Rho激酶信号与神经再生

Central nervous system (CNS) disorders, such as stroke and neurodegenerative diseases, are becoming a serious problem to the human health, in which neurological rehabilitation can offer all patients great help subsequently. Restitution of function by replacement or regeneration of lost neurons has long been regarded as impossible in adult mammalian brain, but it is now accepted that damaged but surviving neurons can re-establish at least some functional connections through processes of axonal and dendritic sprouting and synaptogenesis. Synapse is a basic structure of the nerve regeneration, and is a breakthrough for functional rehabilitation of CNS disorders. The microenvironment within the CNS influences synapse formation and function. It is clear that there exists at least three important synaptic regeneration inhibitory proteins (such as Nogo, MAG and OMgp), which can inhibit the synaptic growth of cultured neurons, in which Nogo protein may present a key element in the nerve regeneration. In addition, glial cells can regulate synapse formation and function under disease or health condition. It is postulated that synaptic connection remains incomplete if without glial cells. . Rho kinase regulates neural cell migration, proliferation and survival, dendritic spine morphology, and axon guidance and regeneration. Rho kinase signaling plays an important role in mediating inhibitory signals to block the inhibition process of axon regeneration in neuron. Numerous studies have demonstrated that abnormal activation of Rho kinas has been involved in many neurological diseases. The inhibition of Rho kinase can promote neuroprotection, synapse formation and functional recovery, displaying a promising drug target for neurological disorders. Therefore, the investigation of cellular and molecular mechanisms between Rho kinase and neurological rehabilitation exhibits theoretical and social significance. .In this issue, we first detect the activity of Rho kinase in serum of different neurodegenerative diseases, observe its effect for the formation of synapses in primary cultured neurons, and utilize Rho kinase inhibitor to improve the generation of synapses. Secondly, we establish several experimental animal models （MCAO,EAE,PD and AD）to explore the activation of Rho kinase and the interpretation with the neurological dysfunction or synaptic plasticity under different pathological conditions. Next, we explore whether disease-related molecules can trigger Rho kinase as upstream signals, and whether Rho kinase inhibitor can improve the formation of synapses as downstream molecules in vitro models. Finally, we try to screen highly selective and effective Rho kinase inhibitor for potential clinical application.

大脑神经功能再生和修复，可通过轴突和树突的发芽和突触的形成重新建立功能连接，突触可塑性研究已成为神经康复领域研究的热点和重点。鉴于Rho激酶信号已成为突触障碍重要的分子事件，因此研究Rho激酶和神经康复具有十分重要的理论和社会意义。本课题,我们检测不同疾病血清中Rho激酶活性,考察其对原代神经元突触形成和功能的影响及Rho激酶抑制剂改善神经突触的效果；利用相应动物模型,探讨不同病理状态下,Rho激酶激活过程在不同脑区和细胞的演绎及与神经功能障碍或神经突触损害的关联，探讨Rho激酶抑制剂改善神经功能，促进神经突触形成和功能的潜能；利用细胞模型,探索不同疾病条件下重要致病分子或细胞是否成为Rho激酶活化的上游信号以及Rho激酶抑制剂改善神经突触形成和功能的下游分子；利用细胞和动物模型筛选具有临床应用前景的高效和高选择性Rho激酶抑制剂，该项研究为大脑神经功能再生和修复提供了技术支撑和实验依据。

Rho激酶（ROCK）是机体普遍存在的一条信号转导通路，其异常激活已涉及包括MS、AD、PD在内的诸多神经系统（NS）变性疾病。神经突触是神经再生的必要结构，是研究NS疾病机制和功能康复的突破口。因此，本课题首先研究了ROCK激活和神经突触抑制。结果显示，Fasudil可抑制CRMP-2、GSK-3β的磷酸化和AMPAR的表达，增强突触素及神经营养因子的表达；C10可抑制ROCK的活性，促进神经元和小胶质细胞突触增长，增加脑内BDNF、GDNF和NT-3的表达。MS患者血清ROCK活性升高，可抑制神经元突触的形成和突触素的表达，增强p-CRMP-2的水平，Fasudil可抑制ROCK的激活和突触的断裂，并促进突触素的表达。.ROCK信号通路是NS疾病很有希望的药物靶点。因此，本课题进一步探讨不同病理状态下ROCK抑制剂的治疗效果。结果显示，C10、WAR-5、FaD-1、C11及Fasudil修饰的免疫细胞可减轻EAE临床症状和病理表现，调节外周免疫应答，改善中枢免疫炎症微环境；MSCs联合Fasudil治疗EAE可发挥协同和叠加的效果。Fasudil可改善AD鼠在认识和记忆方面的缺陷，抑制Aβ、BACE、p-NF-κB/p65、TLR-4、MyD88的表达，减少p-Tau/Ser396细胞数量，促进PSD-95的表达。Fasudil联合NSCs有助于改善PD鼠的焦虑和紧张，减少TH神经元的丢失，抑制胶质细胞在黑质和纹状体的聚集，影响NMDA和AMPA的动态平衡。在OGD实验中，Fasudil可抑制5H5Y-5Y神经元和BV-2小胶质细胞ROCKII、p-MYPT1的表达及5H5Y-5Y神经元PSD-95、synaptophysin的表达。.最后，筛选了高选择性ROCK抑制剂。结果显示，C10对神经元和小胶质细胞毒性最低，细胞活性最佳，并能抵抗较高浓度引起的反应；Fasudil、C10、C12可有效诱导神经元和小胶质细胞突触形成；Fasudil、C10、C13具有较高的ROCK抑制效果。进一步探讨ROCK抑制剂治疗EAE的可能性，结果显示，Fasudil、C6、C11、WAR5对EAE小鼠有很好的治疗效果。