Na-K-Cl Contransporter in Cerebral Ischemia

描述(申请人提供)：过度刺激Na+-K+-2Cl-辅酶转运体1(NKCC1)活性导致脑缺血损伤。NKCC1将1Na+、1K+和2Cl-离子转运到细胞内，在生理条件下对细胞内Na+和Cl-、细胞体积和K+摄取具有重要的调节作用。在缺血条件下，NKCC1的激活导致星形胶质细胞和神经元细胞内Na+和Cl-超载。细胞内Na+超载随后刺激Na+/Ca~(2+)交换的反向模式操作，导致内质网和线粒体延迟的继发性胞浆Ca~(2+)升高和钙调节失调。更重要的是，无论是在体局灶性脑缺血模型还是在体外脑缺血模型中，NKCC1的药物抑制或基因消融都显示出明显的神经保护作用。尽管阻断NKCC1活性在缺血性脑损伤中具有神经保护作用，但目前尚不清楚NKCC1蛋白在缺血脑中是如何被刺激的，以及其上游调控机制是什么。最近的研究表明，一个新的WNK激酶家族(没有赖氨酸=K)及其两个关键的下游底物SPAK(Ste20/SPS1相关的脯氨酸/富含丙氨酸的激酶)及其同系物OSR1(氧化应激反应蛋白1)是通过改变其净磷酸化状态而在进化上保守的离子转运体调节因子。我们的初步研究表明，短暂性局灶性脑缺血在再灌流6-72小时内可显著刺激脑梗塞周围区神经元和白质少突胶质细胞中的关键蛋白(p-SPAK、p-OSR1和p-NKCC1)。最重要的是，用siRNA或转基因敲除方法抑制WNK-Spak/OSR1信号通路可以保护缺血细胞的死亡。此外，自发性高血压大鼠(SHR)对NKCC1抑制表现出更高的敏感性。这些新的发现使我们推测：1)WNK-SPAK/OSR1信号通路在脑缺血后被激活，并通过蛋白磷酸化作为NKCC1的上游调节因子；2)WNK-SAPK/OSR1-NKCC1信号级联的激活参与了脑缺血后灰质和白质的损伤；3)WNK-SPAK/OSR1-NKCC1信号通路的增强在一定程度上导致了高血压脑损伤的加重。这些假设将在四个具体目标上得到检验。该项目的积极成果将产生关于WNK-SPAK/OSR1-NKCC1信号通路是否为开发更有效的中风治疗的新靶点的新知识。这将为未来WNK-Spak/OSR1的新型抑制剂在卒中治疗中的应用奠定基础。

DESCRIPTION (provided by applicant): Over-stimulation of Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) activity contributes to cerebral ischemic damage. NKCC1 transports 1Na+, 1K+, and 2Cl- ions into cells and is important in regulation of intracellular Na+ and Cl-, cell volume, and K+ uptake in the central nervous system under physiological conditions. Under ischemic conditions, NKCC1 activation causes intracellular Na+ and Cl- overload in astrocytes and neurons. The intracellular Na+ overload subsequently stimulates the reverse mode operation of Na+/Ca2+ exchange and leads to a delayed, secondary cytosolic Ca2+ rise and Ca2+ dysregulation in ER and mitochondria. Most importantly, either pharmacological inhibition or genetic ablation of NKCC1 shows significant neuroprotective effects in in vivo focal ischemia model and in vitro ischemia model. Despite of the neuroprotective effects in ischemic brain damage by blocking NKCC1 activity, it remains unknown how NKCC1 protein is stimulated in ischemic brains and what are the up- stream regulatory mechanisms. The recent research reveals that a novel WNK kinase family (with no lysine = K) and its two key down-stream substrates SPAK (Ste20/SPS1-related proline/alanine-rich kinase) and its homolog OSR1 (oxidative stress-responsive kinase 1) are evolutionarily conserved regulators of ion transporters by altering their net phosphorylation state. Our preliminary study shows that triansient focal ischemia triggered a significant stimulation of the key proteins (p-SPAK, p-OSR1 and p-NKCC1) in neurons and in white matter oligodendrocytes of peri-infarct regions during 6- 72 h reperfusion. Most importantly, inhibition of the WNK-SPAK/OSR1 signaling pathway with siRNA or transgenic knockout approaches is protective against ischemic cell death. In addition, spontaneously hypertensive rats (SHRs) exhibited higher sensitivity to NKCC1 inhibition. These new findings led us to hypothesize that: 1) the WNK-SPAK/OSR1 signaling pathway is activated following cerebral ischemia and functions as up-stream regulators of NKCC1 through protein phosphorylation; 2) the activation of the WNK-SAPK/OSR1-NKCC1 signaling cascade contributes to both grey and white matter damage after ischemia; 3) augmentation of the WNK- SPAK/OSR1-NKCC1 signaling pathway in hypertensive brains is in part responsible for the worsened ischemic brain damage in hypertension. These hypotheses will be tested in four Specific Aims. A positive outcome of this project will generate new knowledge on whether the WNK-SPAK/OSR1-NKCC1 signaling pathway is a novel target for developing more effective stroke therapy. This will pave a foundation for testing future novel inhibitors of WNK- SPAK/OSR1 in stroke therapy.