Analysis of the functions of proteases related to demyelination

本课题主要研究了神经蛋白酶和蛋白酶M这两种丝氨酸蛋白酶在脱髓鞘过程中的作用。结果表明：1.通过双标记实验，发现成熟的少突胶质细胞表达neuropsin和protease M。2.通过诱导小鼠实验性变态反应性脑病（experimental allergic encephalopathy，EAE），建立多发性硬化动物模型。在该模型中，neuropsin和蛋白酶M在少突胶质细胞中表达较强。在髓鞘碱性蛋白（MBP）基因缺陷的颤抖小鼠中，蛋白酶M的表达明显受到抑制。选择性变性少突胶质细胞的铜腙饮食降低蛋白酶M的表达，而不含铜腙的正常饮食诱导的髓鞘再生增加蛋白酶M的表达。3.脊髓损伤后存活的少突胶质细胞数量在neuropsin敲除（KO）小鼠中显著多于野生型。在neuropsin-KO小鼠中，后肢的所得运动活性显著更好。Neuropsin-KO小鼠在行为测试中也得分更高。4.当蛋白酶M在原代少突胶质细胞中过表达时，没有观察到明显的变化。而蛋白酶M的表达受到RNAi的抑制后，MBP的表达量下降。5.建立小鼠脊髓损伤模型。neuropsin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，这两种蛋白酶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.