Analyses on beta-site APP cleaving enzyme (BACE1) of amyloid β protein deposited in brains of patients with Alzheimer's disease

点击翻译按钮获取中文摘要

Alzheimer's disease (AD) is characterized by the extensive deposition of amyloid β protein (Aβ) in brain cortex. Aβ is produced from β-amyloid precursor protein (APP) by β-secretase and γ-secretase. Recently, β-secretase was identified as beta-site APP cleaving enzyme 1 (BACE1). Inhibition against BACE1 activity could decrease Aβ generation, indicating the possibility that antagonistic drugs for BACE1 are therapeutic tools for AD.We produced rabbit polyclonal antibodies against synthetic peptides of BACE1. Using these antibodies (aniti-BACE1 antibodies), BACE1 was characterized in human brains (temporal lobes) by Western blotting and immunohistochemistry.All brain fractions extracted by Tris-saline, 1% Triton X-100 and 0.5% SDS sequentialy were revealed to contain BACE1. In order to compare amounts of BACE1 between AD and control brains, brain samples were directly extracted by 0.5% SDS and analyzed by Western blotting and densitometer. Although the mean level of amounts of BACE1 per m … More g protein in AD brains was significantly decreased, the ratio of BACE1 to MAP2 was significantly increased compared to control brains, indicating that remaining small numbers of neurons in AD brains might generate more amounts of BACE1 than control brain neurons. Digestion of both human brains and recombinant BACE1 by N-glycosidase F altered the molecular weight of BACE1 from 70 to 50kDa, consisting with calculated molecular weight of BACE1 depending on its amino acid residues, suggesting that human brains contained both unmodified BACE1 and its glycosylated form. Immunocytochemical studies employing anti-GFAP and anti-MAP2 antibodies as well as anti-BACE1 antibodies have shown that BACE1 was expressed exclusively in neurons.Taking all these findings together in consideration, remaining neurons after neuronal loss in AD brains might generate more amounts of BACE1 than in controls, indicating that increased activities of BACE1 could be one of the causes of AD, which could justify the development of anti-BACE1 drugs for AD treatment. Less