Evidence for differential changes of junctional complex proteins in murine neurocysticercosis dependent upon CNS vasculature

doi:10.1016/j.brainres.2007.07.010

Brain Research

Volume 1169, 12 September 2007, Pages 98-111

https://doi.org/10.1016/j.brainres.2007.07.010 Get rights and content

Abstract

The delicate balance required to maintain homeostasis of the central nervous system (CNS) is controlled by the blood–brain barrier (BBB). Upon injury, the BBB is disrupted compromising the CNS. BBB disruption has been represented as a uniform event. However, our group has shown in a murine model of neurocysticercosis (NCC) that BBB disruption varies depending upon the anatomical site/vascular bed analyzed. In this study further understanding of the mechanisms of BBB disruption was explored in blood vessels located in leptomeninges (pial vessels) and brain parenchyma (parenchymal vessels) by examining the expression of junctional complex proteins in murine brain infected with Mesocestoides corti. Both pial and parenchymal vessels from mock infected animals showed significant colocalization of junctional proteins and displayed an organized architecture. Upon infection, the patterned organization was disrupted and in some cases, particular tight junction and adherens junction proteins were undetectable or appeared to be undergoing proteolysis. The extent and timing of these changes differed between both types of vessels (pial vessel disruption within days versus weeks for parenchymal vessels). To approach potential mechanisms, the expression and activity of matrix metalloproteinase-9 (MMP-9) were evaluated by in situ zymography. The results indicated an increase in MMP-9 activity at sites of BBB disruption exhibiting leukocyte infiltration. Moreover, the timing of MMP activity in pial and parenchymal vessels correlated with the timing of permeability disruption. Thus, breakdown of the BBB is a mutable process despite the similar structure of the junctional complex between pial and parenchymal vessels and involvement of MMP activity.

Introduction

The BBB protects the CNS from compositional fluctuations within the blood stream by limiting the paracellular flux of hydrophilic molecules and the transmigration of leukocytes into the CNS (Wolburg and Lippoldt, 2002). The BBB is composed of a network of highly specialized endothelial cells surrounded by a basal lamina in both pial and parenchymal vessels. In the latter, however, astrocytic endfeet processes ensheath the abluminal surface of these cells playing an important role in the maintenance of vessel impermeability (Allt and Lawrenson, 1997, Gloor et al., 2001, Prat et al., 2001). The paracellular cleft between adjacent lateral endothelial membranes is almost completely sealed because of the presence of a junctional complex that includes tight junctions (TJs) (Kniesel and Wolburg, 2000, Vorbrodt and Dobrogowska, 2003, Wolburg and Lippoldt, 2002) and adherens junctions (AJs) (Schulze and Firth, 1993).

Tight junctions (TJs) are the most distinctive molecules in the paracellular cleft because their presence results in a high transendothelial electrical resistance that restricts pinocytic activity and diffusion of polar solutes through the endothelium (Harhaj and Antonetti, 2004, Huber et al., 2001). TJs are located in the most apical section of the plasma membrane, and they are composed of a combination of transmembrane (occludin and claudins) and cytoplasmic molecules including zonula occludens proteins ZO-1, ZO-2, ZO-3 and others (Petty and Lo, 2002). The transmembrane members form heterodimeric bridges that block paracellular diffusion, and these TJ proteins mediate the gate function of the BBB (Tsukita et al., 1999). The TJs are linked and stabilized via the endothelial cell cytoskeleton by complex interactions between transmembrane and cytoplasmic proteins. This linkage is mediated by ZO proteins that serve as a bridge between the cytoplasmic tails of occludin and claudins with the actin cytoskeleton (Hawkins and Davis, 2005). In the CNS, claudins-1, -3 and -5, together with occludin, have been found in endothelial TJs forming the BBB (Liebner et al., 2000, Morita et al., 1999). Another TJ-associated protein is the junctional adhesion molecule (JAM) (Martin-Padura et al., 1998). Three of these proteins (JAM-A, JAM-B and JAM-C) have been identified, and they belong to the immunologlobulin superfamily (Petty and Lo, 2002). JAM-A is expressed in blood vessels and mediates contact between neighboring cells via homophilic interactions (Aurrand-Lions et al., 2001, Bazzoni et al., 2000a). JAM-A is a ligand for LFA-1 and is involved in monocyte and neutrophil transmigration (Ostermann et al., 2002).

