Zhang Jing, Zhang Zheng Gang, Li Yi, Ding Xiaoshuang, Shang Xia, Lu Mei, Elias Stanton B, Chopp Michael
Department of Neurology, Henry Ford Health System, Detroit, MI 48202, USA.
Department of Neurology, Henry Ford Health System, Detroit, MI 48202, USA.
Neurobiol Dis. 2015 Apr;76:57-66. doi: 10.1016/j.nbd.2015.01.006. Epub 2015 Feb 11.
Multiple sclerosis (MS) is a major demyelinating disease of the central nervous system (CNS) leading to functional deficits. The remyelination process is mediated by oligodendrocyte progenitor cells (OPCs). In this study, we tested the hypothesis that Fingolimod, a sphingosine 1-phosphate (S1P) receptor modulator, stimulates OPC differentiation into mature oligodendrocytes, in addition to its well-known anti-inflammatory effect. Using an animal model of MS, experimental autoimmune encephalomyelitis (EAE), we performed a dose-response study of Fingolimod (0.15 or 0.3mg/kg bw), which was initiated on the day of EAE onset. The neurological function was tested to determine the optimal dose of Fingolimod. Immunofluorescent staining was performed to measure the profile of OPC proliferation and differentiation. The mechanistic premise underlying the therapeutic effect of Fingolimod, was that Fingolimod stimulates the sonic hedgehog (Shh) pathway, and this pathway promotes OPC differentiation. To test this hypothesis, a loss-of-function study using cyclopamine, an inhibitor of the sonic hedgehog (Shh) pathway, was employed in vivo. Protein levels of the Shh pathway were measured by Western blot analysis. We found that Fingolimod treatment (0.3mg/kg bw) significantly decreased cumulative disease score compared to the EAE control group. Concurrently, OPCs and proliferation of OPCs were significantly increased in the white matter of the brain and spinal cord at day 7 and day 30 after EAE onset, and oligodendrocytes, myelination and differentiation of OPCs were significantly increased at day 30 compared with the EAE control group. EAE mice treated with Fingolimod exhibited substantially elevated levels of Shh, its receptor Smoothened and effector Gli1 in the white matter of the CNS. However, combination treatment of EAE mice with cyclopamine-Fingolimod decreased Fingolimod monotherapy elevated protein levels of Smoothened and Gli1, and abolished the effect of Fingolimod on OPC proliferation and differentiation, as well as on neurological function outcome. Together, these data demonstrate that Fingolimod is effective as a treatment of EAE by promoting OPC proliferation and differentiation, which facilitate remyelination. In addition, the Shh pathway likely contributes to the therapeutic effects of Fingolimod on OPCs.
多发性硬化症（MS）是一种导致功能缺陷的中枢神经系统（CNS）主要脱髓鞘疾病。髓鞘再生过程由少突胶质前体细胞（OPC）介导。在本研究中，我们验证了如下假设：芬戈莫德作为一种1-磷酸鞘氨醇（S1P）受体调节剂，除了具有其众所周知的抗炎作用外，还能刺激OPC分化为成熟的少突胶质细胞。使用多发性硬化症的动物模型——实验性自身免疫性脑脊髓炎（EAE），我们对芬戈莫德（0.15或0.3mg/kg体重）进行了剂量反应研究，该研究在EAE发病当天开始。测试神经功能以确定芬戈莫德的最佳剂量。进行免疫荧光染色以测量OPC增殖和分化情况。芬戈莫德治疗效果的机制前提是，芬戈莫德刺激音猬因子（Shh）信号通路，且该信号通路促进OPC分化。为验证这一假设，在体内采用了使用环杷明（一种音猬因子（Shh）信号通路抑制剂）的功能丧失研究。通过蛋白质印迹分析测量Shh信号通路的蛋白质水平。我们发现，与EAE对照组相比，芬戈莫德治疗（0.3mg/kg体重）显著降低了累积疾病评分。同时，在EAE发病后第7天和第30天，脑和脊髓白质中的OPC及其增殖显著增加，与EAE对照组相比，在第30天时少突胶质细胞、OPC的髓鞘形成和分化显著增加。用芬戈莫德治疗的EAE小鼠在CNS白质中表现出Shh、其受体Smo和效应器Gli1水平大幅升高。然而，用环杷明-芬戈莫德联合治疗EAE小鼠降低了芬戈莫德单药治疗使Smo和Gli1升高的蛋白质水平，并消除了芬戈莫德对OPC增殖和分化以及对神经功能结果的影响。总之，这些数据表明，芬戈莫德通过促进OPC增殖和分化来有效治疗EAE，这有助于髓鞘再生。此外，Shh信号通路可能有助于芬戈莫德对OPC的治疗作用。
