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

虽然组织蛋白酶E （CE）是一种细胞内天冬氨酸蛋白酶，在小胶质细胞活化后主要作为成熟酶存在于核内体样结构中，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，我们开始利用天冬氨酸蛋白酶抑制剂胃抑素A来研究CE在小胶质细胞中的可能功能。当原代培养的大鼠小胶质细胞被胃抑素A处理后，小胶质细胞数量呈时间依赖性和剂量依赖性增加。同时，我们注意到具有变形虫和双极形状的小胶质细胞转变为杆状细胞。胃抑素A也以剂量依赖性的方式增加溴脱氧尿苷的摄取。胃抑素A对小胶质细胞的这些作用被斯陶孢素显著抑制。在第二个系列的实验中，我们检查了活化的小胶质细胞对神经元存活的影响，因为活化的小胶质细胞与神经细胞死亡的更多调节有关。当神经元PC12细胞与干扰素- γ /脂多糖处理的小胶质细胞共培养时，神经元PC12细胞凋亡并伴有caspase-3样蛋白酶活化和DNA断裂。用caspase-3样蛋白酶抑制剂n -乙酰基- asp - glu - val - asp -醛预处理并不能逆转这种细胞死亡，尽管它可以完全抑制细胞中caspase-3样蛋白酶的活性。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。虽然Bcl-2在神经元细胞中过表达可明显抑制caspase-3样蛋白酶活性和DNA断裂，但不能抑制活化的小胶质细胞诱导的神经元死亡。电镜下，高Bcl-2水平的神经元PCl2退行性细胞表现为染色质轻微凝聚形成不规则团块，核周严重解体，明显的空泡化。提示激活的小胶质细胞可诱导过表达BcI-2的神经元细胞发生非凋亡性细胞死亡。总之，我们的研究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，即阻断caspase-3蛋白酶级联，可能导致坏死死亡。少

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