Research on proteases related to demyelination expressed by oligodendrocytes

脊髓损伤是一个医学和社会问题，因为在大多数情况下恢复期较差。恢复有限主要是由于损伤轴突的再生潜力不足。最近的研究进展表明，细胞外环境，特别是髓鞘蛋白，可能是抑制再生的主要因素。我们已经证明少突胶质细胞在中枢神经系统损伤后表达细胞外蛋白酶。本研究观察脊髓损伤及多发性硬化症（MS）动物模型实验性变应性脑炎（EAE）后neuropsin和蛋白酶M的表达变化。此外，还研究了神经素可能底物的变化和神经再生。所有的实验都得到了研究所动物伦理委员会的批准。未经治疗的对照组小鼠显示神经递质mRNA的表达仅限于运动神经元。蛋白酶M mRNA在白质中广泛表达。脊髓损伤后，神经球蛋白mRNA在脊髓前侧索孔细胞中表达。在损伤后1-14天观察到上调，在第4天达到峰值。蛋白酶M蛋白在下中枢神经系统中大量表达，其表达与CNPase免疫反应性（成熟少突胶质细胞的标志）共定位。电镜分析显示髓磷脂和少突胶质细胞体具有免疫反应性。kainic酸损伤后，髓鞘对蛋白酶M的免疫反应性增强。为了探索可能的底物，髓磷脂蛋白与神经磷脂孵育，并进行电泳和免疫印迹分离。与髓鞘碱性蛋白（myelin basic protein， MBP）相对应的条带经neuropsin孵育后变弱，免疫印迹分析证实这些条带为髓鞘碱性蛋白。注射MOG后3 ~ 4周EAE达到高峰。苏木精-伊红染色可见脊髓基底下区浸润细胞。炎症区周围细胞表达Neuropsin和蛋白酶M mRNA。这些结果表明细胞外蛋白酶在中枢神经系统的生理和病理状态中的重要性。少

Spinal cord injury is medical and social problem because in most cases the recovery is poor. The limited recovery is mostly caused by scant potential of regeneration of the injured axons. Recent progress of research has shown that the extracellular environment, particularly myelin proteins, may be the primary factor for the inhibition of regeneration. We have shown that oligodendrocytes express extracellular proteases after injury to the central nervous system. In the current study we observed the change of expression of neuropsin and protease M after spinal cord injury and experimental allergic encephalitis (EAE), animal model of multiple sclerosis (MS). Furthermore, the change of possible substrates of neuropsin and nerve regeneration were also studied.All the experiments were approved by the Institute's Animal Ethical Committee.Untreated control mice showed the expression of neuropsin mRNA limited to motoneurons. Protease M mRNA was broadly expressed in white matter. After injury to … More the spinal cord, neuropsin mRNA was expressed by cells in the ventral and lateral funiculi. The upregulation was observed at 1-14 days after injury peaking at 4 day. Protease M protein was abundantly expressed in the lower CNS and the expression was colocalized with CNPase immunoreactivity, marker of mature oligodendrocytes. Electron microscopic analysis revealed that myelins were immunoreactive as well as oligodendrocyte cell bodies. After kainic acid injury, the immunoreactivity to protease M in myelin sheaths was stronger. To explore possible substrate for neuropsin, myelin protein was incubated with neuropsin and separated by electrophoresis and immunoblot was performed. The bands corresponding to myelin basic protein (MBP) became weakened after incubation with neuropsin and immunoblot analysis confirmed that these bands were MBP. EAE peaked 3-4 weeks after injection of MOG. With hematoxylin-eosin staining, infiltrating cells were observed subpial regions of the spinal cord. Neuropsin and protease M mRNA was expressed by the cell around the inflammatory regions.These results show the importance of extracellular proteases in physiological and pathological condition of the CNS. Less