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  • br Materials and methods br Results br Discussion A previous


    Materials and methods
    Discussion A previous study showed that extracts from AD SQ 29,548 australia tissue demonstrate protease activity to ApoE at an acidic pH, which was inhibited by pepstatin A, suggesting that aspartic proteases are involved in the extracts [27]. Cathepsin D is also implicated in the pathogenesis and pathology of AD [28], [29], as it showed hydrolytic activity for both lipid-free and lipidated ApoE. In addition, the major ApoE fragment with cathepsin D had a similar molecular weight to that found in human brain lysates [30]. Furthermore, an immunohistochemical study revealed the colocalization of cathepsin D in neuritic plaques in brains with AD [31]. Other reports have suggested ApoE4 cleavage by a chymotrypsin-like protease, which is contained in human brain tissue or cultured neuronal cell lysates [32]. The hydrolytic activity of the chymotrypsin-like protease was inhibited only by phenylmethylsulfonyl fluoride, and not by other protease inhibitors including ethylene glycol tetraacetic acid, pepstatin, E-64, and iodoacetamide. These studies suggest that the key proteases in the extracts are serine proteases, and it might be a chymotrypsin-like protease, as determined by the substrate specificity of the protease with synthetic peptides [33]. However, these studies did not use the chymotrypsin-specific inhibitor, chymostatin. The involvement of metalloproteases in activated microglial cells has also been reported. Activated microglial cells secrete metalloproteases of 200kDa, which is different from the molecular weights of neutral endopeptidases and macrophage elastases, thus metalloproteases could degrade Aβ [34]. However, in many of the above mentioned studies, the NaCl concentrations in the extraction buffers were different from those used here. For instance, in the study that performed cathepsin D extraction from human AD brain tissue, the concentration was 150mM [30], while that for the extraction of the chymotrypsin-like protease from mouse neuroblastoma cells or human brain tissue was 0–500mM [32]. For the metalloproteases in the soluble cell extracts, an NaCl concentration of 500mM was employed [34]. In the present study, we detected the hydrolytic activity of ApoE in the extracts of human monocytes and BV-2 cells by extraction with an NaCl buffer of over 1M. The amount of extracted protein depends on the NaCl concentration, which resulted in the different protein contents in the extracts. Western blots indicated that the high-salt extracts showed hydrolytic activity for ApoE4, with the highest activity being exhibited for the elastase substrate. Furthermore, the activity increased along with the increasing NaCl concentrations. High activity for the trypsin substrate was detected using the extracts without NaCl and may have derived from the lymphocytes that contaminated the material. Furthermore, ApoE degradation by the high-salt extracts was completely inhibited by Elastase Inhibitor IV, an elastase-specific inhibitor. These findings indicate that elastases are the key proteases for ApoE4 degradation, which was clarified via LC–MS/MS analysis.