Luo Tianfei, Roman Philip, Liu Chunli, Sun Xin, Park Yujung, Hu Bingren
Neurochemistry Laboratory of Brain Injury, Shock Trauma & Anesthesiology Research Center, University of Maryland School of Medicine, Baltimore, MD 21201, United States; Department of Neurology, The First Teaching Hospital, Jilin University, Changchun, China.
Neurochemistry Laboratory of Brain Injury, Shock Trauma & Anesthesiology Research Center, University of Maryland School of Medicine, Baltimore, MD 21201, United States.
Exp Neurol. 2015 Jan;263:306-13. doi: 10.1016/j.expneurol.2014.10.016. Epub 2014 Oct 29.
The microtubule-dependent GEF-H1 pathway controls synaptic re-networking and overall gene expression via regulating cytoskeleton dynamics. Understanding this pathway after ischemia is essential to developing new therapies for neuronal function recovery. However, how the GEF-H1 pathway is regulated following transient cerebral ischemia remains unknown. This study employed a rat model of transient forebrain ischemia to investigate alterations of the GEF-H1 pathway using Western blotting, confocal and electron microscopy, dephosphorylation analysis, and pull-down assay. The GEF-H1 activity was significantly upregulated by: (i) dephosphorylation and (ii) translocation to synaptic membrane and nuclear structures during the early phase of reperfusion. GEF-H1 protein was then downregulated in the brain regions where neurons were destined to undergo delayed neuronal death, but markedly upregulated in neurons that were resistant to the same episode of cerebral ischemia. Consistently, GTP-RhoA, a GEF-H1 substrate, was significantly upregulated after brain ischemia. Electron microscopy further showed that neuronal microtubules were persistently depolymerized in the brain region where GEF-H1 protein was downregulated after brain ischemia. The results demonstrate that the GEF-H1 activity is significantly upregulated in both vulnerable and resistant brain regions in the early phase of reperfusion. However, GEF-H1 protein is downregulated in the vulnerable neurons but upregulated in the ischemic resistant neurons during the recovery phase after ischemia. The initial upregulation of GEF-H1 activity may contribute to excitotoxicity, whereas the late upregulation of GEF-H1 protein may promote neuroplasticity after brain ischemia.
微管依赖性鸟嘌呤核苷酸交换因子H1（GEF-H1）通路通过调节细胞骨架动力学来控制突触重新联网和整体基因表达。了解缺血后的这一通路对于开发恢复神经元功能的新疗法至关重要。然而，短暂性脑缺血后GEF-H1通路是如何被调节的仍不清楚。本研究采用短暂性前脑缺血大鼠模型，运用蛋白质免疫印迹法、共聚焦和电子显微镜技术、去磷酸化分析以及下拉分析来研究GEF-H1通路的变化。在再灌注早期，GEF-H1活性通过以下方式显著上调：（i）去磷酸化；（ii）转位至突触膜和核结构。随后，在注定要发生迟发性神经元死亡的脑区，GEF-H1蛋白下调，但在对同一脑缺血事件具有抗性的神经元中显著上调。同样，GEF-H1的底物GTP- RhoA在脑缺血后也显著上调。电子显微镜进一步显示，在脑缺血后GEF-H1蛋白下调的脑区，神经元微管持续解聚。结果表明，在再灌注早期，GEF-H1活性在易损和抗性脑区均显著上调。然而，在缺血后恢复阶段，GEF-H1蛋白在易损神经元中下调，而在缺血抗性神经元中上调。GEF-H1活性的最初上调可能导致兴奋性毒性，而GEF-H1蛋白的后期上调可能促进脑缺血后的神经可塑性。
