Tucker Budd A, Rahimtula Masuma, Mearow Karen M
Schepens Eye Institute, Harvard University, Boston, MA, United States.
Cell Signal. 2008 Jan;20(1):241-57. doi: 10.1016/j.cellsig.2007.10.014. Epub 2007 Oct 18.
Axonal regeneration is influenced by factors in the extracellular environment, including neurotrophins, such as NGF, and adhesion molecules, such as laminin. The provision of both NGF and a permissive substrate to cultured adult NGF-responsive DRG neurons results in enhanced levels of neurite growth not achievable by either factor alone. In this study, we have investigated the early signalling events that contribute to NGF and laminin-induced neurite growth. Adult NGF-responsive DRG neurons were plated on poly-d-lysine for 2 h then stimulated with NGF, laminin, or laminin plus NGF for 10 min, 1 h, or 6 h. Signalling pathways were subsequently analysed using Western blotting and pharmacological inhibition of specific signalling components. While activation of the various signalling intermediates (Src, FAK, Akt, MAPK) could be detected as early as 10 min-1 h after stimulation, significant neurite growth was observed mainly at the 6 h time point. The results of the time course experiments showed differential activation of the signalling intermediates. Src was activated by all treatments (NGF, laminin and the combination) at the earliest time point analysed, 10 min. NGF stimulation also resulted in detectable activation of FAK, Akt and MAPK by 10 min. However, laminin stimulation alone did not result in detectable activation of FAK, Akt or MAPK until the 1 h time point. Inhibition of either Src or FAK activity attenuated both laminin and/or NGF-induced PI 3-K/Akt and MEK/MAPK signalling pathways, as well as neurite growth. Downstream inhibition of Akt by Akt knockdown also blocked observed neurite growth, while inhibition of MEK/MAPK had no significant effect. Together, these results demonstrate that signalling underlying neurite growth can be detected within minutes of stimulation and provide a mechanism for the observed enhancement of neurite growth when both NGF and the permissive substrate, laminin, are provided.
轴突再生受细胞外环境中的多种因素影响，包括神经营养因子（如神经生长因子，NGF）和黏附分子（如层粘连蛋白）。为培养的成年NGF反应性背根神经节（DRG）神经元同时提供NGF和一个允许性底物，可导致神经突生长水平增强，这是单独任何一种因子都无法实现的。在本研究中，我们调查了促成NGF和层粘连蛋白诱导神经突生长的早期信号事件。将成年NGF反应性DRG神经元接种在聚-D-赖氨酸上2小时，然后用NGF、层粘连蛋白或层粘连蛋白加NGF刺激10分钟、1小时或6小时。随后使用蛋白质免疫印迹法和对特定信号成分的药理学抑制来分析信号通路。虽然在刺激后10分钟至1小时内即可检测到各种信号中间体（Src、黏着斑激酶，FAK、蛋白激酶B，Akt、丝裂原活化蛋白激酶，MAPK）的激活，但显著的神经突生长主要在6小时时间点观察到。时间进程实验结果显示信号中间体的激活存在差异。在最早分析的时间点，即10分钟时，所有处理（NGF、层粘连蛋白及其组合）均激活了Src。NGF刺激在10分钟时也导致可检测到的FAK、Akt和MAPK激活。然而，单独的层粘连蛋白刺激直到1小时时间点才导致可检测到的FAK、Akt或MAPK激活。抑制Src或FAK活性会减弱层粘连蛋白和/或NGF诱导的PI 3-K/Akt和MEK/MAPK信号通路以及神经突生长。通过敲低Akt对Akt进行下游抑制也会阻断观察到的神经突生长，而抑制MEK/MAPK则没有显著影响。总之，这些结果表明，在刺激后数分钟内即可检测到神经突生长的信号，并为当同时提供NGF和允许性底物层粘连蛋白时观察到的神经突生长增强提供了一种机制。
