Occludin oligomeric assemblies at tight junctions of the blood–brain barrier are altered by hypoxia and reoxygenation stress

Abstract

Hypoxic (low oxygen) and reperfusion (post-hypoxic reoxygenation) phases of stroke promote an increase in microvascular permeability at tight junctions (TJs) of the blood–brain barrier (BBB) that may lead to cerebral edema. To investigate the effect of hypoxia (Hx) and reoxygenation on oligomeric assemblies of the transmembrane TJ protein occludin, rats were subjected to either normoxia (Nx, 21% O2, 60 min), Hx (6% O2, 60 min), or hypoxia/reoxygenation (H/R, 6% O2, 60 min followed by 21% O2, 10 min). After treatment, cerebral microvessels were isolated, fractionated by detergent-free density gradient centrifugation, and occludin oligomeric assemblies associated with plasma membrane lipid rafts were solubilized by perfluoro-octanoic acid (PFO) exclusively as high molecular weight protein complexes. Analysis by non-reducing and reducing sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis/western blot of PFO-solubilized occludin revealed that occludin oligomeric assemblies co-localizing with ‘TJ-associated’ raft domains contained a high molecular weight ‘structural core’ that was resistant to disassembly by either SDS or a hydrophilic reducing agent ex vivo, and by Hx and H/R conditions in vivo. However, exposure of PFO-solubilized occludin oligomeric assemblies to SDS ex vivo revealed the non-covalent association of a significant amount of dimeric and monomeric occludin isoforms to the disulfide-bonded inner core, and dispersal of these non-covalently attached occludin subunits to lipid rafts of higher density in vivo was differentially promoted by Hx and H/R. Our data suggest a model of isoform interaction within occludin oligomeric assemblies at the BBB that enables occludin to simultaneously perform a structural role in inhibiting paracellular diffusion, and a signaling role involving interactions of dimeric and monomeric occludin isoforms with a variety of regulatory molecules within different plasma membrane lipid raft domains.

Oxidative Stress Increases Blood–Brain Barrier Permeability and Induces Alterations in Occludin during Hypoxia–Reoxygenation

“…Our data showing that tempol prevents the increase in BBB permeability to 14 Csucrose after HR treatment suggest that oxidative stress may also affect the molecular structure of the TJs. Previous work by our laboratory has indicated that the TJ protein occludin is affected by HR (McCaffrey et al, 2009), and its ability to oligomerize is redox sensitive (Walter et al, 2009). These observations led us to investigate the effects of oxidative stress on occludin at both the cellular and molecular level using confocal microscopy and density gradient subcellular fractionation followed by western blot analysis.…”

“…Occludin oligomers are held together covalently by disulfide bonds (McCaffrey et al, 2009;Walter et al, 2009). These bonds can be disrupted by pathologic conditions such as HR and inflammatory pain, leading to structural changes of the oligomers (McCaffrey et al, 2008;McCaffrey et al, 2009). Changes in the structure of occludin oligomers could potentially influence the ability of the TJ complexes to limit paracellular diffusion of molecules from the blood into the brain.…”

“…Multiple domains of occludin have previously been shown to regulate the diffusion of solutes across the TJ (Balda et al, 2000). Occludin oligomers are held together covalently by disulfide bonds (McCaffrey et al, 2009;Walter et al, 2009). These bonds can be disrupted by pathologic conditions such as HR and inflammatory pain, leading to structural changes of the oligomers (McCaffrey et al, 2008;McCaffrey et al, 2009).…”

“…This increase in BBB permeability correlates with alterations in the oligomeric assembly of the critical TJ protein occludin (McCaffrey et al, 2009). Occludin is a transmembrane protein that exists as oligomers in lipid raft regions at the plasma membrane of the BBB (McCaffrey et al, 2007).…”

Reactive Oxygen Species in Inflammation and Tissue Injury

“…During inflammation, enhanced oxidative stress can drastically alter the GSH/GSSG ratio toward glutathione GSSG, which prevents oligomeric assembly of occludin. Along this line, inflammation or hypoxia/reoxygenation-induced oxidative stress resulted in reduced occludin oligomerization (279,281). Mice and rat deficient in SOD1 also have decreased expression of occludin, claudin-5, and ZO-1 correlating with excessive ROS generation (260,261,472).…”