GSK3β-MOB2-NDR2轴调控神经元轴突伸长修复脊髓损伤的机制研究

How to effectively repair spinal cord injury (SCI) is a hotspot and difficult point in the field of neuroscience. We found that MOB2, as a new neuronal factor, was involved in neural regeneration, which was closely related to PI3K/Akt and NDR2 signaling pathways. However, the role of MOB2 in repairing SCI is currently unclear. Based on literature research and pre-research results, we hypothesized that GSK3β couldn phosphorylate MOB2 at Ser75 site, and MOB2 was degraded by ubiquitination system. So we will inhibit this site, stabilize MOB2 protein in neurons, activate NDR2 kinase, and promote axonal elongation of neuron via increasing the level and spatial distribution of axon skeleton protein microfilaments, and ultimately promote the recovery of SCIc. Therefore, this project will estabish in vitro and in vivo experiments to focus on : (1) the mechanism of MOB2 promoting neuronal axon elongation ; (2) the mechanism of GSK3β regulating the stabilization of MOB2 protein ; (3) the role of GSK3β-MOB2-NDR2 axis in regulation of axonal elongation after SCI . The aim of this grant is to uncover the role and mechanism of GSK3β-MOB2-NDR2 axis in regulation of repair SCI via promoting neuronal axon elongation, and further provide a theoretical basis for drug development for clinical treatment of SCI.

如何有效地修复脊髓损伤是神经科学领域的研究热点和难点。研究发现MOB2作为新的神经因子参与到神经再生，与PI3K/Akt和NDR2信号通路密切相关。然而，MOB2在脊髓损伤中修复作用目前不明确。根据文献调研和前期的预研结果，我们推测GSK3β能够使MOB2 Ser75位点磷酸化，使MOB2经泛素化途径降解，故抑制该位点，稳定神经元中MOB2蛋白，激活NDR2激酶，改变神经元轴突骨架蛋白微丝的表达和空间分布，促进神经元轴突的伸长，最终促进脊髓损伤恢复。因此，本项目拟通过体内外实验重点分析：（1）MOB2促进神经元轴突伸长机制；（2）GSK3β调控MOB2蛋白稳定机制；（3）GSK3β-MOB2-NDR2轴驱动脊髓损伤神经元轴突伸长作用。本研究在理论上将阐明GSK3β-MOB2-NDR2轴调控神经元轴突伸长修复脊髓损伤的作用及机制，并进一步为临床治疗脊髓损伤药物研发提供理论依据。

寻找脊髓损伤后促进神经元轴突伸长的有效基因一直是本课题组研究的重点方向。本项目首次发现MOB2作为新的神经元轴突伸长因子参与到小鼠脊髓损伤后神经再生过程，研究发现MOB2在小鼠正常脊髓和损伤脊髓内均主要表达在神经元细胞内。体内外实验均证实MOB2能够促进小鼠脊髓损伤后神经元轴突伸长、促进损伤组织结构恢复和促进小鼠后肢运动功能恢复。进一步发现MOB2和其下游的NDR2蛋白表达水平在小鼠大脑和脊髓发育过程、小鼠脊髓损伤过程中均成正相关性，且两者在正常和损伤脊髓的神经元内均富集表达，并在脊髓损伤后两者表达量明显增加。在机制方面，研究发现在体外培养的神经元中MOB2蛋白能够与NDR2蛋白发生结合作用，并使NDR2蛋白磷酸化，从而减少NDR2经蛋白泛素化途径降解，使其稳定在神经元细胞内，从而发挥促进神经元轴突伸长作用。我们进一步研究发现NDR2可能通过影响神经元轴突的微管蛋白β3-tubulin的表达来调控神经元轴突伸长作用。在体内实验，敲减损伤区域的NDR2表达水平能够逆转过表达MOB2促进小鼠脊髓损伤后神经元轴突伸长、促进损伤组织结构恢复和促进小鼠后肢运动功能恢复的作用。为了进一步明确MOB2在体内神经元中作用，本课题组采用Cre/Loxp系统已经成功构建MOB2条件性敲除小鼠（B6/JGpt-Mob2em1Cflox/Gpt），目前正常与神经元特异性标记Synapsin蛋白的Cre的工具鼠（H11-Synapsin-iCre-polyA）进行了杂交，为最终得到在神经元中特异性敲除MOB2的转基因小鼠（Mob2-/-;Synapsin-Cre）并在后续实验中进一步开展。综上我们初步研究已经明确MOB2促进小鼠脊髓损伤后神经元轴突伸长的作用及机制，为进一步探讨其在神经损伤内源性神经修复中的作用及靶向药物的开发提供依据。