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

doi:10.13523/j.cb.2212013

China Biotechnology

2023, Vol. 43

Issue (5): 1-10 DOI: 10.13523/j.cb.2212013

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

HAN Yu-ying^1,²,NING Hui-min^1,²,ZHANG Yi-zhe²,SONG Xiao-peng²,XU Cheng-fang²,CAI Yun-ting²,YANG Xiao^1,^2,^**(

),WANG Jun^2,^**(

)

1 School of Basic Medicine, Qingdao University, Qingdao 266071, China
2 State Key Laboratory of Proteomics, Beijing National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing 102206, China

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Abstract

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.

Key words： Microglia Cerebral vascular endothelial cell Prmt5 Blood-brain barrier

Received: 06 December 2022 Published: 01 June 2023

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	HAN Yu-ying
	NING Hui-min
	ZHANG Yi-zhe
	SONG Xiao-peng
	XU Cheng-fang
	CAI Yun-ting
	YANG Xiao
	WANG Jun

Cite this article:

HAN Yu-ying, NING Hui-min, ZHANG Yi-zhe, SONG Xiao-peng, XU Cheng-fang, CAI Yun-ting, YANG Xiao, WANG Jun. Mice Lacking Prmt5 in Cerebral Vascular Endothelial Cells Showing Microglial Activation and Protecting Blood-brain Barrier Integrity. China Biotechnology, 2023, 43(5): 1-10.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2212013 OR https://manu60.magtech.com.cn/biotech/Y2023/V43/I5/1

Table 1 Real-time PCR primer sequences

Fig.1 Knockout of Prmt5 gene in cerebrovascular endothelial cells led to activation of microglia (a) Expression of microglia in cerebrovascular endothelial cells Prmt5 gene knockout mice. IBA1 (green) was used to label microglia, and DAPI (blue) was used to label nucleus. Scale bar was 100 μm (b)Quantitative analysis of IBA1⁺cell. n=3, ^* P< 0.05, ^** P< 0.01, ^*** P<0.001 (c) Western blot analysis of microglia expression in different brain regions of Prmt5 gene knockout mice. n=3 (d) Quantitative analysis of Western blot. n=4, ^* P<0.05 (e) Immunostaining analysis of brain sections for CD68(green) and IBA1(red). Scale bar was 50 μm

Fig.2 Knockout of Prmt5 gene in cerebrovascular endothelial cells upregulate M1 and M2 markers of microglia Real-time PCR(a) and Western blot(c) were used to detect the expression level of M1 markers, n=3 or n=4, ^* P<0.05, ^** P<0.01, ^*** P<0.001 Real-time PCR (b) and Western blot (e) were used to detect the expression levels of M2 markers, n=3 or n=4, ^* P< 0.05, ^** P<0.01, ^*** P<0.001 (d),(f)Quantitative analysis of Western blot, n=3 or n=4, ^* P<0.05, ^** P<0.01

Fig.3 PLX5622 treatment resulted in microglial depletion and worsened the BBB leakage phenotype of Prmt5 gene knockout mice (a) Real-time PCR was used to analyze the depletion of microglia after PLX5622 treatment. n=3, ^** P<0.01 (b) Immunofluorescence staining of microglia in Prmt5 gene knockout mice after PLX5622 treatment. Scale bar was 100 μm (c) Immunofluorescence staining of brain tissue sections from Prmt5 gene knockout mice mice after Sulfo-NHS-Biotin injections, green represents Sulfo-NHS-Biotin. Scale bar was 1 000 μm


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