Molecular mechanisms of GPR124/RECK/WNT7 signaling

• Applicant: Dr. Mario Vallon
• Subject Area: Anatomy and Physiology ; Cell Biology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 440925599

Endothelial WNT7 signaling critically regulates developmental CNS angiogenesis and blood-brain barrier (BBB) formation. In brain endothelium the WNT7 signaling pathway is highly specialized and requires the WNT7-specific co-activators GPR124 and RECK as well as the classical Wnt co-receptors FZD and LRP. Intriguingly, we recently found that this signaling pathway is similarly specialized in non-CNS hyaloid endothelial cells of the developing eye, where it mediates physiologic regression of hyaloid blood vessel. Persistence of hyaloid blood vessels after birth, called persistent hyperplastic primary vitreous (PHPV), can lead to impaired vision and blindness. The molecular mechanisms of GPR124/RECK/WNT7 signaling in brain and hyaloid endothelium are still not completely understood and actively investigated by us. Our findings will advance basic research and may be beneficial for the development of drugs with the potential to (1) modulate BBB integrity and/or to (2) prevent/mitigate PHPV. The project has three specific aims: Aim 1 is to elucidate the molecular mechanisms of GPR124/RECK/WNT7-mediated hyaloid blood vessel regression in mice. We will perform single cell RNA sequencing to gain insights into GPR124/RECK/WNT7-induced apoptosis and target genes in hyaloid endothelial cells and potential crosstalk with other primary vitreous cells (pericytes, smooth muscle cells, macrophages). Aim 2 is to identify GPR124/WNT7-dependent interactions of RECK with other proteins on the surface of brain endothelial cells. During the first funding period we established a protocol for isolation of RECK and RECK-associated proteins from bEnd.3 brain endothelial cells. Using this protocol, we identified 43 RECK-associated proteins. 21 of these proteins were extracellular/transmembrane proteins, including the known RECK binding partners GPR124 and ADAM10. The other 19 proteins might be novel, direct or indirect binding partners of RECK involved in WNT7 signaling. In the second funding period we will validate these newly identified RECK-associated proteins and continue to use our system to potentially identify GPR124/WNT7-dependent RECK interactions. Aim 3 is to elucidate which FZD receptor isoforms (FZD1-10) transduce the GPR124/RECK/WNT7 signal into brain endothelial cells. In the first funding period we already generated Fzd4, 5, 6, and 8 single knockout bEnd.3 cells. Interestingly, neither single knockout affected WNT7 signaling. In the second funding period we will generate single knockouts of the other Fzd genes and different Fzd combination knockouts. To identify other relevant FZD isoforms, we will perform co-pull-down assays using purified recombinant FZD ectodomains (FZD1-10) and WNT7 conditioned medium. Based on the results, single and combination knockouts of all FZD receptors that (1) directly bind to WNT7 and (2) are expressed in bEnd.3 cells will be generated. Knockout cells will be assayed for WNT7 signaling activity using a Wnt reporter gene assay.

• DFG Programme: Research Grants