Deubiquitinase USP9X regulates Wnt-signaling to control CNS vascular formation and barrier maintenance

Deubiquitinating enzymes play crucial roles in various cellular activities, yet their involvement in central nervous system (CNS) vascularization and barrier function remains elusive. Canonical Wnt signaling is essential for proper CNS vascularization and barrier maintenance. Using a loss-of-function screening for Wnt signaling activity, we identified ubiquitin-specific peptidase 9 X-linked (USP9X) as a key regulator in brain endothelial cells (BECs). Endothelium-specific Usp9x knockout reduces Wnt-signaling activity, compromising CNS vascularization and barrier function during development. Activation of Wnt signaling rescues these defects. Mechanistically, we uncovered β-catenin as a direct substrate of USP9X, with USP9X catalyzing K48 polyubiquitin chains to stabilize β-catenin. In pathological models of impaired CNS vascular barrier function, including an intracerebral hemorrhage model and an oxygen-induced retinopathy model, loss of Usp9x intensifies barrier disruption, accentuating defects. This finding implicates USP9X as a critical regulator of CNS vascularization and barrier function through Wnt signaling, offering insights into CNS disease implications.