胚胎小鼠脊髓腹侧中线Nogo-B对交叉后连合轴突的排斥导向作用及其机制

Nogo-B is a member of the reticulon family of protein and expressed widely in the developing central nervous system (CNS), but the function of Nogo-B in the developing CNS is mainly unknown. Our previous studies have showed that in embryonic spinal cord, Nogo is mainly expressed by neurons and radial glial cells and its receptor (NgR) is expressed at the low level in pre-commissural axons but upregulated on post-commissural axons (Wang et al., 2010). Functional studies find that Nogo is involved in guiding commissural axons, but knockout of Nogo-A cannot disturb the pathfinding of commissural axons and optic axons, which indicate that Nogo-A is not involved in axon guidance. Further studies showed Nogo-B is mainly expressed by radial glia in the midline (the floor plate and the optic chiasm) (Wang et al., 2015), which give the hint that Nogo-B might be the isoform involved in midline crossing. In this project, we will investigate the repellent function of midline Nogo-B on post-commissural axons using shRNA to knockdown Nogo-B in vitro and in vivo. Furthermore, how NgR expression is regulated will be also studied. Results of this study will provide insights into the role of Nogo-B in axon guidance, which in turn may improve Nogo related treatments to promote regeneration in diseased and damaged nervous system.

Nogo-B是浆膜蛋白超家族4的成员之一，在发育中枢神经系统中广泛表达，但对其功能知之甚少。我们前期研究发现，胚胎脊髓神经元及放射状胶质细胞表达Nogo，连合轴突交叉前低表达/交叉后高表达NgR，功能研究发现Nogo参与连合轴突导向过程，但Nogo-A基因敲除后轴突导向没有被改变，提示Nogo-A并非轴突导向的关键亚型。进一步研究发现轴突导向中继站（脊髓神经管底板/视交叉中线）放射状胶质细胞主要表达Nogo-B (Wang et al., 2015)，说明B亚型很可能在轴突导向中起重要作用，但尚缺乏足够证据且机制不明。本项目拟利用shRNA体内、体外下调Nogo-B表达，证实其对交叉后连合轴突的排斥导向作用，运用培养的连合神经元研究连合轴突腹侧交叉前后差异表达NgR的分子机制。研究结果将阐明Nogo-B在轴突导向中的作用及机制，对中枢神经损伤后新生轴突与靶组织建立正确连接有重要意义。

Nogo是中枢神经损伤后轴突再生的抑制因子，中枢神经系统主要表达两种Nogo亚型：A和B。这两种亚型在发育中枢神经系统中也有广泛表达，多项研究表明Nogo-A参与神经生长发育的多个过程，但Nogo-B在发育神经系统中的相关功能研究报道甚少。.本课题发现神经管底板表达的Nogo-B可以帮助高表达NgR的脊髓连合轴突离开神经管底板。Nogo-A敲除的胚胎小鼠中，脊髓连合纤维的走向及视神经在视交叉处的导向均未受到影响。缺乏Nogo-A的神经管底板与脊髓组织共培养，抑制交叉后连合轴突的生长作用依然存在。分析发现神经管底板分泌分子量为37 kDa的Nogo-B亚型。用Nogo-A/B shRNA转染脊髓神经管底板发现其对交叉后连合纤维的抑制作用消失。.此外我们发现NogoBR并非是介导Nogo-B在连合轴突导向中的主要受体。NogoBR主要分布于脊髓腹侧神经元以及轴突行走的腹侧索、侧索以及背侧索。此外NogoBR也表达于交叉前后的连合纤维，但表达未见差异。加入重组NogoBR胞外结构域蛋白（NgBR-Ecto）竞争性抑制NogoBR功能，神经管底板依然抑制交叉后连合纤维的生长，“open-book preparation”实验也并未影响连合纤维的导向。.本课题还探讨了脊髓连合轴突交叉前低表达/交叉后高表达NgR的可能分子机制。条件性培养基（FPcm）可以上调体外连合神经元生长锥中的NgR表达水平，此作用可被金属蛋白水解酶抑制剂BB-94模拟。然而神经管底板上调NgR表达水平并非是由Slit、L1CAM、Shh及Nogo等蛋白所调控。此外本课题还发现在视神经通路中，Lingo-1表达具有动态性，提示其有可能协同NgR参与视轴突在视神经盘和视交叉中线处的导向。.本课题明确Nogo-B是引导轴突导向的关键分子，而介导此作用的是NgR而非NogoBR。NgR在连合轴突交叉前低表达是由于金属蛋白酶的剪切，而在交叉后高表达并非由神经管底板特异性表达的相关分子所调控。本研究结果为中枢神经损伤后再生轴突的正确导向提供新的临床途径。