转录因子Fezf2重编程皮层投射神经元重建脑梗死小鼠神经网络的研究

Neuronal regeneration and motor function reconstruction after ischemic infarction are hot spots of present research. In our previous study, we found out that multiple damaging mechanisms hindered rehabilitation process in the infarcted hemisphere. However, remodeling of the contralesional hemisphere accompanied by reconnecting the disconnected fibers, are correlated to motor function recovery. Lineage reprogramming, which can be defined as direct conversion of specific differentiated cell type into another lineage, has been a potential approach for producing neurons. However, whether reprogramming neurons could contribute to functional recovery after ischemic infarction has not been reported. Therefore, we plan to apply a CaMKIIα-CreER transgenic mouse model of ischemic infarction, and use direct reprogramming of callosal neurons into coticofugal neurons by force expression of Fezf2, which is an intrinsic master gene capable of instructing identity of corticospinal motor neurons. The properties of induced corticospinal motor neurons will be determined by using a combination of morphological, molecular, eletrophysiological and ultrastuctural imaging approaches. To test whether induced corticospinal motor neurons could reshape the local neocortical microcircuit, patch clamp recordings will be performed. By investigating the underlying molecular mechanisms, our study may provide an experimental basis for a novel interventional target and a promising approach to cure ischemic infarction.

如何促进脑梗死后运动功能的重建是目前临床治疗的难点。本课题组前期研究发现，脑梗死后患侧皮层受多重组织损害机制影响，严重影响了神经功能恢复，而健侧运动皮层神经重塑重建了上下运动神经元之间的联系并促进运动功能恢复，因此被认为可能是个潜在的干预靶点。关键转录因子过表达可在体内重编程神经元，但能否通过重编程的方式增加梗死后健侧皮层皮质脊髓运动神经元的数量，实现神经网络重建和功能代偿目前尚不清楚。本项目拟采用CaMKIIα-CreER转基因小鼠建立脑梗死模型，用AAV腺相关病毒构建调控皮质脊髓运动神经元分化的关键因子Fezf2表达载体，将Ⅱ/Ⅲ层胼胝体投射神经元重编程为皮质脊髓运动神经元，鉴定重编程神经元的形态学、表观遗传学和电生理特性并计算出转化率，解析其能否诱导中间神经元构建新的神经网络，评估其对神经功能症状的改善效果，进而探讨其可能的分子机制，为寻找脑梗死治疗新靶点和新策略提供实验依据。

脑梗死后的神经元再生和运动功能重建是目前脑卒中研究领域的热点。我们前期研究已经发现，脑梗死后受损侧皮层受到多重组织损害机制的影响，这严重阻碍神经功能恢复，而健侧运动皮层神经重塑重建了上下运动神经元之间的联系并促进运动功能恢复，可能是有潜力的干预靶点。前人研究表明，关键转录因子的过表达可以在体内重编程神经元，但这种方式能否应用于脑梗死后修复重建尚未见报道。我们选择能定时使病毒特异性感染皮层兴奋性神经元细胞的转基因工具鼠 CaMKIIα-CreER，利用内皮素颅内注射建立稳定的运动皮层脑梗死小鼠模型，并观察脑梗死小鼠大脑健侧皮层神经元的重塑情况。通过在脑梗死小鼠健侧皮层植入转录因子 Fezf2 将健侧皮层胼胝体投射神经元重编程为皮质脊髓投射神经元，观察脑梗死小鼠的运动功能恢复情况。此项目目前主要完成的内容包括：1. 脑梗死健侧皮层神经元可塑性检测；2.脑梗死小鼠健侧皮层投射神经元重编程改变；3.行为学实验评估脑梗死小鼠运动恢复情况。实验主要结果： 脑梗死后健侧皮层的 GAP-43 表达升，表明了神经元具有可塑性；脑梗死大脑健侧皮层注射过表达病毒 Fezf2 后，皮质脊髓投射神经元标记物 CRYM 的表达表达增多；在行为学实验中，网格错步实验观察到脑梗死健侧皮层注射病毒后第 29 天开始腺相关病毒 Fezf2 过表达组小鼠患侧上肢的错步率较腺相关病毒空载对照组小。