Abstract
Background: Recent studies suggest that regulator of G-protein signaling 5 (RGS5), a negative modulator of the G-protein coupled receptors (GPCR) signaling pathways, may be an important modulator of glutamate receptor activation and calcium overload in endothelial cells after ischemic stroke. While glutamate may cause neuronal damage, it is unknown how glutamate affects blood-brain barrier (BBB) permeability. Methods: To investigate the effects of RGS5 on stroke severity, wild type (WT) and RGS5 -/- mice were subjected to 30 minutes of middle cerebral artery occlusion (MCAO). Cerebral infarct volume was evaluated by TTC and Nissl staining, while neurological deficit was assessed using a 5-point motor grading scale and adhesive tape removal test. BBB permeability was analyzed by measuring brain edema with the wet/dry method and by injecting 40kDA FITC-Dextran into the femoral vein. The role of RGS5 in regulating ischemia/glutamate-induced Rho/ROCK signaling in human brain microvasculare cells (HBMEC) was investigated using lentiviral transduction of RGS5 shRNA. To mimic brain ischemic conditions in vitro , we used a model consisting of 3-hour oxygen-glucose deprivation (OGD) with L-glutamate. RhoA/ROCK activity was determined by the phosphorylation MYPT1. In vitro permeability was assessed by Transwell permeability assays. Formation of actin stress fibers was observed with Phalloidin staining. Redistribution of important tight junction (TJ) proteins was analyzed by immunostaining. Results: Following MCAO, RGS5 -/- mice exhibited larger cerebral infarct volume and greater neurological deficits compared to controls. Brain water content and FITC-Dextran leakage were higher in the ipsilateral hemisphere of RGS5 -/- mice compared to controls. Furthermore, the Transwell permeability assay demonstrated increased permeability of FITC-Dextran in RGS5-deficient HBMEC, higher Rho/ROCK activation, greater actin-stress fiber formation, and redistribution of TJ proteins. Conclusion: These findings indicate that RGS5 plays an important role in actin cytoskeleton remodeling and BBB permeability following focal cerebral ischemia, in part, through the upregulation of RhoA/ROCK pathway.
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