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

Gwen McCaffrey, Colin L. Willis, William D. Staatz, Nicole Nametz, Carolyn A. Quigley, Sharon Hom, Jeffrey J. Lochhead, Thomas P Davis

Research output: Contribution to journalArticle

56 Citations (Scopus)

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% O 2, 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.

Original languageEnglish (US)
Pages (from-to)58-71
Number of pages14
JournalJournal of Neurochemistry
Volume110
Issue number1
DOIs
StatePublished - Jul 2009

Fingerprint

Occludin
Tight Junctions
Blood-Brain Barrier
Sodium Dodecyl Sulfate
Protein Isoforms
Cell membranes
Membrane Lipids
Molecular Weight
Molecular weight
Cell Membrane
Hypoxia
Tight Junction Proteins
Density Gradient Centrifugation
Centrifugation
Reducing Agents
Brain Edema
Capillary Permeability
Microvessels
Electrophoresis
Disulfides

Keywords

  • Blood-brain barrier
  • Density gradient
  • Lipid raft
  • Occludin
  • Tight junction

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

Cite this

Occludin oligomeric assemblies at tight junctions of the blood-brain barrier are altered by hypoxia and reoxygenation stress. / McCaffrey, Gwen; Willis, Colin L.; Staatz, William D.; Nametz, Nicole; Quigley, Carolyn A.; Hom, Sharon; Lochhead, Jeffrey J.; Davis, Thomas P.

In: Journal of Neurochemistry, Vol. 110, No. 1, 07.2009, p. 58-71.

Research output: Contribution to journalArticle

McCaffrey, Gwen ; Willis, Colin L. ; Staatz, William D. ; Nametz, Nicole ; Quigley, Carolyn A. ; Hom, Sharon ; Lochhead, Jeffrey J. ; Davis, Thomas P. / Occludin oligomeric assemblies at tight junctions of the blood-brain barrier are altered by hypoxia and reoxygenation stress. In: Journal of Neurochemistry. 2009 ; Vol. 110, No. 1. pp. 58-71.
@article{faa83ee055ee493799154d31a13378f2,
title = "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{\%} O 2, 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.",
keywords = "Blood-brain barrier, Density gradient, Lipid raft, Occludin, Tight junction",
author = "Gwen McCaffrey and Willis, {Colin L.} and Staatz, {William D.} and Nicole Nametz and Quigley, {Carolyn A.} and Sharon Hom and Lochhead, {Jeffrey J.} and Davis, {Thomas P}",
year = "2009",
month = "7",
doi = "10.1111/j.1471-4159.2009.06113.x",
language = "English (US)",
volume = "110",
pages = "58--71",
journal = "Journal of Neurochemistry",
issn = "0022-3042",
publisher = "Wiley-Blackwell",
number = "1",

}

TY - JOUR

T1 - Occludin oligomeric assemblies at tight junctions of the blood-brain barrier are altered by hypoxia and reoxygenation stress

AU - McCaffrey, Gwen

AU - Willis, Colin L.

AU - Staatz, William D.

AU - Nametz, Nicole

AU - Quigley, Carolyn A.

AU - Hom, Sharon

AU - Lochhead, Jeffrey J.

AU - Davis, Thomas P

PY - 2009/7

Y1 - 2009/7

N2 - 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% O 2, 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.

AB - 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% O 2, 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.

KW - Blood-brain barrier

KW - Density gradient

KW - Lipid raft

KW - Occludin

KW - Tight junction

UR - http://www.scopus.com/inward/record.url?scp=67449128444&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=67449128444&partnerID=8YFLogxK

U2 - 10.1111/j.1471-4159.2009.06113.x

DO - 10.1111/j.1471-4159.2009.06113.x

M3 - Article

C2 - 19457074

AN - SCOPUS:67449128444

VL - 110

SP - 58

EP - 71

JO - Journal of Neurochemistry

JF - Journal of Neurochemistry

SN - 0022-3042

IS - 1

ER -