|Budget Amount *help
¥3,100,000 (Direct Cost : ¥3,100,000)
Fiscal Year 1998 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1997 : ¥2,200,000 (Direct Cost : ¥2,200,000)
Although cathepsin E (CE), an intracellular aspartic proteinase, is existed predominantly as the mature enzyme in the endosome-like structures following cellular activation of microglia, functions of CE in activated microglia are currently unknown. In the present study, we have initiated to investigate possible functions of CE in microglia by using the aspartic proteinases inhibitor pepstatin A.When primaiy cultured rat microglia were treated with pepstatin A, the number of microglia was increased in both time- and dose-dependent manners. At the same time, it was noted that microglia with ameboid and bipolar-shape was changed into rod-like cells. The uptake of bromodeoxyuridine was also increased by pepstatin A in a dose-dependent manner. These effects of pepstatin A on microglia were significantly suppressed by staurosporine.
In the second series of experiments, we have examined effects of activated microglia on neuronal survival, since activated microglia have been implicated in the r
egulation of neuronal cell death. When neuronal PC12 cells were cocultured with microglia that were treated with interferon-gamma /lipopolysaccharide, neuronal PC12 cells caused apoptosis accompanied by caspase-3-like protease activation and DNA fragmentation. Pretreatment with the caspas-3-like protease inhibitor N-acetyl-Asp-Glu-Val-Asp-aldehyde did not reverse this cell death, although it resulted in complete inhibition of the caspase-3-like protease activity in the cells. This suggests that the inhibition of caspase-3-like protease activity is not sufficient to inhibit the activated microglia-induced neuronal death. Although Bcl-2 overexpression in the neuronal cells caused the apparent inhibition of caspase-3-like protease activity and DNA fragmentation, it could not suppress the activated microglia-induced neuronal death. At electronmicroscopic level, the degenerating neuronal PCl2 cells with high levels of Bcl-2 were characterized by slightly condensed chromatins forming irregular shaped masses, severely disintegrated perikarya, and marked vacuolation. These results suggest that activated microglia induce non- apoptotic cell death in the neuronal cells overexpressing BcI-2. Altogether, our study provides an alternative death pathway for the activated microglia-induced neuronal death by blockage of the caspase-3 protease cascade, probably leading to necrotic death. Less