BTK通过NEK7-NLRP3信号通路对小鼠阿尔茨海默样病变的调控作用

Regulatory effect of BTK on mouse Alzheimer disease-like pathology via NEK7-NLRP3 signaling pathway

目的:本研究探讨布鲁顿酪氨酸激酶(BTK)对阿尔茨海默样病变及NIMA相关激酶7(NEK7)-核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)信号通路的调控作用。方法:选取2、4、6月龄的5xFAD阿尔茨海默病(AD)模型小鼠和野生型(WT)小鼠,采用Western blot检测脑内BTK、NEK7及NLRP3等蛋白水平,免疫荧光染色法检测BTK、NEK7及NLRP3等蛋白的表达,免疫沉淀法检测NEK7和NLRP3的相互作用;选取3月龄5xFAD小鼠,随机分为BTK抑制剂依鲁替尼处理组和溶剂对照组,腹腔注射依鲁替尼(10 mg/kg)或溶剂连续14 d,随后进行Morris水迷宫和Y迷宫行为学实验分析各组小鼠学习与记忆能力;采用β-淀粉样蛋白42(Aβ_(42))侧脑室注射法构建AD模型,注射14 d后进行Morris水迷宫实验,Western blot检测脑内BTK蛋白水平;在BV2细胞中分别使用依鲁替尼预处理脂多糖(LPS)诱导的神经炎症模型或者Aβ_(42)刺激的AD细胞模型,Western blot检测细胞NEK7、NLRP3、BTK以及p-BTK(Y223)蛋白水平的变化,免疫荧光染色法检测炎症小体,包括ASC、caspase-1、NEK7及NLRP3等蛋白的表达。结果:与WT组小鼠相比,5xFAD小鼠脑内炎症小体通路相关蛋白表达增加,NEK7蛋白表达增加,NEK7与NLRP3之间存在相互作用,AD模型小鼠脑内BTK蛋白围绕4G8斑块表达,表达量增加,且与Iba1存在共定位现象;与AD模型组小鼠相比,BTK抑制剂依鲁替尼处理组小鼠学习记忆功能有所改善;细胞实验结果显示,依鲁替尼预处理有效的抑制了Aβ_(42)刺激后BV2细胞NLRP3炎症小体通路相关蛋白以及NEK7的表达。结论:BTK能够通过NEK7-NLRP3相互作用介导神经炎症,抑制BTK可减轻神经炎症、从而改善阿尔茨海默样病变小鼠的学习记忆功能。

AIM:To investigate the impact of Bruton tyrosine kinase(BTK)on Alzheimer disease(AD)-like pathology through the NIMA(never in mitosis gene A)-related kinase 7(NEK7)-nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)pathway.METHODS:5xFAD and wild-type(WT)mice aged 2,4 and 6 months were utilized to assess the expression of BTK,NEK7 and NLRP3 proteins in the hippocampus and cortex via Western blot and immunofluorescence.Co-immunofluorescence was conducted to identify the interaction between NEK7 and NLRP3 in the brains of 4-month-old mice.Three-month-old mice were divided into a control group and an ibrutinib treatment group,receiving intraperitoneal injections of ibrutinib(10 mg/kg)or solvent for 14 d,and were then subjected to behavioral assessments including learning and memory tests using the Morris water maze and Y-maze.Wild-type mice were induced with an AD model by intracerebroventricular injection of Aβ_(42).Morris water maze tests were performed after 14 d to evaluate learning and memory,followed by measurement of BTK protein levels in the brain via Western blot.BV2 microglial cells were treated with ibrutinib,followed by LPS or Aβ_(42) stimulation.Western blot analysis was conducted to measure the protein levels of NEK7,NLRP3,BTK and p-BTK(Y223),while immunofluorescence was used to assess the protein expression of ASC,caspase-1,NEK7 and NLRP3.RESULTS:The levels of BTK,NEK7 and NLRP3 in the brains of 5×FAD mice were significantly elevated compared to WT mice,with observed interaction between NEK7 and NLRP3 in the 5xFAD mouse brains.Ibrutinib treatment significantly improved learning and memory functions in mice compared to the AD group.In BV2 cells,pre-treatment with ibrutinib effectively suppressed the NLRP3 inflammasome pathway and NEK7 proteins in response to Aβ_(42) stimulation.CONCLUSION:BTK plays a regulatory role in AD-like pathology through the NEK7-NLRP3 pathway both in vivo and in vitro.