Variants of WNT7A and GPR124 are associated with hemorrhagic transformation following intravenous thrombolysis in ischemic stroke

Song Ta, Xianfang Rong, Zhen-Ni Guo, Hang Jin, Peng Zhang, Fenge Li, Zhihuan Li, Lilong Lin, Chenqing Zheng, Qingquan Gu, Yuan Zhang, Wenlan Liu, Yi Yang, Junlei Chang, Song Ta, Xianfang Rong, Zhen-Ni Guo, Hang Jin, Peng Zhang, Fenge Li, Zhihuan Li, Lilong Lin, Chenqing Zheng, Qingquan Gu, Yuan Zhang, Wenlan Liu, Yi Yang, Junlei Chang

Abstract

Aims: The canonical Wnt signaling pathway plays an essential role in blood-brain barrier integrity and intracerebral hemorrhage in preclinical stroke models. Here, we sought to explore the association between canonical Wnt signaling and hemorrhagic transformation (HT) following intravenous thrombolysis (IVT) in acute ischemic stroke (AIS) patients as well as to determine the underlying cellular mechanisms.

Methods: 355 consecutive AIS patients receiving IVT were included. Blood samples were collected on admission, and HT was detected at 24 hours after IVT. 117 single-nucleotide polymorphisms (SNPs) of 28 Wnt signaling genes and exon sequences of 4 core cerebrovascular Wnt signaling components (GPR124, RECK, FZD4, and CTNNB1) were determined using a customized sequencing chip. The impact of identified genetic variants was further studied in HEK 293T cells using cellular and biochemical assays.

Results: During the study period, 80 patients experienced HT with 27 parenchymal hematoma (PH). Compared to the non-PH patients, WNT7A SNPs (rs2163910, P = .001, OR 2.727; rs1124480, P = .002, OR 2.404) and GPR124 SNPs (rs61738775, P = .012, OR 4.883; rs146016051, P < .001, OR 7.607; rs75336000, P = .044, OR 2.503) were selectively enriched in the PH patients. Interestingly, a missense variant of GPR124 (rs75336000, c.3587G>A) identified in the PH patients resulted in a single amino acid alteration (p.Cys1196Tyr) in the intracellular domain of GPR124. This variant substantially reduced the activity of WNT7B-induced canonical Wnt signaling by decreasing the ability of GPR124 to recruit cytoplasmic DVL1 to the cellular membrane.

Conclusion: Variants of WNT7A and GPR124 are associated with increased risk of PH in patients with AIS after intravenous thrombolysis, likely through regulating the activity of canonical Wnt signaling.

Keywords: blood-brain barrier; intracerebral hemorrhage; ischemic stroke; signaling pathway; single-nucleotide polymorphism.

Conflict of interest statement

The authors declare no conflict of interest.

© 2020 The Authors. CNS Neuroscience & Therapeutics Published by John Wiley & Sons Ltd.

Figures

Figure 1
Figure 1
Schematic diagram of the study design. A, Schematic illustration of the canonical Wnt signaling in brain endothelium. B, Summary of Wnt signaling‐related genes assessed in this study. Either both exon sequence variations and known SNPs (left column) or only known SNPs (right column) were determined and analyzed
Figure 2
Figure 2
Shift distribution of patient numbers by GPR124 c.3587G>A mutation or by the top five Wnt signaling genetic variants. Number of patients (%) in each group with GPR124 c.3587G>A mutation (A) or with alleles of the top five Wnt signaling genetic variants enriched in the PH patients (WNT7A rs2163910 and rs1124480, GPR124 rs61738775, rs146016051, and rs75336000; 2 alleles/variant, 10 alleles totally) (B) was calculated
Figure 3
Figure 3
The GPR124 C1196Y mutation reduces Wnt signaling activity. A, Schematic illustration of the C1196Y alteration in the intracellular domain (ICD) of GPR124. B, Wild‐type (WT) GPR124 and GPR124 C1196Y were cloned into pcDNA3.1‐3xFLAG construct and overexpressed in HEK 293T cells. Protein subcellular location was determined by confocal immunofluorescence staining. C, Expression levels of GPR124 WT, GPR124 C1196Y, and GPR124 with ICD deletion (GPR124 △ICD) were determined by Western blotting. D‐E, Wnt signaling activity was measured by AXIN2 qRT‐PCR (D) and β‐catenin‐dependent transcription assay TOP‐Flash (E) after transfecting HEK 293T cells with the indicated plasmids. Data are presented as mean ± SE. *P < .05, **P < .01. NS, not significant, Student's t test
Figure 4
Figure 4
The GPR124 C1196Y mutation decreases the interaction between DVL1 and GPR124. A, 3xFLAG‐GPR124 WT or 3xFLAG‐GPR124 C1196Y was co‐expressed with DVL1‐His in HEK 293T cells. GPR124 was precipitated with anti‐FLAG antibody, and DVL1 protein bound to GPR124 was detected with anti‐6xHis antibody. This experiment was repeated three times and quantified in (B). Data are presented as mean ± SE. ***P < .01, Student's t test. C, Interaction between GPR124 (WT or C1196Y) and DVL1 was determined by co‐immunofluorescence staining (co‐IF) in HEK 293T cells. D‐F, A series of truncated DVL1 mutants were constructed, and co‐IP was performed to map the binding sites of DVL1 with GPR124 protein. Experiments were repeated three times, and representative results were presented. G, Proposed working model

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