WNK as therapeutic targets for ischemic stroke

 描述(由申请人提供)： 高血压研究的最新突破表明，一个新的WNK激酶家族及其下游两个关键底物SPAK(Ste20/SPS1相关的脯氨酸/富丙氨酸激酶)及其同系物OSR1(氧化应激反应激酶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 在肾盐处理和高血压的发病机制中起重要作用。Na+-K+-2Cl-共转运体1(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+、Cl-、细胞体积和K+摄取具有重要的调节作用。我们实验室和其他实验室以前的研究清楚地表明，NKCC1活性的过度刺激有助于脑缺血损伤。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，无论是药物抑制还是基因消融，NKCC1在大鼠局灶性脑缺血(MCAO)模型和体外脑缺血模型上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。我们的初步研究显示，WNK-Spak/OSR1信号通路在缺血脑中得到了强有力的刺激。本研究的目的是探讨WNK3-SPAK-NKCC1信号通路是否参与缺血性脑损伤，以及用新发现的WNK-SPAK通路抑制剂STOCK1S-50699或转基因敲除WNK3(WNK3KO)或SPAK(SPAKKO)阻断WNK3/SPAK是否具有神经保护作用。我们的初步研究表明，WNK3KO小鼠表现出显著的脑梗塞体积缩小，较少的轴突脱髓鞘，并加速神经行为恢复。这次重新提交的新数据表明，Spak KO小鼠的脑梗塞体积减少了约70%。这些数据为离子转运蛋白及其调节蛋白在缺血性神经胶质损伤中的作用提供了新的见解。该项目的完成将帮助我们确定WNK3-SPAK激酶复合体是否为治疗缺血性脑损伤的新的神经保护策略提供了一个引人注目的靶点。我们的研究将为开发新的WNK-SPAK抑制剂治疗缺血性脑损伤奠定基础。

 DESCRIPTION (provided by applicant): The recent breakthrough in hypertension 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 and play an important role in renal salt handling and in the pathogenesis of hypertension. Na+-K+-2Cl- cotransporter isoform 1 (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 (CNS) under physiological conditions. Previous studies from our lab and others' clearly indicate that over-stimulation of NKCC1 activity contributes to cerebral ischemic damage. 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 (middle cerebral artery occlusion, MCAO) 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. Our pilot study revealed robust stimulation of the WNK-SPAK/OSR1 signaling pathway in ischemic brains. The goal of this project is to investigate whether the cerebral WNK3-SPAK-NKCC1 signaling pathway contributes to ischemic brain damage and whether blocking the WNK3/SPAK kinases with a newly discovered WNK-SPAK pathway inhibitor STOCK1S-50699 or transgenic knockout of WNK3 (WNK3 KO), or SPAK (SPAK KO) is neuroprotective. Our preliminary study shows that WNK3 KO mice exhibited significantly reduced infarct volume, less axonal demyelination, and accelerated neurobehavioral recovery. New data in this resubmission illustrates a ~70% reduction in infarct volume in SPAK KO mice. These data provide fresh insight into the role of ion transporters and their regulatory kinases in ischemic neuroglial injury. Completion of this project will help us to determine whether the WNK3-SPAK kinase complex presents a compelling target for novel neuroprotective strategies for ischemic brain injury. Our study will pave a foundation for developing new WNK-SPAK inhibitors for ischemic brain damage therapy.