Qi Chenxing, Wang Yeping, Li Xuhang, Zheng Cheng, Gu Yi, Hu Junxiao, Qiu Yiming, Xie Guomin, Xu Shujun, Zheng Yuyin, Lv Zhongyue, Zheng Wu
Department of Neurology, Ningbo Medical Center Lihuili Hospital, Ningbo University, Ningbo, Zhejiang, 315040, China.
Rehabilitation Medicine Center, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, 325027, China.
J Neuroinflammation. 2025 Mar 13;22(1):80. doi: 10.1186/s12974-025-03407-5.
Disease-modifying therapies (DMTs) that prevent immune cell infiltration into the brain have demonstrated efficacy in multiple sclerosis (MS) treatment. However, their unpredictable adverse effects necessitate the development of safer therapeutic alternatives. The choroid plexus (ChP) functions as a crucial barrier against immune cell invasion, and previous studies have shown that preventing immune cell infiltration across the ChP reduces brain lesion in MS animal models. Understanding ChP barrier regulation is therefore essential for identifying novel therapeutic targets for MS. Here, we explored the role of Ste20-related proline/alanine-rich kinase (SPAK) in experimental autoimmune encephalomyelitis (EAE).
We examined the expression patterns of SPAK signaling in ChP using immunofluorescence in the EAE model. To investigate the roles of SPAK, matrix metalloproteinase (MMP) 2 and MMP9 in EAE pathology, we performed ChP-specific gene manipulation via intracerebroventricular (ICV) injection of recombinant adeno-associated virus 2/5 (rAAV2/5). T cell infiltration into the central nervous system (CNS) was analyzed using CD4 immunostaining and flow cytometry. We employed cell immunofluorescence, transwell assays, and rescue experiments in vitro to study SPAK's effects on ChP epithelial barrier integrity. We also evaluated the protective effects of SPAK-Na-K-2Cl cotransporter-1 (NKCC1) inhibitors (ZT-1a and bumetanide) on immune invasion and demyelination during EAE using pharmacological approaches.
Following EAE induction, we observed progressive increases in both total and phosphorylated SPAK levels in ChP epithelium. Notably, ChP-specific SPAK knockdown significantly reduced T cell invasion and ameliorated EAE pathology, while SPAK overexpression exacerbated these effects. Bulk RNA sequencing and subsequent qPCR validation revealed that SPAK knockdown decreased the expression of MMP2 and MMP9, MMPs that compromise barrier integrity by degrading tight junction proteins. In vitro studies demonstrated that SPAK overexpression impaired ChP barrier function through the activator protein-1 (AP-1)-MMP2/9-zonula occludens-1 (ZO-1) axis. Furthermore, ChP-specific knockdown of either MMP2 or MMP9 protected against EAE pathology. Additionally, we identified SPAK-NKCC1 antagonists (bumetanide and ZT-1a) as promising therapeutic candidates for MS/EAE treatment.
Our findings demonstrate that targeting ChP-SPAK signaling represents a novel therapeutic strategy for MS treatment.
可防止免疫细胞浸润入脑的疾病修饰疗法（DMTs）已在多发性硬化症（MS）治疗中显示出疗效。然而，其不可预测的不良反应使得有必要开发更安全的治疗选择。脉络丛（ChP）作为抵御免疫细胞入侵的关键屏障，先前的研究表明，防止免疫细胞穿过ChP浸润可减少MS动物模型中的脑损伤。因此，了解ChP屏障调节对于确定MS的新治疗靶点至关重要。在此，我们探讨了丝氨酸/苏氨酸蛋白激酶20相关富含脯氨酸/丙氨酸激酶（SPAK）在实验性自身免疫性脑脊髓炎（EAE）中的作用。
我们在EAE模型中使用免疫荧光检查了ChP中SPAK信号的表达模式。为了研究SPAK、基质金属蛋白酶（MMP）2和MMP9在EAE病理中的作用，我们通过脑室内（ICV）注射重组腺相关病毒2/5（rAAV2/5）进行ChP特异性基因操作。使用CD4免疫染色和流式细胞术分析T细胞向中枢神经系统（CNS）的浸润。我们在体外采用细胞免疫荧光、Transwell实验和拯救实验来研究SPAK对ChP上皮屏障完整性的影响。我们还使用药理学方法评估了SPAK-钠-钾-2-氯共转运体-1（NKCC1）抑制剂（ZT-1a和布美他尼）对EAE期间免疫侵袭和脱髓鞘的保护作用。
在诱导EAE后，我们观察到ChP上皮中总SPAK水平和磷酸化SPAK水平均逐渐升高。值得注意的是，ChP特异性SPAK敲低显著减少了T细胞浸润并改善了EAE病理，而SPAK过表达则加剧了这些影响。批量RNA测序及随后的qPCR验证显示，SPAK敲低降低了MMP2和MMP9的表达，MMPs通过降解紧密连接蛋白损害屏障完整性。体外研究表明，SPAK过表达通过激活蛋白-1（AP-1）-MMP2/9-闭合小带蛋白-1（ZO-1）轴损害ChP屏障功能。此外，ChP特异性敲低MMP2或MMP9均可预防EAE病理。此外，我们确定SPAK-NKCC1拮抗剂（布美他尼和ZT-1a）为MS/EAE治疗有前景的候选药物。
我们的研究结果表明，靶向ChP-SPAK信号代表了一种新的MS治疗策略。
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