Hwang Jinyeon, Namgung Uk
Department of Korean Medicine, Daejeon University, Daejeon, Korea.
Int Neurourol J. 2020 May;24(Suppl 1):S19-27. doi: 10.5213/inj.2040158.080. Epub 2020 May 31.
The goal of this study is to investigate the role of cyclin-dependent kinase 5 (Cdk5) in axonal regeneration in dorsal root ganglion (DRG) neurons after peripheral nerve injury.
Crush injury was given on the sciatic nerve in rats. The DRG tissues were prepared 1, 3, and 7 days after injury and used for western blotting and immunofluorescence staining experiments. Primary DRG neurons were prepared and treated with Cdk5 inhibitor roscovitine or used for transfections with plasmid constructs. After immunofluorescence staining, neurite length of DRG neurons was analyzed and compared among experimental groups. In addition, roscovitine was injected into the DRG in vivo, and the sciatic nerve after injury was prepared and used for immunofluorescence staining to analyze axonal regeneration in nerve sections.
Levels of Cdk5 and p25 were increased in DRG neurons after sciatic nerve injury (SNI). Levels of S727-p-STAT3, but not Y705-p-STAT3, were increased in the DRG. Immunofluorescence staining revealed that Cdk5 and STAT3 proteins were mostly colocalized in DRG neurons and Y705-p-STAT3 signals were localized within the nucleus area of DRG neurons. A blockade of Cdk5 activity by roscovitine or by transfection with dominant negative Cdk5 (dn-Cdk5) and nonphosphorylatable forms of STAT3 (S727A or Y705F) resulted in significant reductions of the neurite outgrowth of cultured DRG neurons. In vivo administration of roscovitine into the DRG markedly attenuated distal elongation of regenerating axons in the sciatic nerve after injury.
Our study demonstrated that Cdk5 activity induced from DRG neurons after SNI increased phosphorylation of STAT3. The activation of Cdk5-STAT3 pathway may be involved in promoting axonal regeneration in the peripheral nerve after injury.
本研究旨在探讨细胞周期蛋白依赖性激酶5（Cdk5）在周围神经损伤后背根神经节（DRG）神经元轴突再生中的作用。
对大鼠坐骨神经进行挤压损伤。在损伤后1、3和7天制备DRG组织，用于蛋白质免疫印迹和免疫荧光染色实验。制备原代DRG神经元，用Cdk5抑制剂罗可维汀处理或用质粒构建体进行转染。免疫荧光染色后，分析并比较各实验组DRG神经元的神经突长度。此外，将罗可维汀注射到体内的DRG中，制备损伤后的坐骨神经并用于免疫荧光染色，以分析神经切片中的轴突再生情况。
坐骨神经损伤（SNI）后DRG神经元中Cdk5和p25水平升高。DRG中S727-p-STAT3水平升高，但Y705-p-STAT3水平未升高。免疫荧光染色显示，Cdk5和STAT3蛋白在DRG神经元中大多共定位，且Y705-p-STAT3信号定位于DRG神经元的核区域内。用罗可维汀或用显性负性Cdk5（dn-Cdk5）和非磷酸化形式的STAT3（S727A或Y705F）转染阻断Cdk5活性，导致培养的DRG神经元神经突生长显著减少。在体内将罗可维汀注射到DRG中，可显著减弱损伤后坐骨神经中再生轴突的远端延伸。
我们的研究表明，SNI后DRG神经元诱导的Cdk5活性增加了STAT3的磷酸化。Cdk5-STAT3通路的激活可能参与促进损伤后周围神经的轴突再生。
