Agrin在脑卒中后运动促进突触再生中的作用及机制研究

Stroke lead to a significant loss of neurons and synapses, thus the synaptogenesis is critical for recovery of function after stroke. Our previous researches have shown that early exercise after stroke could significantly promoted the recovery of learning and memory, and coordinated locomotor function.The results of our prepare experiment shown that exercise after stroke upregulated the expression of BDNF and synaptophysin in infarct region, which indicated that exercise could promoted the synaptogenesis after stroke. However, the underlying mechanisms are poorly understood.Agrin is a glycoprotein expressed in whole neural system, and is crital for synaptogenesis and stabilization of synapse. Exercise after stroke improve the synaptogenesis, for the underlying mechanisms , we hypothesized that exercise might upregulate the expression of Agrin, active the transcription factor CREB, and then upregualte some protein that improve the synaptogenesis, such as BDNF, promote the recovery of function. To verify the hypothesis,firstly， we test the expression of Agrin after stroke and the effect of exercise on the expression of Agrin，CREB and BDNF；secondly，determine the quantity of synaptogenesis through testing the expression of Syn1 and GAP43；finally，evaluate the function of synaptogenesis through electrophysiology, electron microscope and behavioral methods. Meanwhile, through culturing ischemic neuron by OGD in vitro, we explore the pathway of Agrin→CREB→BDNF→synaptogenesis. Thereafter, we confirm this pathway in vivo through adding recombinant Agrin and antisene-Agrin. Finally, the molecular mechanism for synaptogenesis by early exercise can be demonstrated through these verification research in vivo and vitro.

脑卒中引起大量神经元和突触损失，因此卒中后突触再生对功能恢复意义重大。我们前期研究表明：康复运动能促进卒中后学习记忆和运动功能恢复、上调BDNF和突触素表达，提示运动能促进突触再生，但对其机制尚不清楚。Agrin是一个对突触形成和维持起关键作用的蛋白，我们推测运动促进突触再生可能是通过运动上调Agrin，激活CREB继而上调BDNF表达，增强突触再生。为验证这一假说，拟首先检测卒中后Agrin、CREB和BDNF表达谱及运动对其影响；其次通过检测Syn1、GAP43、免疫荧光和电镜分析突触再生数量；并通过电生理和行为学评估这些突触的功能。同时建立体外神经元缺血模型，通过添加Agrin及Knock down其表达探明缺血后突触再生是否存在Agrin→CREB→BDNF→突出再生通路。最后通过动物实验验证运动是否通过该通路促进突触再生。通过体内外研究阐明脑卒中后运动训练促进突触再生的分子机制。

急性缺血性脑中风破坏神经网络是脑中风后神经功能障碍的关键原因，神经网络的恢复和重建是脑中风治疗的最终目的。中风半影区神经网络的重建主要由缺血半影区内伴有突触损伤的神经元、周围脑组织的完整神经元、动员的内源神经干细胞分化为神经元以及各种胶质细胞和胞外基质参与，在整个过程中突触的重新形成至关重要。本项目的研究发现，大鼠缺血性脑中风后早期进行强度逐步增加的运动康复训练显著促进行为功能恢复、减少梗死体积和抑制脑水肿，并通过western blot、免疫荧光、电镜和电生理技术证实了脑中风早期运动康复训练明显促进了脑梗死半影区内神经突触的数量和质量，并发现突触再生与神经元分泌的聚集蛋白Agrin有密切关系。为进一步探明损伤神经元突触再生的分子机制，建立了体外神经元缺血缺氧模型，通过病毒介导的Agrin过表达证实Agrin可促进缺血缺氧损伤神经元的突触再生，通过抑制CREB可减少Agrin的表达，进而抑制损伤神经元的突触再生。最后通过体内实验证实了运动训练可上调Agrin的表达，进而促进突触再生相关蛋白BDNF和GAP43的表达上调。通过本项目的研究明确了脑中风早期运动训练的神经保护作用，其保护机制与促进突触再生和神经网络重建有关，并初步探明运动训练通过CREB-BDNF通路促进Agrin的表达，进而促进突触再生的分子机制。本项目根据任务书计划，顺利完成了申请书计划研究内容，形成的主要成果有①正式发表相关的学术论文11篇，其中SCI收录8篇，中文核心期刊3篇；②获得授权专利5项；③培养硕士研究生5名；④通过本项目的实施延伸申请新的国家自然科学基金3项，其中本项目负责人主持1项，课题组成员主持2项。