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激活后内体样结构中的成熟酶存在，但 CE 在激活的小胶质细胞中的功能目前尚不清楚。使用天冬氨酸蛋白酶抑制剂胃酶抑素 A 研究 CE 在小胶质细胞中的可能功能。当用胃酶抑素 A 处理原代培养的大鼠小胶质细胞时，小胶质细胞的数量在时间和浓度上均增加。同时，人们注意到，胃酶抑素A也以剂量依赖性方式增加了阿米巴样和双极形状的小胶质细胞对溴脱氧尿苷的摄取。小胶质细胞上的 A 被星形孢菌素显着抑制。在第二个系列的实验中，我们研究了激活的小胶质细胞对神经存活的影响，因为激活的小胶质细胞与神经存活有关……神经元细胞死亡的更多调节当神经元 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 细胞引起细胞凋亡，并伴有 caspase-3 样蛋白酶激活和 caspas-3- 预处理。像蛋白酶抑制剂 N-乙酰基-天冬氨酸-谷氨酸-缬氨酸-天冬氨酸-醛一样不能逆转这种细胞死亡，尽管它导致完全抑制细胞中的 caspase-3 样蛋白酶活性，这表明尽管 Bcl-2 在神经元中过度表达，但抑制 caspase-3 样蛋白酶活性不足以抑制激活的小胶质细胞诱导的神经元死亡。细胞引起caspase-3样蛋白酶活性和DNA断裂的明显抑制，但不能抑制激活的小胶质细胞诱导的神经元死亡。在电镜水平上，退化的神经元PCl2细胞。具有高水平 Bcl-2 的特征是染色质稍微浓缩，形成不规则形状的团块、严重解体的核周和明显的空泡化。这些结果表明，活化的小胶质细胞诱导过度表达 Bcl-2 的神经元细胞死亡。研究通过阻断 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，可能导致坏死死亡。