Zhao J, Paganini L, Mucke L, Gordon M, Refolo L, Carman M, Sinha S, Oltersdorf T, Lieberburg I, McConlogue L
Athena Neurosciences, Inc., South San Francisco, California 94080, USA. amyloid!
J Biol Chem. 1996 Dec 6;271(49):31407-11. doi: 10.1074/jbc.271.49.31407.
Alzheimer's disease is characterized by the extracellular deposition of beta-amyloid peptide (Abeta) in cerebral plaques and evidence is accumulating that amyloid is neurotoxic. Abeta is derived from the beta-amyloid precursor protein (APP). Proteolytic processing of APP by the enzyme, beta-secretase, produces the N terminus of Abeta, and releases a secreted ectodomain of APP (beta-s-APP). To develop animal models for measuring beta-secretase activity in specific brain cells in vivo, we have targeted the expression of the full-length human APP to either neurons or astrocytes in transgenic mice using the neuron- specific enolase (NSE) promoter or a modified glial fibrillary acidic protein (GFAP) gene, respectively. The APP cDNAs expressed were mutated (KM to NL at 670/671) to encode amino acid substitutions that enhance amyloidogenic processing in vitro. Western analyses revealed abundant production of beta-s-APP in the brains of NSE-APP mice and enzyme-linked immunosorbent assay analyses showed production of Abeta in fetal primary mixed brain cultures and brain homogenates from these transgenic animals. Because the NSE promoter drives expression primarily in neurons, this provides in vivo evidence that the beta-secretase cleavage necessary for generation of beta-s-APP and Abeta is efficiently performed in neurons. In contrast, only little beta-s-APP was detected in brain homogenates of GFAP-APP mice, indicating that astrocytes show very little beta-secretase activity in vivo. This provides strong in vivo evidence that the major source of Abeta in brain is from neurons and not from astrocytes.
阿尔茨海默病的特征是β-淀粉样肽（Aβ）在脑斑中的细胞外沉积，并且越来越多的证据表明淀粉样蛋白具有神经毒性。Aβ来源于β-淀粉样前体蛋白（APP）。APP经β-分泌酶的蛋白水解加工产生Aβ的N端，并释放APP的分泌型胞外结构域（β-s-APP）。为了建立在体内测量特定脑细胞中β-分泌酶活性的动物模型，我们分别使用神经元特异性烯醇化酶（NSE）启动子或修饰的胶质纤维酸性蛋白（GFAP）基因，将全长人APP的表达靶向转基因小鼠的神经元或星形胶质细胞。所表达的APP cDNA发生了突变（670/671位的KM突变为NL），以编码增强体外淀粉样生成加工的氨基酸替代。蛋白质印迹分析显示NSE-APP小鼠脑中大量产生β-s-APP，酶联免疫吸附测定分析表明在这些转基因动物的胎儿原代混合脑培养物和脑匀浆中产生了Aβ。由于NSE启动子主要驱动神经元中的表达，这提供了体内证据，表明产生β-s-APP和Aβ所需的β-分泌酶切割在神经元中有效进行。相比之下，在GFAP-APP小鼠的脑匀浆中仅检测到少量的β-s-APP，表明星形胶质细胞在体内显示出非常低的β-分泌酶活性。这提供了强有力的体内证据，表明脑中Aβ的主要来源是神经元而非星形胶质细胞。
