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

脂筏是膜微结构域，其中胆固醇是主要成分之一。体外和体内研究表明，细胞内胆固醇水平对淀粉样蛋白前体蛋白（APP）向淀粉样蛋白β肽（a β）的加工有很大影响，淀粉样蛋白β肽是老年斑的主要成分。由于APP和淀粉样蛋白源性APP切割酶（β-和γ-分泌酶）都在脂筏中被发现，微结构域将是胆固醇依赖性调节a β产生的关键位点。我们假设APP在脂筏中的分配是由APP和筏蛋白之间的相互作用控制的，并且它调节淀粉样蛋白分泌酶的获取。为了寻找筏中的候选蛋白，我们从小鼠大脑或原代培养的皮质神经元中分离脂质筏，然后对含app的筏进行免疫分离。免疫分离筏的蛋白质成分表征表明，含有app的筏在神经元细胞内囊泡运输中具有功能特化。一些富含反式高尔基网络/内体的蛋白质在含有app的细胞器和含有γ-分泌酶的细胞器中被发现，这表明这些细胞器中的脂质筏可能参与了胆固醇依赖性的Aβ产生调节。对免疫分离筏及其蛋白质成分的进一步分析将为神经元中APP加工的筏相关调控机制提供新的见解。

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.