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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

虽然组织蛋白酶E（CE）是一种细胞内天冬氨酸蛋白酶，主要以成熟酶的形式存在于小胶质细胞活化后的内体样结构中，但CE在活化的小胶质细胞中的功能目前尚不清楚。在本研究中，我们已经开始调查CE在小胶质细胞中可能的功能，通过使用天冬氨酸蛋白酶抑制剂胃酶抑素A。当原代培养的大鼠小胶质细胞用胃酶抑素A处理，小胶质细胞的数量增加，在时间和剂量依赖性的方式。同时，还观察到变形双极小胶质细胞向杆状细胞转变。胃蛋白酶抑制剂A也以剂量依赖性方式增加溴脱氧尿苷的吸收。在第二组实验中，我们检测了活化的小胶质细胞对神经元存活的影响，因为活化的小胶质细胞与神经元存活有关。 ...更多信息神经元细胞死亡的调节。当神经元型PC 12细胞与用干扰素-γ/脂多糖处理的小胶质细胞共培养时，神经元型PC 12细胞引起凋亡，伴随着caspase-3样蛋白酶激活和DNA片段化。用半胱天冬酶-3样蛋白酶抑制剂N-乙酰基-Asp-Glu-Val-Asp-醛预处理不能逆转这种细胞死亡，尽管它导致细胞中半胱天冬酶-3样蛋白酶活性的完全抑制。这表明抑制半胱天冬酶-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。虽然Bcl-2在神经元细胞中的过表达引起caspase-3样蛋白酶活性和DNA片段化的明显抑制，但它不能抑制活化的小胶质细胞诱导的神经元死亡。电镜下，Bcl-2高表达的PCl-2细胞变性，染色质轻度浓缩，形成不规则团块，胞体严重崩解，空泡化明显。这些结果表明，激活的小胶质细胞诱导过表达Bcl-2的神经元细胞的非凋亡性细胞死亡。总之，我们的研究提供了一个替代的死亡途径激活小胶质细胞诱导的神经元死亡的caspase-3蛋白酶级联反应的阻断，可能导致坏死性死亡。少