WNK as therapeutic targets for ischemic stroke

 描述（由申请人提供）： 近年来高血压研究的突破性进展表明，一个新的WNK激酶家族[（with-no- lysine（K）]及其两个关键的下游底物SPAK（Ste 20/SPS 1-related proline/alanine-rich kinase））及其同源物OSR 1（oxidative stress-responsive kinase 1）通过改变离子转运蛋白的净磷酸化状态，是进化上保守的离子转运蛋白调节因子 并在肾盐处理和高血压的发病机制中起重要作用。Na ~+-K ~+-2 Cl ~-协同转运蛋白亚型1（NKCC 1）在生理条件下转运1 Na ~+、1 K ~+和2 Cl ~-离子进入细胞，并在调节细胞内Na ~+和Cl ~-、细胞体积和中枢神经系统（CNS）K ~+摄取中起重要作用。我们实验室和其他人的先前研究清楚地表明，过度刺激NKCC 1活性有助于脑缺血性损伤。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NKCC 1显示出显着的神经保护作用，在体内局灶性缺血模型（大脑中动脉闭塞，MCAO）和体外缺血模型。尽管通过阻断NKCC 1活性对缺血性脑损伤具有神经保护作用，但NKCC 1蛋白在缺血性脑中是如何被激活的以及其上游调控机制仍不清楚。 我们的初步研究揭示了缺血性脑中WNK-SPAK/OSR 1信号通路的强刺激。本项目的目的是研究大脑WNK 3-SPAK-NKCC 1信号通路是否有助于缺血性脑损伤，以及用新发现的WNK-SPAK通路抑制剂STOCK 1 S-50699或转基因敲除WNK 3（WNK 3 KO）或SPAK（SPAK KO）阻断WNK 3/SPAK激酶是否具有神经保护作用。我们的初步研究表明，WNK 3基因敲除小鼠表现出显著减少梗死体积，轴突脱髓鞘较少，神经行为恢复加快。此次重新提交的新数据表明SPAK KO小鼠的梗死体积减少约70%。这些数据提供了新的洞察离子转运蛋白和它们的调节激酶在缺血性神经胶质细胞损伤中的作用。该项目的完成将帮助我们确定WNK 3-SPAK激酶复合物是否为缺血性脑损伤的新神经保护策略提供了一个引人注目的靶点。本研究为开发新的WNK-SPAK抑制剂治疗缺血性脑损伤奠定了基础。

 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.