Molecular aspects of WNT/Frizzled signalling in brain angiogenesis

The family of WNT lipoglycoproteins consists of 19 secreted proteins that are important for multiple cellular processes including cell proliferation, migration and fate. WNTs induce signalling cascades by binding the seven transmembrane receptors named Frizzleds (FZDs) classified as G protein-coupled receptors (GPCRs). Binding of WNT proteins to FZDs leads to the activation of either β-catenin-dependent and/or β-catenin–independent pathways. Mutations or misregulation in the WNT signalling components can cause severe developmental defects, and disorders such as cancer. During embryonic development WNT/FZD signalling is one of the key regulators of vascular development in the central nervous system (CNS). Ablation of the transcriptional activator β-catenin resulted in haemorrhage throughout the entire CNS. Moreover, deletion of one of the WNT receptors, FZD4, also exhibited vascular defects, which were mainly localised to the retina.

In this thesis we investigated the process leading to CNS haemorrhage by creating a novel mouse model where β-catenin was inhibited. By overexpressing AXIN1, one of the major components of the β-catenin destruction complex, we could conclude that inhibition of β-catenin in CNS endothelium increased vessel regression and remodelling. Furthermore, we identified an extracellular matrix protein, ADAMTS-Like Protein 2 (ADAMTSL2), to be important for proper vascularisation of the brain. In addition, RNA sequencing data of the embryonic forebrain endothelial cells provided us with the FZD expression profile revealing that FZD4 and its close homolog FZD10 are present during early blood vessels development.

Even though FZDs are classified as GPCRs, the question as to whether they can activate G proteins has been under the debate. Despite sequence homology indicating a structure similar to other classical GPCRs, the evidence that they can signal through G proteins has been limited due to a lack of pharmacological tools and robust read-out assays. Here, we show for the first time that the two WNT receptors, FZD4 and FZD10 belonging to the same FZD homology cluster, form inactive-state complexes with the Gα12/13 proteins. Moreover, we show that FZD4, the receptor previously connected to vascular malformations is able to induce Gα12/13-dependent recruitment of p115-RHOGEF, suggesting a novel FZD4/Gα12/13/RHO signalling axis. In addition, we demonstrate that FZD10 has a selective preference for Gα13 over other G protein family members. While FZD10 is able to activate β-catenin transcriptional activity in the presence of overexpressed LRP6, FZD10 triggers Gα12/13-dependent transcriptional activity of Yes Associated Protein 1 (YAP) and Tafazzin (TAZ) in the absence of its co-receptor. Furthermore, we confirm by in situ hybridisation that as indicated in the RNA sequencing data, FZD10 mRNA is indeed present in the developing CNS endothelium.

Much like WNT/β-catenin signalling also Gα12/13 dependent signalling is crucial for proper vascular development. With that in mind and with the data presented in this thesis, I would like to propose a parallel route for WNT/FZD induced signalling during CNS vascular development, mediated through the Gα12/13 proteins.

List of scientific papers

I. The WNT/ß-catenin axis modulates early BBB development by fine-tuning TGF-ß signalling through ADAMTSL2. B. Salagic Hot, L. Dahl Jensen, D. Ramsköld, C. Yokota, S. Giatrellis, V.M. Lauschke, M.X. Li, M. Hupe, T.D. Arnold, R. Sandberg, J. Frisén, A. Martowicz, J. Wisniewska-Kruk, D. Nyqvist, R.H. Adams, J.M, Stenman and J. Kele. [Manuscript]

II. WNT Stimulation Dissociates a Frizzled 4 Inactive-State Complex with Gα12/13. Elisa Arthofer, Belma Hot, Julian Petersen, Katerina Strakova, Stefan Jäger, Manuel Grundmann, Evi Kostenis, J. Silvio Gutkind, and Gunnar Schulte. Mol Pharmacol. 2016 Oct;90(4):447-59.
https://doi.org/10.1124/mol.116.104919

III. FZD10-Gα13 signalling axis points to a role of FZD10 in CNS angiogenesis. Belma Hot, Jana Valnohova, Elisa Arthofer, Katharina Simon, Jaekyung Shin, Mathias Uhlén, Evi Kostenis, Jan Mulder, Gunnar Schulte. Cell Signal. 2017 Apr;32:93-103.
https://doi.org/10.1016/j.cellsig.2017.01.023