The adherens junctions (AJs) are ubiquitous in the vasculature and mediate the adhesion of endothelial cells to each other and partially regulate paracellular permeability (Bazzoni and Dejana, 2004, Brown and Davis, 2002). They consist of the transmembrane proteins cadherins and the cytoplasmic proteins catenins. Catenins are linked with the actin cytoskeleton and support the formation of adhesive contacts between cells. AJs assemble via homophilic interactions between the extracellular domains of calcium-dependent cadherin on the surface of adjacent cells (Ballabh et al., 2004). TJ and AJ components are known to interact, particularly ZO-1 and catenins, and such interplay is known to influence TJ assembly (Matter and Balada, 2003).

BBB disruption is a critical process associated with development and progression of several CNS pathologies involving inflammation (Petty and Lo, 2002). Neurocysticercosis (NCC) is one such disease caused by the parasite T. solium where inflammatory infiltrates cause a range of symptoms including epileptic seizures, headaches, hydrocephalus and even death (White, 2000). It is the leading cause of epilepsy worldwide. Recently, we have shown that the kinetics of increased BBB permeability in a murine model of NCC caused by the closely related parasite Mesocestoides corti depend upon several parameters including the anatomical area, type of vascular bed, cytokine microenvironment and the subsets of infiltrating leukocytes (Alvarez and Teale, 2006).

The aim of this study was to investigate changes in the expression and structural organizations of TJ and AJ proteins in pial and parenchymal vessels during CNS infection using a murine model of NCC. Brain sections were obtained from mice infected with the cestode M. corti. Pial vessels and parenchymal vessels were studied using in situ immunofluorescent staining to detect expression of junctional complex proteins. Disruption of junctional complex proteins positively correlated with specific areas of leukocyte extravasation in parasite infected animals. As matrix metalloproteinases (MMPs), particularly MMP-9, have been associated with barrier disruption (Gidday et al., 2005, Justicia et al., 2003, Sellebjerg and Sorensen, 2003), in situ zymography was used to determine MMP-9 activity in compromised vessels. The findings suggest that there is a direct correlation between the distinct temporal changes in the structural alterations of junctional complex proteins in pial and parenchymal vessels and the timing of detectable MMP-9 activity in these vessels.

Section snippets

Alterations in the structural organization of tight junction proteins upon infection

Upon infection, analysis of whole brain sections (n = 5) revealed that 36% ± 5 of pial vessels and 1% ± 0.1 of parenchymal vessels have perivascular immune infiltrates by 1 and 5 weeks post-infection (n = 5), respectively. To test if the expression and spatial pattern of TJ molecules change during the infectious process, several antibodies directed against TJ proteins including ZO-1, occludin, claudin-3, claudin-5 and JAM-A were used for in situ immunofluorescence microscopy. Pial and parenchymal

Discussion

Under basal physiological conditions the BBB limits the entrance of leukocytes supporting CNS immunosurveillance. But upon infections such as NCC, bacterial meningitis or HIV encephalitis, disruption of the BBB integrity is associated with infiltration of antigen presenting cells, neutrophils, B cells, αβ T cells and γδ T cells (Alvarez and Teale, 2006, Bell, 2004, Cardona et al., 1999, Koedel et al., 2002). Our data show that the marked differences in the kinetics of leukocyte extravasation

Animals

Female BALB/c mice 3–5 weeks old were purchased from the National Cancer Institute Animal Program (Bethesda, MD). Wild-type FVB mice and MMP-9 null (MMP-9 deficient) mice 3–5 weeks old were purchased from Jackson Laboratory (Bar Harbor, ME). Animal experiments were conducted under the guidelines of the University of Texas System, the U.S. Department of Agriculture, and the National Institutes of Health.

