting lipid rafts in such organelles may be involved in cholesterol-dependent regulation of Aβ production.Further analyses of immuno-isolated rafts and their protein components will provide new insights into raft-related regulatory mechanisms of APP processing in neurons.