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

项目总结 血管对认知损害和痴呆的贡献(VCID)目前被认为是 导致痴呆症的主要原因。VCID的主要特征是弥漫性白质病变(WML)和 海马区损伤，包括髓鞘丢失、轴突破坏和星形胶质细胞增多症。然而，潜在的 白细胞性白血病、海马区损伤和认知障碍的分子和细胞机制尚不清楚 明白了。高血压和动脉粥样硬化是痴呆流行的最重要的危险因素。vbl.使用 两种慢性双侧颈动脉狭窄(BCAS)的VCID小鼠模型，我们检测到进展 WNK-SPAK-NKCC1蛋白复合体在白质束和海马区的激活，这是 与脑部损伤和认知缺陷有关。脑内Na+-K+-Cl-共转运体1(NKCC1)参与 细胞内Na+、Cl-超载，细胞毒性水肿，兴奋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)]及其下游的Spak(与STE20/SPS1相关) 富含脯氨酸/丙氨酸的激酶)通过蛋白磷酸化激活NKCC1活性。在我们的初步研究中，我们发现 BCAS触发了Spak-NKCC1蛋白复合体的时间依赖性激活，特别是在语料库中 胶体(CC)和海马区GFAP阳性反应星形胶质细胞。WNK-Spak-NKCC1蛋白复合体是怎样的 在BCAS诱导的WML和海马区损伤的发病机制中的作用尚不清楚。 我们检测了BCAS诱导的白介素18受体1(IL-18R1)表达和细胞核的上调 PNF-κB在胶质纤维酸性蛋白阳性反应星形胶质细胞中的移位，与核因子-kB在星形胶质细胞中的募集增加有关 WNK/SPAK/Nkcc1基因启动子。重要的是，用一种新型的选择性Spak抑制剂阻断Spak功能 ZT-1a可显著降低WML、海马CA1区神经变性和认知功能减退 功能受损。因此，我们认为WNK-SPAK-NKCC1复合体的激活在 在BCAS诱导的发病机制中起重要作用。我们假设(1)BCAS引起的低灌流 刺激IL-18/IL-18R-MyD88-NF-κB级联反应上调WNK-SPAK-NKCC1复合体的反应性 (2)星形胶质细胞WNK-Spak-NKCC1信号增强和星形胶质细胞增生症参与了星形胶质细胞的死亡。 少突胶质细胞、脱髓鞘、CA1神经元丢失和认知障碍；以及(3)BCAS术后 给予新型Spak激酶抑制剂ZT-1a可通过以下方式减少脑损伤和认知障碍 减轻星形胶质细胞增生症和OL和CA1神经元变性。这些假设将在 以下是具体目标： 目的1.明确WNK-Spak-NKCC1级联激活和星形胶质细胞增生的分子机制 BCAS术后脑白质束和海马区。 目的2.确定BCAS诱导的脑星形细胞WNK-Spak-NKCC1级联激活的致病作用 损害和认知功能损害。 目的：研究新型SPAK抑制剂ZT-1a在减少星形胶质细胞增生症、脑损伤和脑损伤中的作用。 BCAS小鼠的认知功能损害。

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.