Epac1 and PDZ-GEF cooperate in Rap1 mediated endothelial junction control.

Epac1 and its effector Rap1 are important mediators of cAMP induced tightening of endothelial junctions and consequential increased barrier function. We have investigated the involvement of Rap1 signalling in basal, unstimulated, barrier function of a confluent monolayer of HUVEC using real time Electric Cell-substrate Impedance Sensing. Depletion of Rap1, but not Epac1, results in a strong decrease in barrier function. This decrease is also observed when cells are depleted of the cAMP independent Rap exchange factors PDZ-GEF1 and 2, showing that PDZ-GEFs are responsible for Rap1 activity in control of basal barrier function. Monolayers of cells depleted of PDZ-GEF or Rap1 show an irregular, zipper-like organization of VE-cadherin and live imaging of VE-cadherin-GFP reveals enhanced junction motility upon depletion of PDZ-GEF or Rap1. Importantly, activation of Epac1 increases the formation of cortical actin bundles at the cell-cell junctions, inhibits junction motility and restores barrier function of PDZ-GEFs depleted, but not Rap1 depleted cells. We conclude that PDZ-GEF activates Rap1 under resting conditions to stabilize cell-cell junctions and maintain basal integrity. Activation of Rap1 by cAMP/Epac1 induces junctional actin to further tighten cell-cell contacts.

Glia-specific activation of all pathways of the unfolded protein response in vanishing white matter disease.

Leukoencephalopathy with vanishing white matter (VWM) is a childhood white matter disorder with an autosomal-recessive mode of inheritance. The clinical course is chronic progressive with episodes of rapid neurologic deterioration after febrile infections. The disease is caused by mutations in the genes encoding the subunits of eukaryotic initiation factor 2B (eIF2B), a protein complex that is essential for protein synthesis. In VWM, mutations in the eIF2B genes are thought to impair the ability of cells to regulate protein synthesis under normal and stress conditions. It has been suggested that the pathophysiology of VWM involves inappropriate activation of the unfolded protein response (UPR). The UPR is a protective mechanism activated by an overload of unfolded or malfolded proteins in the endoplasmic reticulum. Activation of one pathway of the UPR, in which eIF2B is involved, has already been described in brain tissue of patients with VWM. In the present study, we demonstrate activation of all 3 UPR pathways in VWM brain tissue using real-time quantitative polymerase chain reaction and immunohistochemistry. We show that activation occurs exclusively in the white matter, predominantly in oligodendrocytes and astrocytes. The selective involvement of these cells suggests that inappropriate UPR activation may play a key role in the pathophysiology of VWM.