Inhibition of WNK3 Kinase Signaling Reduces Brain Damage and Accelerates Neurological Recovery After Stroke.
Inhibition of WNK3 Kinase Signaling Reduces Brain Damage and Accelerates Neurological Recovery After Stroke.
复制标题
WNK3激酶信号传导的抑制可减少脑损伤并加速中风后神经系统恢复。
DOI：
10.1161/strokeaha.115.008939
复制
发表时间：
2015-07
期刊：
影响因子：
8.3
通讯作者：
Sun D
中科院分区：
文献类型：
--
作者：
Begum G;Yuan H;Kahle KT;Li L;Wang S;Shi Y;Shmukler BE;Yang SS;Lin SH;Alper SL;Sun D
包括WNK3在内的WNK蛋白及其下游相关的Spak和OSR1蛋白，构成了一个重要的信号通路，调节着阳离子-氯离子共转运蛋白。缺血诱导的布美他尼敏感的Na+-K+-Cl-协同转运体(NKCC1)在实验性卒中的病理生理过程中起重要作用，但其调控机制尚不清楚。在这里，我们研究了WNK3-SPAK/OSR1通路作为NKCC1刺激的调节因子及其在缺血性脑损伤中的共同作用。野生型WNK3(WT)和WNK3基因敲除(KO)小鼠采用短暂性大脑中动脉(MCA)闭塞的方法建立缺血性卒中模型。对脑梗塞体积、脑水肿、血脑屏障(BBB)损害、白质脱髓鞘和神经功能缺陷进行评估。免疫印迹和免疫染色检测WNK3和Spak/OSR1的总形式和磷酸化形式。体外培养的神经元和未成熟的少突胶质细胞采用缺氧-葡萄糖剥夺/复氧法进行缺血研究。与WNK3WT小鼠相比，WNK3KO小鼠的脑梗塞体积和轴索脱髓鞘显著减少，脑水肿减轻，神经行为恢复加快。WNK3 KO提供的神经保护表型与Spak/OSR1催化T环和NKCC1刺激位点Thr203/Thr207/Thr212的刺激性过度磷酸化减少以及NKCC1细胞表面表达的减少有关。基因抑制WNK3或Spak/OSR1 siRNA敲除可增加原代培养神经元和少突胶质细胞对体外缺血的耐受性。这些数据证实了WNK3-Spak/OSR1-NKCC1信号通路在缺血性神经胶质细胞损伤中的新作用，并提示WNK3-Spak/OSR1信号通路是缺血性卒中后神经保护的治疗靶点。
WNK kinases, including WNK3, and the associated downstream SPAK and OSR1 kinases, comprise an important signaling cascade that regulates the cation-chloride cotransporters. Ischemia-induced stimulation of the bumetanide-sensitive Na+-K+-Cl- cotransporter (NKCC1) plays an important role in the pathophysiology of experimental stroke, but the mechanism of its regulation in this context is unknown. Here, we investigated the WNK3-SPAK/OSR1 pathway as a regulator of NKCC1 stimulation and their collective role in ischemic brain damage. Wild-type WNK3 (WT) and WNK3 knockout (KO) mice were subjected to ischemic stroke via transient middle cerebral artery (MCA) occlusion. Infarct volume, brain edema, blood brain barrier (BBB) damage, white matter demyelination, and neurological deficits were assessed. Total and phosphorylated forms of WNK3 and SPAK/OSR1 were assayed by immunobloting and immunostaining. In vitro ischemia studies in cultured neurons and immature oligodendrocytes were conducted using the oxygen-glucose deprivation/reoxygenation method. WNK3 KO mice exhibited significantly decreased infarct volume and axonal demyelination, less cerebral edema, and accelerated neurobehavioral recovery compared to WNK3 WT mice subjected to MCA occlusion. The neuroprotective phenotypes conferred by WNK3 KO were associated with a decrease in stimulatory hyper-phosphorylations of the SPAK/OSR1 catalytic T-loop and of NKCC1 stimulatory sites Thr203/Thr207/Thr212, as well as with decreased cell surface expression of NKCC1. Genetic inhibition of WNK3 or siRNA knockdown of SPAK/OSR1 increased the tolerance of cultured primary neurons and oligodendrocytes to in vitro ischemia. These data identify a novel role for the WNK3-SPAK/OSR1-NKCC1 signaling pathway in ischemic neuroglial injury, and suggest the WNK3-SPAK/OSR1 kinase pathway as a therapeutic target for neuroprotection following ischemic stroke.