However in many of the above mentioned studies

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 IC261 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. Microglia represent approximately 5–10% of all glia found in the brain. Various proteases have been detected in the conditioned medium from microglial cell cultures including the metalloproteinase for Aβ40 degradation [34], the cysteine protease cathepsin B for neurodegeneration in the central nervous system (CNS) [35], and the serine proteases plasmin and urokinase-type plasminogen activator [36]. A secretory protease in the conditioned medium of cultured rat microglia was characterized as an elastase-like protease using myelin basic protein as a substrate. The molecular weight of the protease was 25kDa, the optical pH was 7–8, and its activity was inhibited by 60% with PMSF and by 50% with elastatinal [37], [38]. The elastase-like proteases from the human monocytes or BV-2 cells observed in our study showed different responses to the protease inhibitors, suggesting that they were different proteases from the elastase-like proteases reported by Nakajima et al. [37]. However, to the best of our knowledge, there are no reports on ApoE degradation by microglial proteases. Though monocytes of peripheral origin are far from being microglia in the brain, they are derived from the same myeloid lineage. Therefore, we used human peripheral monocytes instead of microglial cells as a material for identifying the proteases responsible for ApoE degradation in the brain. The present materials consisted of 80% monocytes, but the remaining 20% was contaminated with lymphocytes. The hydrolytic activity for ApoE was detected in the crude extracts with a high concentration of salt (2.0M NaCl). The extraction step with the high-salt solution may be useful for removing neutral serine proteases in the crude extracts from the material [24], [39], but not the proteases for ApoE degradation. ApoE degradation was completely inhibited by Pefabloc-Sc, indicating the possibility of proteolysis by serine proteases. Various artificial substrates for serine proteases were tested to identify the serine protease responsible for the degradation contained in the high-salt extracts. The extracts showed high activity only to the elastase substrate, while it showed no activity to the substrate of trypsin, chymotrypsin and cysteine protease. The microglia found within and adjacent to the amyloid plaques in the brains with AD exhibited a reactive phenotype, which produces inflammatory components including cytokines and nitric oxide that are directly and indirectly neurotoxic [40]. ApoE was also observed adjacent to microglial cells. The microglial cells were stained with an anti-neutrophilic elastase antibody in the immunohistochemical examinations, suggesting that a microglial elastase is the protease responsible for ApoE degradation.