Analysis of the functions of proteases related to demyelination

在本研究项目中，我们研究了两种丝氨酸蛋白酶、神经蛋白酶和蛋白酶M在脱髓鞘中的作用。我们获得了以下结果：1.我们进行了双标记实验，发现成熟的少突胶质细胞表达神经蛋白酶和蛋白水解酶M。2.我们建立了实验性变态反应性脑病(EAE)的小鼠多发性硬化动物模型。在此模型中，神经蛋白酶和蛋白酶M在少突胶质细胞中表达较强。在缺乏髓鞘碱性蛋白(MBP)基因的寒战小鼠中，蛋白酶M的表达明显受到抑制。选择性降解少突胶质细胞的铜必酮饮食可降低少突胶质细胞的蛋白水解酶M的表达，而正常饮食诱导的再髓鞘形成增加了少突胶质细胞M3的表达。神经蛋白酶基因敲除(KO)小鼠脊髓损伤后存活的少突胶质细胞数明显多于野生型。Neuropsin-KO组小鼠的后肢运动活动明显好于对照组。Neuropsin-KO小鼠在行为学测试中的得分也较高。4.当原代少突胶质细胞中蛋白酶M过表达时，没有明显的变化。然而，RNAi抑制蛋白水解酶M的表达降低了MBP.5的表达。神经蛋白酶基因敲除小鼠存活的轴突数量明显多于野生型。上述结果表明，这两种蛋白在损伤后的少突胶质细胞中表达，并参与脱髓鞘。

In this research project, we have investigated on the function of 2 serine proteases, neuropsin and protease M to demyelination. We obtained following results :1.We performed double labeling experiments and found that neuropsin and protease M were expressed by mature oligodendrocytes.2.We induced experimental allergic encephalopathy (EAE), an animal model of multiple sclerosis in mice. In this model, neuropsin and protease M expressed stronger in oligodendrocytes. In shiverer mice, which defect myelin basic protein (MBP) gene, protease M expression was markedly suppressed. Cuprizone diet, which selectively degenerates oligodendrocytes, decreased protease M expression and remyelination induced by normal diet without cuprizone increased the expression of protease M.3.The number of survived oligodendrocytes after spinal cord injury was significantly more in neuropsin-knockout (KO) mice than in the wild type. The resultant motion activity of the hind limb was significantly better in neuropsin-KO mice. Neuropsin-KO mice also scored better in behavioral tests.4.When protease M was overexpressed in primary oligodendrocytes, no prominent change was observed. However, suppression of protease M expression with RNAi decreased the expression of MBP.5.We made model mice of spinal cord injury. The number of survived axons was significantly more in neuropsin knockout mice than wild type.The above results indicate that these two proteases are expressed in oligodendrocytes after injury and are involved in demyelination.