Inhibition of Glycogen Synthase Kinase 3β Promotes Tight Junction Stability in Brain Endothelial Cells by Half-Life Extension of Occludin and Claudin-5

Neuroinflammatory conditions often involve dysfunction of the Blood-Brain Barrier (BBB). Therefore, identifying molecular targets that can maintain barrier fidelity is of clinical importance. We have previously reported on the anti-inflammatory effects that glycogen synthase kinase 3 beta (GSK3 beta) inhibition has on primary human brain endothelial cells. Here we show that GSK3 beta inhibitors also promote barrier tightness by affecting tight junction (TJ) protein stability. Transendothelial electrical resistance (TEER) was used to evaluate barrier integrity with both pharmacological inhibitors and mutants of GSK3 beta. Inhibition of GSK3 beta produced a gradual and sustained increase in TEER (as much as 22% over baseline). Analysis of subcellular membrane fractions revealed an increase in the amount of essential tight junction proteins, occludin and claudin-5, but not claudin-3. This phenomenon was attributed to a decrease in TJ protein turnover and not transcriptional regulation. Using a novel cell-based assay, inactivation of GSK3 beta significantly increased the half-life of occludin and claudin-5 by 32% and 43%, respectively. A correlation was also established between the enhanced association of beta-catenin with ZO-1 as a function of GSK3 beta inhibition. Collectively, our findings suggest the possibility of using GSK3 beta inhibitors as a means to extend the half-life of key tight junction proteins to promote re-sealing of the BBB during neuroinflammation.