EXPRESSION AND FUNCTION OF CATHEPSIN E IN ACTIVATED MICROGLIA FOLLOWING FACIAL NERVE INJURY

面神经损伤后激活的小胶质细胞中组织蛋白酶 E 的表达和功能

基本信息

批准号：
09671898
负责人：
NAKANISHI Hiroshi
金额：
$ 1.98万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

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 … More 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

尽管组织蛋白酶E（CE）是一种细胞内天冬氨酸蛋白酶，主要作为小胶质细胞活化后内体样结构中的成熟酶存在，但目前尚不清楚CE在活化的小胶质细胞中的功能。在本研究中，我们启动了通过使用天冬氨酸蛋白酶抑制剂pepstatin A. Primaiy培养的大鼠小胶质细胞用pepstatin A处理的小胶质细胞中CE的可能功能，小胶质细胞的数量在时间和剂量依赖性依赖性的方式中都增加了。同时，注意到肌胶质细胞和双极形状的小胶质细胞变成棒状细胞。 pepstatin a也以剂量依赖性方式增加了溴脱氧尿苷的摄取。倍霉素A对小胶质细胞的影响得到了星形孢菌素的显着抑制。在第二系列实验中，我们检查了活化的小胶质细胞对神经元存活的影响，因为在R的R.经济的神经元细胞死亡中，已激活的小胶质细胞受到了激活的小胶质细胞的影响。当将神经元PC12细胞与用干扰素γ /脂多糖处理的小胶质细胞共培养时，神经元PC12细胞会导致通过caspase-3样蛋白酶激活和DNA片段化完成的凋亡。通过CASPAS-3样蛋白酶抑制剂N-乙酰基-ASP-GLU-VAL-ASP-醛来预处理并没有逆转这种细胞死亡，尽管它导致细胞中caspase-3样蛋白酶的活性完全抑制。这表明抑制caspase-3样蛋白酶活性不足以抑制活化的小胶质细胞诱导的神经元死亡。尽管神经元细胞中的Bcl-2过表达引起了caspase-3样蛋白酶活性和DNA片段化的明显抑制作用，但它无法抑制活化的小胶质细胞诱导的神经元死亡。在电子显微镜水平上，具有高水平BCl-2的退化神经元PCL2细胞的特征是略微凝结的染色质，形成不规则形状的肿块，严重崩解的周围核糖核酸和显着的真空吸尘器。这些结果表明，过表达BCI-2的神经元细胞中活化的小胶质细胞诱导的非凋亡细胞死亡。总的来说，我们的研究通过阻塞Caspase-3蛋白酶级联反应为激活的小胶质细胞诱导的神经元死亡提供了另一种死亡途径，这可能导致坏死死亡。较少的