Parasites and inoculations

M. corti metacestodes were maintained by serial intraperitoneal inoculation of 8 to

References (76)

G. Allt et al.
Is the pial microvessel a good model for blood–brain barrier studies?
Brain Res Brain Res. Rev.
(1997)
J.I. Alvarez et al.
Breakdown of the blood brain barrier and blood–cerebrospinal fluid barrier is associated with differential leukocyte migration in distinct compartments of the CNS during the course of murine NCC
J. Neuroimmunol.
(2006)
M. Aurrand-Lions et al.
Heterogeneity of endothelial junctions is reflected by differential expression and specific subcellular localization of the three JAM family members
Blood
(2001)
P. Ballabh et al.
The blood–brain barrier: an overview: structure, regulation, and clinical implications
Neurobiol. Dis.
(2004)
G. Bazzoni et al.
Homophilic interaction of junctional adhesion molecule
J. Biol. Chem.
(2000)
G. Bazzoni et al.
Interaction of junctional adhesion molecule with the tight junction components ZO-1, cingulin, and occludin
J. Biol. Chem.
(2000)
S.M. Gloor et al.
Molecular and cellular permeability control at the blood–brain barrier
Brain Res. Brain Res. Rev.
(2001)
K.J. Gurney et al.
Blood–brain barrier disruption by stromelysin-1 facilitates neutrophil infiltration in neuroinflammation
Neurobiol. Dis.
(2006)
N.S. Harhaj et al.
Regulation of tight junctions and loss of barrier function in pathophysiology
Int. J. Biochem. Cell Biol.
(2004)
J.D. Huber et al.
Molecular physiology and pathophysiology of tight junctions in the blood–brain barrier
Trends Neurosci.
(2001)

M.A. Kelly et al.

Matrix metalloproteinase activation and blood–brain barrier breakdown following thrombolysis

Exp. Neurol.

(2006)

A. Khandoga et al.

Junctional adhesion molecule-A deficiency increases hepatic ischemia–reperfusion injury despite reduction of neutrophil transendothelial migration

Blood

(2005)

U. Koedel et al.

Pathogenesis and pathophysiology of pneumococcal meningitis

Lancet Infect. Dis.

(2002)

C.H. Lai et al.

Critical role of actin in modulating BBB permeability

Brain Res. Brain Res. Rev.

(2005)

S. Liebner et al.

Correlation of tight junction morphology with the expression of tight junction proteins in blood–brain barrier endothelial cells

Eur. J. Cell Biol.

(2000)

K.J. Mandell et al.

The JAM family of proteins

Adv. Drug Deliv. Rev.

(2005)

K. Matter et al.

Functional analysis of tight junctions

Methods

(2003)

M.S. McAllister et al.

Mechanisms of glucose transport at the blood–brain barrier: an in vitro study

Brain Res.

(2001)

Y. Persidsky et al.

Rho-mediated regulation of tight junctions during monocyte migration across the blood–brain barrier in HIV-1 encephalitis (HIVE)

Blood

(2006)

M.A. Petty et al.

Junctional complexes of the blood–brain barrier: permeability changes in neuroinflammation

Prog. Neurobiol.

(2002)

F. Sellebjerg et al.

Chemokines and matrix metalloproteinase-9 in leukocyte recruitment to the central nervous system

Brain Res. Bull.

(2003)

S.K. Shaw et al.

Reduced expression of junctional adhesion molecule and platelet/endothelial cell adhesion molecule-1 (CD31) at human vascular endothelial junctions by cytokines tumor necrosis factor-alpha plus interferon-gamma does not reduce leukocyte transmigration under flow

Am. J. Pathol.

(2001)

S. Tsukita et al.

Structural and signalling molecules come together at tight junctions

Curr. Opin. Cell Biol.

(1999)

P.E. Van den Steen et al.

Neutrophil gelatinase B potentiates interleukin-8 tenfold by aminoterminal processing, whereas it degrades CTAP-III, PF-4, and GRO-alpha and leaves RANTES and MCP-2 intact

Blood

(2000)

A.W. Vorbrodt et al.

Molecular anatomy of intercellular junctions in brain endothelial and epithelial barriers: electron microscopist's view

Brain Res. Brain Res. Rev.

(2003)

H. Wolburg et al.

Tight junctions of the blood–brain barrier: development, composition and regulation

Vasc. Pharmacol.

(2002)

I.E. Andras et al.

