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

Stroke is the second leading cause of death worldwide (Donnan et al. 2008). On average, every 40 s someone suffers a stroke, and stroke is a leading cause of serious, long-term disability in the United States (http://www.strokeassociation.org). Stroke involves a cerebral blood vessel blockage, the consequence of which is that a particular region of the brain is deprived for a period of time of oxygen and nutrients. During the ischemic (hypoxic) and reperfusion (reoxygenation) phases of stroke there is a breach (i.e., leak) of the blood–brain barrier (BBB) (Sandoval and Witt 2008). The BBB is the critical interface between the CNS and the periphery. Anatomically comprised of approximately 20 m2 of cerebral microvascular endothelial cells (per 1.3 kg brain), the BBB forces water-soluble substances to pass from the systemic circulation to the brain by either a transcellular route (through microvascular endothelial cells) or a paracellular route (between microvascular endothelial cells) (Abbott et al. 2006). Paracellular diffusion of solutes, water, and ions between adjacent microvascular endothelial cells is severely restricted by tight junctions (TJs), and changes in TJ integrity during stroke directly promote the cerebral edema that is a leading cause of death subsequent to ischemic stroke (Bounds et al. 1981; Heo et al. 2005; Sandoval and Witt 2008). TJs are large, multiprotein complexes that extend into the interendothelial space to create a physical barrier to paracellular diffusion. Current understanding of the molecular composition of BBB TJs describes a framework of integral transmembrane proteins that interacts with cytoplasmic accessory, signaling, and regulatory proteins to generate a barrier to paracellular diffusion which is capable of rapid disassembly in response to extracellular stressors, such as pain, inflammation, and hypoxia (Hx) (Huber et al. 2001; Wolka et al. 2003; Hawkins and Davis 2005; Balda and Matter 2008; Forster 2008; Paris et al. 2008). The transmembrane protein occludin is critical for BBB TJ function (Harhaj and Antonetti 2004; Hawkins and Davis 2005). Its M-shaped topology, characterized by a four transmembrane helix architecture with cytoplasmic N- and C- termini (Furuse et al. 1993; Sanchez-Pulido et al. 2002), facilitates both structural and signaling roles at the BBB. Through its two extracellular loops, it interacts with homologous segments of occludin molecules on adjacent microvascular endothelial cell membranes to enable the fusion of the apposing cell membranes that creates a tight, interendothelial (TJ) seal to restrict paracellular diffusion (Lacaz-Vieira et al. 1999; Feldman et al. 2005). Through its C-terminus, it interacts with accessory proteins, zonula occludens (ZO-1, ZO-2 and ZO-3), thereby establishing a link to the underlying actin cytoskeleton (Furuse et al. 1994; Fanning et al. 1998). Also through its C-terminus, occludin interacts not only with other occludin molecules to form dimers (Blasig et al. 2006) but also with serine–threonine protein kinase C-zeta, tyrosine kinase c-Yes, regulatory (p85) subunit of phosphatidylinositol 3-kinase, gap junction component connexin-26 (Nusrat et al. 2000a), tyrosine kinase c-Src (Elias et al. 2009), casein kinase I epsilon (McKenzie et al. 2006), and casein kinase II (Smales et al. 2003). Other signaling and regulatory proteins reported to interact with occludin include Rab13 (Morimoto et al. 2005), Rho kinase (Yamamoto et al. 2008), caveolin (Nusrat et al. 2000b), clathrin (Ivanov et al. 2004), 33 kDa Vamp-associated protein (Lapierre et al. 1999), protein phosphatases PP2A and PP1 (Seth et al. 2007), E3 ubiquitin-protein ligase itch (Traweger et al. 2002), and transforming growth factor beta receptors I and II (Barrios-Rodiles et al. 2005). A relationship between microvascular endothelial paracellular permeability and occludin has been demonstrated in numerous in vivo and in vitro studies that employed a variety of Hx model systems and examined occludin localization and/or expression and post-translational modification (Mark and Davis 2002; Witt et al. 2003; Kago et al. 2006; Wang et al. 2007; Bangsow et al. 2008). Hypoxic stress-induced changes in occludin have been shown to be influenced by matrix metalloproteinases (Lohmann et al. 2004; Reijerkerk et al. 2006; Rosenberg and Yang 2007; Yang et al. 2007), hepatocyte growth factor (Date et al. 2006), phospholipase C gamma, phosphatidylinositol 3-kinase and protein kinase G (Fischer et al. 2004), antioxidants (Martin et al. 2006; Xu et al. 2007; Handa et al. 2008), clusterin (Kim et al. 2007), calcium (Park et al. 1999; Brown and Davis 2005), mitogen-activated protein kinase (Kevil et al. 2000; Fischer et al. 2005; Krizbai et al. 2005; Reijerkerk et al. 2008), chemokine (C-C motif) ligand 2 (Dimitrijevic et al. 2006), interleukin-1beta (Bolton et al. 1998; Yamagata et al. 2004), and reactive oxygen species (Kevil et al. 2000; Lee et al. 2004; Schreibelt et al. 2007). How occludin performs on a molecular level, both structural and signaling roles at the BBB TJ, is incompletely understood. Previously, we demonstrated the use of a neutral pH, detergent-free, isosmotic OptiPrep density gradient method to fractionate intact cerebral microvessels to isolate oligomeric occludin within the context of its normal plasma membrane lipid raft environment (McCaffrey et al. 2007). In this study, we employ this subcellular fractionation technique to investigate the effect of Hx and H/R on occludin oligomeric assemblies at the TJ. Using perfluoro-octanoic acid (PFO) to solubilize occludin oligomeric assemblies so that hydrophobic interactions are maintained (Ramjeesingh et al. 1999; Mitic et al. 2003), we used non-reducing and reducing SDS–PAGE/western blot analysis to investigate if Hx and H/R promote changes in the structural integrity and isoform composition of occludin multiprotein complexes at TJs of the BBB. We incorporated the use of an in vivo rat model of global H/R in which anesthetized animals were subjected for 1 h to an acute, moderate-hypoxic stress (inhaled 6% O2), and then exposed for a brief period (10 min) to normal atmospheric conditions (21% O2). Previous work in our laboratory showed that anesthetized animals subjected to these conditions of global Hx and reoxygenation exhibited increased paracellular permeability (i.e., leak) to [14C]sucrose, vasogenic brain edema, and altered expression of occludin isoforms in cerebral micro-vessel homogenates (Witt et al. 2003, 2008). Our data reveal, for the first time, that treatment of animals with Hx and H/R promotes a dynamic reorganization of occludin oligomeric assemblies in which changes occur in the appearance/disappearance of occludin isoforms of different molecular weights, in the association with different occludin isoforms with lipid rafts of different density, and in the extent of non-covalent (hydrophobic) and covalent (disulfide bond) interactions between occludin subunits within higher order structures. In addition, our data support the hypothesis that occludin oligomeric assemblies at TJs of the BBB in vivo are comprised of a backbone of disulfide-bonded subunits (engaged in a critical structural role) that is adorned with non-covalently attached dimeric and monomeric subunits whose greater accessibility to signaling and regulatory molecules, and ease of disassociation from the parent oligomeric core and subsequent association with different raft domains, allows them to perform a signaling role that incorporates diversity and amplification in the case of hypoxic stress.