Inhibition of Glycogen Synthase Kinase 3[beta] 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.