Na-K-Cl Contransporter in Cerebral Ischemia

描述（由申请人提供）：过度刺激Na+- k +- 2cl -共转运体异构体1 （NKCC1）活性有助于脑缺血损伤。NKCC1将1Na+、1K+和2Cl-离子转运到细胞内，在生理条件下中枢神经系统细胞内Na+和Cl-、细胞体积和K+摄取的调节中起重要作用。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-过载。细胞内Na+超载随后刺激Na+/Ca2+交换的反向模式操作，并导致延迟，继发性胞质Ca2+上升和内质网和线粒体中的Ca2+失调。最重要的是，无论是药物抑制还是基因消融，NKCC1在体内局灶性缺血模型和体外缺血模型中都显示出显著的神经保护作用。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蛋白在缺血性脑中的刺激机制及其上游调控机制。最近的研究表明，一个新的WNK激酶家族（不含赖氨酸= K）及其两个关键的下游底物SPAK （Ste20/ sps1相关脯氨酸/富含丙氨酸激酶）及其同源物OSR1（氧化应激反应激酶1）通过改变离子转运体的净磷酸化状态而成为进化上保守的调节剂。我们的初步研究表明，短暂局灶性缺血在再灌注6- 72 h时，可引起神经元和梗死周围白质少突胶质细胞中关键蛋白（p-SPAK、p-OSR1和p-NKCC1）的显著刺激。最重要的是，用siRNA或转基因敲除方法抑制WNK-SPAK/OSR1信号通路可防止缺血性细胞死亡。此外，自发性高血压大鼠（SHRs）对NKCC1抑制表现出更高的敏感性。这些新发现使我们推测：1)脑缺血后，WNK-SPAK/OSR1信号通路被激活，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缺血后灰质和白质损伤；3)高血压脑内WNK- SPAK/OSR1-NKCC1信号通路的增强是高血压缺血性脑损伤加重的部分原因。这些假设将在四个具体目标中得到检验。该项目的积极成果将为WNK-SPAK/OSR1-NKCC1信号通路是否是开发更有效的卒中治疗的新靶点提供新的知识。这将为测试未来WNK- SPAK/OSR1在卒中治疗中的新型抑制剂奠定基础。

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.