Project 455103

The brain and retina are protected by barriers, formed by cells called endothelial cells, which prevent molecules and cells that may be harmful to these tissues from leaking from blood vessels. Highly coordinated signaling is required to maintain these barriers between tissues and the blood, and their breakdown is a major cause of dysfunction and disease. Frizzled-4 (FZD4) is a cell surface receptor expressed in endothelial cells that gets turned on by secreted signals, such as WNT7A/B and Norrin (NDP), that bind to the receptor. We have observed that when FZD4 is turned on by either NDP or a synthetic antibody drug that mimics NDP, called F4L5.13, the endothelial cell barrier is less leaky and more stable. In fact, in a mouse model of endothelial barrier dysfunction that mimics human retinopathy (eye disease), F4L5.13 can stabilize the blood-retinal barrier which means that F4L5.13 may be a promising therapy to treat barrier dysfunction. How turning on FZD4 with NDP or F4L5.13 leads to barrier protection is largely unknown, however, we observe that barrier stability is achieved very quickly. This suggests that FZD4 initiates signaling events inside the cell that are responsible for the effects we see. One important signaling event that occurs within cells is called phosphorylation, which results in proteins inside the cell being modified to turn on or off. The goal of this proposal is to use a powerful technique called phosphoproteomics to look at all the changes in protein phosphorylation that occur within the cell following the addition of NDP or F4L5.13. Further, we want to determine which of these signaling events caused by FZD4 leads to barrier protection and stabilization. Since dysfunction in FZD4 signaling is related to stroke, neurodegenerative disease, and retinopathy this research has very high potential to help us understand how FZD4 works and what the best proteins are to target with drugs to help protect our brain and eye barriers.