HIV-1 Tat protein alters tight junction protein expression and distribution in cultured brain endothelial cells

J. Neurosci. Res.

(2003)

M. Asahi et al.

Effects of matrix metalloproteinase-9 gene knock-out on the proteolysis of blood–brain barrier and white matter components after cerebral ischemia

J. Neurosci.

(2001)

C.J. Bachmeier et al.

Comparison of drug efflux transport kinetics in various blood–brain barrier models

Drug Metab. Dispos.

(2006)

G. Bazzoni et al.

Endothelial cell-to-cell junctions: molecular organization and role in vascular homeostasis

Physiol. Rev.

(2004)

J.E. Bell

An update on the neuropathology of HIV in the HAART era

Histopathology

(2004)

R.C. Brown et al.

Calcium modulation of adherens and tight junction function: a potential mechanism for blood–brain barrier disruption after stroke

Stroke

(2002)

A.R. Calabria et al.

Puromycin-purified rat brain microvascular endothelial cell cultures exhibit improved barrier properties in response to glucocorticoid induction

J. Neurochem.

(2006)

A.E. Cardona et al.

Development of an animal model for neurocysticercosis: immune response in the central nervous system is characterized by a predominance of gamma delta T cells

J. Immunol.

(1999)

P.O. Couraud

Infiltration of inflammatory cells through brain endothelium

Pathol. Biol. (Paris)

(1998)

A.G. de Boer et al.

The transference of results between blood–brain barrier cell culture systems

Eur. J. Pharm. Sci.

(1999)

O. Del Brutto

Cysticercosis in encephalitic phase

O.H. Del Brutto et al.

Neurocysticercosis: an update

Rev. Infect. Dis.

(1988)

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^☆: Supported by grants NS 35974 and AI 59703 from the National Institutes of Health.

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Research ReportEvidence for differential changes of junctional complex proteins in murine neurocysticercosis dependent upon CNS vasculature☆

Abstract

Introduction

Section snippets

Alterations in the structural organization of tight junction proteins upon infection

Discussion

Animals

Parasites and inoculations

Brain Res Brain Res. Rev.

J. Neuroimmunol.

Blood

Neurobiol. Dis.

J. Biol. Chem.

J. Biol. Chem.

Brain Res. Brain Res. Rev.

Neurobiol. Dis.

Int. J. Biochem. Cell Biol.

Trends Neurosci.

Exp. Neurol.

Blood

Lancet Infect. Dis.

Brain Res. Brain Res. Rev.

Eur. J. Cell Biol.

Adv. Drug Deliv. Rev.

Methods

Brain Res.

Blood

Prog. Neurobiol.

Brain Res. Bull.

Am. J. Pathol.

Curr. Opin. Cell Biol.

Blood

Brain Res. Brain Res. Rev.

Vasc. Pharmacol.

HIV-1 Tat protein alters tight junction protein expression and distribution in cultured brain endothelial cells

J. Neurosci. Res.

Effects of matrix metalloproteinase-9 gene knock-out on the proteolysis of blood–brain barrier and white matter components after cerebral ischemia

J. Neurosci.

Comparison of drug efflux transport kinetics in various blood–brain barrier models

Drug Metab. Dispos.

Endothelial cell-to-cell junctions: molecular organization and role in vascular homeostasis

Physiol. Rev.

An update on the neuropathology of HIV in the HAART era

Histopathology

Calcium modulation of adherens and tight junction function: a potential mechanism for blood–brain barrier disruption after stroke

Stroke

Puromycin-purified rat brain microvascular endothelial cell cultures exhibit improved barrier properties in response to glucocorticoid induction

J. Neurochem.

Development of an animal model for neurocysticercosis: immune response in the central nervous system is characterized by a predominance of gamma delta T cells

J. Immunol.

Infiltration of inflammatory cells through brain endothelium

Pathol. Biol. (Paris)

The transference of results between blood–brain barrier cell culture systems

Eur. J. Pharm. Sci.

Cysticercosis in encephalitic phase

Neurocysticercosis: an update

Rev. Infect. Dis.

Research Report
Evidence for differential changes of junctional complex proteins in murine neurocysticercosis dependent upon CNS vasculature☆