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

脂筏是以胆固醇为主要成分的膜微区。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)转化为淀粉样蛋白-β肽(A-β)的过程有很大影响，而淀粉样前体蛋白是老年斑的主要成分。由于APP和APP裂解酶(β和γ分泌酶)都在脂筏中被发现，所以微域将是胆固醇依赖的Aβ产生调节的关键部位。我们假设APP在脂筏中的分配是由APP和RAFT蛋白(S)之间的相互作用控制的，并且它调节获得淀粉样分泌酶的途径。为了寻找木筏中的候选蛋白，我们从小鼠脑或原代培养的皮质神经元中分离脂筏，然后对含有APP的木筏进行免疫分离。对免疫隔离木筏的蛋白质成分的表征表明，含有APP的木筏对神经元内的囊泡运输具有功能专一性。在含有APP的RAFT和含有γ分泌酶的RAFT中发现了几种富含反高尔基网络/内体的蛋白质，提示这种细胞器中的脂筏可能参与了胆固醇依赖的Aβ产生的调节。进一步分析免疫隔离的RAFT及其蛋白质组分将为了解RAFT相关的神经元APP处理的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.