脑血管内皮细胞Prmt5基因敲除导致小胶质细胞激活并影响血脑屏障完整性

Mice Lacking Prmt5 in Cerebral Vascular Endothelial Cells Showing Microglial Activation and Protecting Blood-brain Barrier Integrity

目的:研究脑血管内皮细胞Prmt5基因敲除小鼠脑中小胶质细胞是否激活,及其对血脑屏障渗透性的影响。方法:通过免疫荧光和Western blot检测小鼠皮层、丘脑、小脑中小胶质细胞标志分子表达水平,探究脑血管内皮细胞Prmt5基因敲除小鼠脑中小胶质细胞是否激活,并通过实时定量PCR和Western blot检测不同脑区M1型(CD86、CD16、TNF-α)和M2型(Ym1、Arg1、IL-10)小胶质细胞标志物,考察激活小胶质细胞极化类型。利用集落刺激因子1受体(CSF1R)抑制剂PLX5622清除对照和Prmt5基因敲除小鼠脑中的小胶质细胞,通过实时定量PCR、免疫荧光检测小胶质细胞标志物表达水平,评价小胶质细胞耗竭效率;利用N-羟基磺酸基琥珀生物素(Sulfo-NHS-Biotin)染料灌注和示踪的方法评价耗竭小胶质细胞对脑血管内皮细胞Prmt5基因敲除小鼠血脑屏障完整性的影响。结果:脑血管内皮细胞Prmt5基因敲除导致小胶质细胞激活,小胶质细胞M1型标志物(CD16、CD86及TNF-α)及M2型标志物(Ym1、Arg1及IL-10)均表达上调。PLX5622处理导致小胶质细胞耗竭并加重Prmt5基因敲除小鼠BBB渗漏表型。结论:脑血管内皮细胞Prmt5基因敲除导致小胶质细胞激活,小胶质细胞参与保护脑血管内皮细胞Prmt5基因敲除导致的血脑屏障损伤。

Objective:Endothelial cells(ECs)together with pericytes,microglia,astrocytes and neurons,form a neurovascular unit(NVU)that constitutes the blood-brain barrier(BBB).However,the role of microglia in the maintenance of the BBB remains under debate.Protein arginine methyltransferase 5(PRMT5)is the main catalyzing arginine symmetric dimethylation methyltransferase in vivo.Our previous work found that cerebral vascular EC-specific Prmt5 gene knockout resulted in severe cerebrovascular lesions and impaired BBB.Whether microglia are activated in cerebral vascular lesions caused by Prmt5 gene knockout in cerebral vascular ECs is investigated,and microglia's effects on the BBB permeability are explored.Methods:SP-A-Cre transgenic mice were bred with Prmt5~(fl/fl)conditional gene targeting mice,the knock-in mice with Rosa26~(tdTomato)(RFP)reporter lines,to generate cerebral vascular ECs Prmt5 gene knockout(Prmt5~(fl/fl))mice.To examine whether microglia are activated in the cerebral vascular ECs Prmt5~(fl/fl)mice,Ionized Calcium Binding Adaptor Molecule 1(IBA1)expression levels in the cortex,thalamus and cerebellum of the mice in the control group and Prmt5~(fl/fl)mice were detected by immunofluorescence and Western blot.The M1 microglial markers of the cluster of differentiation 86(CD86),the cluster of differentiation 16(CD16),tumor necrosis factor alpha(TNF-α)and M2 microglial markers of chitinase-like protein 3(Chil3/Ym1),arginase 1(Arg1),and Interleukin-10(IL-10)were detected by real-time quantitative PCR and Western blot to evaluate the microglial polarization in different brain regions.CSF1R inhibitor PLX5622 was intraperitoneally injected into the mice in the control group and Prmt5~(fl/fl)mice to deplete microglia.IBA1 expression level was detected by immunofluorescence and real-time quantitative PCR to evaluate the efficiency of microglial depletion.Sulfo-NHS-Biotin was intraperitoneally injected into the mice in the control group and Prmt5~(fl/fl)mice to examine the BBB integrity.Results:The expression level of IBA1 increased significantly in the cortex,thalamus and cerebellum.The IBA1 and CD68 double positive cells accumulated in the thalamic lesion area of Prmt5~(fl/fl)mice showed that knockout of Prmt5 gene in cerebral vascular ECs led to activation of microglia.The expression of M1 markers(CD16,CD86 and TNF-α)and M2 markers(Ym1,Arg1 and IL-10)were up-regulated in the cortex,thalamus and cerebellum in cerebral vascular ECs of Prmt5~(fl/fl)mice.PLX5622 treatment resulted in microglial depletion with a depletion efficiency of more than 70%.Depletion of microglia leads to increased BBB permeability of Prmt5~(fl/fl)mice.Conclusion:Deletion of Prmt5 in cerebrovascular ECs leads to activation of microglia,which may be involved in the maintenance of the BBB integrity.