Spastin Interacts with CRMP2 to Regulate Neurite Outgrowth by Controlling Microtubule Dynamics through Phosphorylation Modifications
Spastin Interacts with CRMP2 to Regulate Neurite Outgrowth by Controlling Microtubule Dynamics through Phosphorylation Modifications
复制标题
Spastin 与 CRMP2 相互作用，通过磷酸化修饰控制微管动力学来调节神经突生长
DOI：
10.2174/1871527319666201026165855
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发表时间：
2021-01-01
影响因子：
3
通讯作者：
Guo, Guoqing
中科院分区：
文献类型：
--
作者：
Li, Sumei;Zhang, Jifeng;Guo, Guoqing
目的：我们的工作旨在揭示神经元发育过程中神经突生长的潜在微管机制，并为神经损伤后神经网络连接重建提供可行的干预途径。背景：微管聚合和切断是神经突生长和分支形成的基础。然而，微管动态不稳定性背后的机制尚不清楚。在这里，我们发现由崩溃反应介质蛋白2 （CRMP2）介导的神经突生长可以被痉挛蛋白增强，这对微管细胞骨架的切断有影响。目的：探讨CRMP2与痉挛素的协同作用是否介导神经突生长。方法：将海马神经元体外培养于24孔培养板中，培养4天后进行转染。用磷酸钙转染CRMP2和spastin构建体及其控制。通过拉下、CoIP和免疫荧光共定位检测CRMP2和spastin之间的相互作用。免疫染色测定神经突形态。结果：我们首次证明CRMP2与spastin相互作用，促进神经突生长和分支形成。然后我们的研究结果发现，CRMP2通过其c端与spastin的微管结合域相互作用，删除这些结合位点会抑制神经突的生长和分支的形成。此外，我们在海马神经元的spastin的S210处确认了一个磷酸化位点。S210位点的Spastin磷酸化不能改变CRMP2的结合亲和力，但抑制了其与微管的结合。进一步的研究表明，S210位点的spastin磷酸化通过下调微管切断活性来抑制CRMP2和spastin相互作用诱导的神经突生长。结论：综上所述，我们的数据表明CRMP2和spastin的相互作用以及spastin的微管切断活性都是神经突起生长和分支形成所必需的。
Aims: Our work aims to revealing the underlying microtubule mechanism of neurites outgrowth during neuronal development and also proposes a feasible intervention pathway for reconstructing neural network connections after nerve injury.Background: Microtubule polymerization and severing form the basis for neurite outgrowth and branch formation. However, the mechanisms that underlie the dynamic instability of microtubules are unclear. Here, we showed that neurite outgrowth mediated by collapsing response mediator protein 2 (CRMP2) can be enhanced by spastin, which had an effect on the severing of microtubule cytoskeleton.Objective: To explore whether neurite outgrowth was mediated by coordination of CRMP2 and spastin.Methods: Hippocampal neurons were cultured in vitro in 24-well culture plates for 4 days before being used to perform the transfection. Calcium phosphate was used to transfect the CRMP2 and spastin constructs and their control into the neurons. An interaction between CRMP2 and spastin was examined by using pull down, CoIP and immunofluorescence colocalization assays. And immunostaining was also performed to determine the morphology of neurites.Results: We first demonstrated that CRMP2 interacted with spastin to promote neurite outgrowth and branch formation. Then our results identified that CRMP2 interacted with the microtubule-binding domain of spastin via its C-terminus, and deleting these binding sites inhibited neurite outgrowth and branch formation. In addition, we confirmed one phosphorylation site at S210 of spastin in hippocampal neurons. Spastin phosphorylation at S210 failed to alter the binding affinity of CRMP2 but inhibited its binding to microtubules. Further study showed that phosphorylation spastin at S210 inhibited the neurite outgrowth induced by CRMP2 and spastin interaction through downregulation of microtubule-severing activity.Conclusion: Taken together, our data demonstrated that both CRMP2 and spastin interaction and the microtubule-severing activity of spastin were required for neurite outgrowth and branch formation.