Na-K-Cl Contransporter in Cerebral Ischemia

描述（由申请方提供）：Na+-K+-2Cl-协同转运蛋白亚型1（NKCC 1）活性的过度刺激可导致脑缺血性损伤。NKCC 1转运1 Na+、1 K+和2Cl-离子进入细胞，并且在生理条件下在中枢神经系统中对细胞内Na+和Cl-、细胞体积和K+摄取的调节中是重要的。在缺血条件下，NKCC 1激活导致星形胶质细胞和神经元中的细胞内Na+和Cl-过载。细胞内Na+超载随后刺激Na+/Ca 2+交换的反向模式操作，并导致延迟的继发性胞质Ca 2+升高和ER和线粒体中的Ca 2+失调。最重要的是，无论是药物抑制或基因消融的NKCC 1显示出显着的神经保护作用，在体内局灶性缺血模型和体外缺血模型。尽管通过阻断NKCC 1活性在缺血性脑损伤中具有神经保护作用，但NKCC 1蛋白在缺血性脑中是如何被激活的以及其上游调节机制仍然未知。 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（Ste 20/SPS 1-related proline/alanine-rich kinase）和它的同源物OSR 1（oxidative stress-responsive kinase 1）是进化上保守的离子转运蛋白调节剂，通过改变它们的净磷酸化状态。我们的初步研究表明，在6- 72小时的再灌注过程中，短暂的局灶性脑缺血触发了脑梗死周围区域神经元和白色少突胶质细胞中关键蛋白（p-SPAK、p-OSR 1和p-NKCC 1）的显著刺激。最重要的是，用siRNA或转基因敲除方法抑制WNK-SPAK/OSR 1信号传导途径可保护免于缺血性细胞死亡。此外，自发性高血压大鼠（SHR）表现出更高的敏感性NKCC 1抑制。这些新的发现使我们推测：1）脑缺血后WNK-SPAK/OSR 1信号通路被激活，并通过蛋白磷酸化作为NKCC 1的上游调节因子发挥作用：2）WNK-SAPK/OSR 1-NKCC 1信号级联反应的激活参与了脑缺血后灰质和白色质的损伤; 3）高血压脑内WNK-SPAK/OSR 1-NKCC 1信号通路的增强是高血压脑缺血性损害加重的部分原因。这些假设将在四个具体目标中进行检验。该项目的积极成果将产生关于WNK-SPAK/OSR 1-NKCC 1信号通路是否是开发更有效的中风治疗的新靶点的新知识。这将为将来WNK-SPAK/OSR 1抑制剂在脑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.