APPLICATION OF B-SECRETASE FOR PRESYMPTOMATIC DIAGNOSIS OF ALZHEIMERS DISEASE

β-淀粉样前体蛋白（APP）的加工和β-淀粉样蛋白（Aβ）的生成与阿尔茨海默病（AD）的病理生理过程密切相关。由于负责人脑中该过程的蛋白酶尚未阐明，我们在人海马中寻找能够切割天然脑APP的活性。对体外降解天然脑APP的40 kDa蛋白水解活性进行纯化和表征，并且随后的分子分析澄清为属于羧肽酶B（CP B）家族的新型蛋白酶。过表达编码该蛋白酶的cDNA的PC 12细胞在胞质溶胶中产生主要的12 kDa的β-淀粉样蛋白承载肽，该肽也在使用纯化的脑APP作为底物的无细胞系统中检测到。虽然该蛋白酶与肝脏中合成的血浆CPB同源，但它具有特定的结构域，如C-末端14个氨基酸残基。Western分析、cDNA克隆过程和北方分析表明该蛋白酶的脑特异性表达。免疫组织化学研究表明，蛋白酶表达在各种神经元胞体，包括海马锥体神经元，室管膜脉络丛细胞，并在正常大脑的小胶质细胞的一部分。在散发性AD患者的脑中，检测到神经元表达减少和一簇小胶质细胞与其细胞外沉积相关的蛋白酶免疫反应性。这些结果表明，脑CPB在APP加工中具有生理功能，并可能在AD病理生理学中具有重要意义。

The processing of β-amyloid precursor protein (APP) and generation of β-amyloid (Aβ) essentially are associated with the pathophysiology of Alzheimer's disease (AD). As the proteases responsible for the process in the human brain have yet to be clarified, we searched for activities capable of cleaving native brain APP in human hippocampus. 40 kDa proteolytic activity that degrades native brain APP in vitro was purified and characterized, and following molecular analysis clarified as being a novel protease belonging to the carboxypeptidase B (CPB) family. PC12 cells overexpressing the cDNA encoding this protease generate a major 12 kDa β-amyloid-bearing peptide in cytosol, which peptide was also detected in a cell-free system using purified brain APP as substrate. although the protease is homologous to plasma CPB synthesized in liver, it has specific domain such as C-terminal 14 amino acid residues. Western analysis, cDNA-cloning process, and Northern analysis suggested a brain-specific expression of this protease. An immunohistochemical study showed that the protease is expressed in various neuronal perikarya, including that of pyramidal neurons of the hippocampus, ependymal-choroid plexus cells, and in a portion of the microglia of normal brains. In brains of patients with sporadic AD, decreased neuronal expression and a cluster of microglia with protease immunoreactivity associated with its extracellular deposition being detected. These findings suggest that brain CPB has a physiological function in APP processing and may have significance in AD pathophysiology.