GIV/Girdin Modulation of Microglial Activation in Ischemic Stroke: Impact of FTO-Mediated m6A Modification

Abstract

Ischemic stroke (IS) is one of the most common causes of death in the world. The lack of effective pharmacological treatments for IS was primarily due to a lack of understanding of its pathogenesis. Gα-Interacting vesicle-associated protein (GIV/Girdin) is a multi-modular signal transducer and guanine nucleotide exchange factor that controls important signaling downstream of multiple receptors. The purpose of this study was to investigate the role of GIV in IS. In the present study, we found that GIV is highly expressed in the central nervous system (CNS). GIV protein level was decreased, while GIV transcript level was increased in the middle cerebral artery occlusion reperfusion (MCAO/R) mice model. Additionally, GIV was insensitive lipopolysaccharide (LPS) exposure. Interestingly, we found that GIV overexpression dramatically restrained microglial activation, inflammatory response, and M1 polarization in BV-2 microglia induced by oxygen-glucose deprivation and reoxygenation (OGD/R). On the contrary, GIV knockdown had the opposite impact. Mechanistically, we found that GIV activated the Wnt/β-catenin signaling pathway by interacting with DVL2 (disheveled segment polarity protein 2). Notably, m⁶A demethylase fat mass and obesity-associated protein (FTO) decreased the N6-methyladenosine (m6A) modification-mediated increase of GIV expression and attenuated the inflammatory response in BV-2 stimulated by OGD/R. Taken together, our results demonstrate that GIV inhibited the inflammatory response via activating the Wnt/β-catenin signaling pathway which expression regulated in an FTO-mediated m⁶A modification in IS. These results broaden our understanding of the role of the FTO-GIV axis in IS development.

Keywords: FTO; GIV/Girdin; Inflammatory response; Ischemic stroke; M6A.

Fig. 1

Expression and distribution of GIV in mouse tissue. A Relative expression of the GIV gene in various tissues generated using Human Protein Atlas (www.proteinatlas.org). B Relative expression of GIV protein in various tissues generated using Human Protein Atlas (www.proteinatlas.org). C mRNA expression of GIV in different organs of mice examined by qRT-PCR. D Protein expression of GIV in different organs of mice detected by Western blotting

Fig. 2

GIV expression was downregulated in the mice model of ischemic stroke and cell model of OGD/R. A, B TTC staining of brains showed that MCAO/R induced an increase in the infarct volume compared with the sham group. C Neurological function was evaluated using the Longa score. D H&E staining of the cerebral cortex region revealed histopathological changes induced by MCAO/R, including loose cytoplasm, edema, and nuclear division. E, F Nissl staining of cerebral cortex region revealed that MCAO/R decreased the percentage of Nissl bodies and promoted neuronal loss compared with the sham group. G GIV protein levels were reduced in the brain periinfarct area from MCAO/R group mice. H Immunofluorescence of GIV in Sham or MCAO/R group mice. I GIV protein levels were reduced in OGD/R group BV-2 cells. J GIV protein levels were reduced in OGD/R HMC3 cells. K LPS exposure did not affect the protein expression of GIV. L GIV mRNA was increased in the brain periinfarct area from MCAO/R group mice and OGD/R group HMC3(M) cells (n ≥ 3, **P < 0.01)

Fig. 3

GIV deficiency aggravates inflammatory response in BV-2 cells induced by OGD/R. A, B Representative images of GIV shRNA knockdown efficiency detected by Western blot. C–G The relative mRNA expression levels of TNF-α, IL-6, IL-1β, iNOS, Cox-2, and NLRP3 after GIV knockdown. H Representative images of Western blot. I–J The relative protein expression levels of iNOS and CD16, CD206, and Arg-1 after GIV knockdown. K The Iba1 protein expression level after GIV knockdown. (n ≥ 3, *P < 0.05, **P < 0.01)

Fig. 4

GIV overexpression inhibits the inflammatory response in BV-2 cells induced by OGD/R. A, B Representative images of GIV overexpression were detected using Western blot. The relative mRNA expression levels of TNF-α (C), IL-6 (D), IL-1β (E), iNOS (F), Cox-2 (G), and NLRP3 (H) were measured after GIV overexpression. The representative images of Western blot are shown in I. The relative protein expression levels of iNOS and CD16 after GIV overexpression are shown in J. The relative protein expression levels of CD206 and Arg-1 after GIV overexpression are shown in K. The Iba1 protein expression level after GIV overexpression is shown in L (n ≥ 3, *P < 0.05, **P < 0.01)

Fig. 5

GIV regulates BV-2 microglia M1/M2 polarization. A The representative images of CD16-positive cells by flow cytometry after GIV knockdown and overexpression. B The CD16-positive cells count after GIV knockdown and overexpression. C The representative images of CD206-positive cells by flow cytometry after GIV knockdown and overexpression. D The CD206-positive cells count after GIV knockdown and overexpression (n = 3, **P < 0.01)

Fig. 6

GIV regulates the Wnt/β-catenin signaling pathway. A comparison of GIV transcript levels in the control and Stroke groups from GEO DataSets (GSE58294) revealed significant differences. B Gene set enrichment analysis indicated an upregulation of the Wnt/β-catenin signaling pathway in the Stroke group. C The mRNA expression of GIV was compared between the control and OGD/R groups in BV-2 microglia. The relative mRNA expression levels of Gsk3b (D), Ctnnb1 (E), Ccnd1 (F), Fzd1 (G), Fzd2 (H), and Axin2 (I) were measured after GIV knockdown or overexpression (n ≥ 3, **P < 0.01)

Fig. 7

GIV interacts with DVL2 and activates Wnt/β-catenin signaling pathway. A Protein-protein interaction network analysis (PPI). B Structure-based protein interaction interface analysis between GIV and DVL2. C Representative images of Western blot after GIV overexpression. D Representative images of western blot after GIV knockdown. E Representative images of immunofluorescent staining. F DVL2, GIV, and IgG antibody, THP-1 lysates were immunoprecipitated using DVL2 antibody and then analyzed by Western blot using the indicated antibodies. G Half-life of DVL2 in BV-2 cells with Vector or overexpression of GIV, treated with cycloheximide (CHX) at the indicated times and analyzed by Western blot (n = 3, **P < 0.01, scale bar = 20 μm)

Fig. 8

FTO suppressed inflammatory response and modulates GIV expression. A BV-2 cells challenged with OGD/R, FTO, and GIV protein expression was reduced. B FTO suppressed COX-2 and IL-1β expression in BV-2 cell challenged with OGD/R. C FTO knockdown increased COX-2 and IL-1β expression. D GIV is a target gene of FTO predicated by RM2Target software. E FTO overexpression promotes GIV expression of BV-2 cells challenged with OGD/R. F FTO Knockdown suppresses GIV protein expression in BV-2 (n = 3, **P < 0.01)

Fig. 9

FTO knockdown increased GIV mRNA stability by m6A modification. A The GIV m⁶A site was predicted using the SRAMP software. B The sequence of GIV m⁶A modification was determined. C Overexpression of FTO resulted in a reduction in m⁶A modification, as confirmed by dot blot assay. D After FTO overexpression, the m⁶A modification of GIV mRNA expression was found to be reduced, as determined by MeRIP-PCR. E The RIP-PCR assay detected the enrichment of GIV mRNA precipitated by the anti-FTO antibody. F RNA stability analysis revealed a decrease in GIV mRNA expression in FTO knockdown BV-2 cells treated with Actinomycin D (Act. D) (n = 3, **P < 0.01)