Abstract
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 3β (GSK3β) inhibition has on primary human brain endothelial cells. Here we show that GSK3β 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 GSK3β. Inhibition of GSK3β 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 GSK3β 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 β-catenin with ZO-1 as a function of GSK3β inhibition. Collectively, our findings suggest the possibility of using GSK3β inhibitors as a means to extend the half-life of key tight junction proteins to promote re-sealing of the BBB during neuroinflammation.
Highlights
The blood brain barrier (BBB) shields the brain parenchyma from immune cells and toxins in the blood, maintaining the adequate environment needed for normal neuronal and glial cell function [1]
To confirm that the effect seen on transendothelial electrical resistance (TEER) was due to inhibition of glycogen synthase kinase 3b (GSK3b) and not other cellular targets, we evaluated the effect of GSK3b mutants on TEER
These analyses suggest that inactivation of GSK3b in brain endothelial cells directly benefits barrier integrity
Summary
The blood brain barrier (BBB) shields the brain parenchyma from immune cells and toxins in the blood, maintaining the adequate environment needed for normal neuronal and glial cell function [1]. One manifestation of BBB dysfunction is evident by increased permeability of blood solutes into the brain parenchyma which is greatly controlled by the tight junction (TJ) complex located between endothelial cells. This physical barrier is mainly responsible for generating the hallmark features of the BBB. The dynamic nature of the TJ in response to cellular stimuli can manifest as disassembly, re-distribution, degradation and remodeling [6]. These events impact barrier genesis, barrier maintenance and barrier dysfunction (seen in neuropathological conditions)
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