Astrocytic WNK-SPAK-NKCC1 Cascade in White Matter Astrogliosis and Injury

项目摘要 血管对认知障碍和痴呆（VCID）的贡献目前被认为是其中一个 痴呆症的主要病因VCID的主要特征是弥漫性白色脑膜病变（WML）， 海马损伤，包括髓鞘丢失、轴突破坏和星形胶质细胞增生。然而，底层 WML、海马损伤和认知障碍的分子和细胞机制尚不清楚 明白高血压和动脉粥样硬化是痴呆症流行的最重要的危险因素。使用 在两种慢性双侧颈动脉狭窄（BCAS）的VCID小鼠模型中，我们检测到进行性 白色物质束和海马中WNK-SPAK-NKCC 1蛋白复合物的激活， 与脑损伤和认知缺陷有关。脑Na+-K+-Cl-协同转运蛋白亚型1（NKCC 1） 细胞内Na+和Cl-超负荷、细胞毒性水肿和兴奋毒性缺血性神经元损伤。丝氨酸- 苏氨酸WNK激酶家族[不含赖氨酸（K）]及其下游激酶SPAK（STE 20/SPS 1相关的 脯氨酸/富含丙氨酸的激酶）通过蛋白质磷酸化激活NKCC 1活性。在我们的初步研究中，我们发现 BCAS触发SPAK-NKCC 1蛋白复合物的时间依赖性激活，特别是在语料库中， 胼胝体（CC）和海马GFAP+反应性星形胶质细胞。WNK-SPAK-NKCC 1蛋白复合物是如何 其在BCAS诱导的WML和海马损伤的发病机制中的作用尚不清楚。 我们检测到BCAS诱导的白细胞介素-18受体1（IL-18 R1）表达升高， pNF-κB在GFAP+反应性星形胶质细胞中的易位，这与NF-κ B募集增加相关。 Wnk/Spak/Nkcc 1基因启动子。重要的是，用一种新的选择性SPAK抑制剂阻断SPAK功能 ZT-1a显著降低WML、海马CA 1区神经变性和减弱认知功能 功能障碍因此，我们认为WNK-SPAK-NKCC 1复合物的激活在调节细胞凋亡中起着重要作用。 在BCAS诱导的发病机制中起重要作用。我们假设（1）BCAS诱导的低灌注 刺激IL-18/IL-18 R-MyD 88-NF-κB级联反应，以上调WNK-SPAK-NKCC 1复合物的反应性表达。 （2）星形胶质细胞WNK-SPAK-NKCC 1信号传导和星形胶质细胞增生的升高导致星形胶质细胞的细胞死亡。 少突胶质细胞（OL）、脱髓鞘、CA 1神经元丢失和认知缺陷;和（3）BCAS后 新型SPAK激酶抑制剂ZT-1a的给药通过以下方式减少脑部病变和认知缺陷 减轻星形胶质细胞增生和OLs和CA 1神经元变性。这些假设将在 具体目标如下： 目标1.确定刺激WNK-SPAK-NKCC 1级联激活和星形胶质细胞增生的分子机制 BCAS后的白色物质束和海马。 目标2.确定BCAS诱导的星形胶质细胞WNK-SPAK-NKCC 1级联激活在脑中的致病作用 损害和认知功能损害。 目标3.研究新型SPAK抑制剂ZT-1a在减少星形胶质细胞增生、脑损伤和 BCAS小鼠的认知功能障碍。

PROJECT SUMMARY Vascular contributions to cognitive impairment and dementia (VCID) are currently considered as one of the leading causes of dementing illness. The key feature of VCID is diffuse white mater lesions (WML) and hippocampal damage, including myelin loss, axonal disruption, and astrogliosis. However, the underlying molecular and cellular mechanisms for WML, hippocampal damage, and cognitive impairment are not well understood. Hypertension and atherosclerosis are the most significant risk factors for dementia epidemics. Using two mouse models of VCID with chronic bilateral carotid artery stenosis (BCAS), we detected progressive activation of the WNK-SPAK-NKCC1 protein complex in white matter tracts and hippocampus, which is associated with brain lesion and cognitive deficits. Brain Na+-K+-Cl- cotransporter isoform 1 (NKCC1) contributes to intracellular Na+ and Cl- overload, cytotoxic edema, and excitotoxic ischemic neuronal damage. The serine- threonine WNK kinase family [with no lysine (K)], and its downstream kinase SPAK (the STE20/SPS1-related proline/alanine-rich kinase) activate NKCC1 activity via protein phosphorylation. In our pilot study, we detected that BCAS triggered a time-dependent activation of the SPAK-NKCC1 protein complex, specifically in corpus collosum (CC) and hippocampal GFAP+ reactive astrocytes. How the WNK-SPAK-NKCC1 protein complex is stimulated and its role in the pathogenesis of BCAS-induced WML and hippocampal lesion remains unknown. We detected BCAS-induced elevation of interleukin-18 receptor 1 (IL-18R1) expression and nuclear translocation of pNF-κB in GFAP+ reactive astrocytes, which are correlated with increased NF-kB recruitment on the Wnk/Spak/Nkcc1 gene promoters. Importantly, blocking SPAK function with a novel, selective SPAK inhibitor ZT-1a significantly reduced WML, hippocampal CA1 region neurodegeneration, and attenuated cognitive function impairment. Therefore, we propose that activation of the WNK-SPAK-NKCC1 complex plays an important role in the BCAS-induced pathogenesis. We hypothesize that (1) BCAS-induced hypoperfusion stimulates the IL-18/IL-18R-MyD88-NF-κB cascade to upregulate the WNK-SPAK-NKCC1 complex in reactive astrocytes; (2) elevated astrocytic WNK-SPAK-NKCC1 signaling and astrogliosis contribute to cell death of oligodendrocytes (OLs), demyelination, CA1 neuronal loss, and cognitive deficits; and (3) Post-BCAS administration of the novel SPAK kinase inhibitor ZT-1a reduces brain lesions and cognitive deficits by attenuating astrogliosis and degeneration of OLs and CA1 neurons. These hypotheses will be tested in the following specific aims: Aim 1. Identify molecular mechanisms that stimulate WNK-SPAK-NKCC1 cascade activation and astrogliosis in white matter tracts and hippocampus after BCAS. Aim 2. Determine causative roles of BCAS-induced astrocytic WNK-SPAK-NKCC1 cascade activation in brain lesion and cognitive function impairment. Aim 3. Investigate the efficacy of the novel SPAK inhibitor ZT-1a in reducing astrogliosis, brain lesion, and cognitive function impairment in the BCAS mice.