Dose-dependent expression of claudin-5 is a modifying factor in schizophrenia.

Schizophrenia is a neurodevelopmental disorder that affects up to 1% of the general population. Various genes show associations with schizophrenia and a very weak nominal association with the tight junction protein, claudin-5, has previously been identified. Claudin-5 is expressed in endothelial cells forming part of the blood-brain barrier (BBB). Furthermore, schizophrenia occurs in 30% of individuals with 22q11 deletion syndrome (22q11DS), a population who are haploinsufficient for the claudin-5 gene. Here, we show that a variant in the claudin-5 gene is weakly associated with schizophrenia in 22q11DS, leading to 75% less claudin-5 being expressed in endothelial cells. We also show that targeted adeno-associated virus-mediated suppression of claudin-5 in the mouse brain results in localized BBB disruption and behavioural changes. Using an inducible 'knockdown' mouse model, we further link claudin-5 suppression with psychosis through a distinct behavioural phenotype showing impairments in learning and memory, anxiety-like behaviour and sensorimotor gating. In addition, these animals develop seizures and die after 3-4 weeks of claudin-5 suppression, reinforcing the crucial role of claudin-5 in normal neurological function. Finally, we show that anti-psychotic medications dose-dependently increase claudin-5 expression in vitro and in vivo while aberrant, discontinuous expression of claudin-5 in the brains of schizophrenic patients post mortem was observed compared to age-matched controls. Together, these data suggest that BBB disruption may be a modifying factor in the development of schizophrenia and that drugs directly targeting the BBB may offer new therapeutic opportunities for treating this disorder.

The chemokine receptor CCR2 mediates the binding and internalization of monocyte chemoattractant protein-1 along brain microvessels.

Previous results from this laboratory revealed the presence of high-affinity saturable binding sites for monocyte chemoattractant protein-1 (MCP-1) along human brain microvessels (Andjelkovic et al., 1999; Andjelkovic and Pachter, 2000), which suggested that CC chemokine receptor 2 (CCR2), the recognized receptor for this chemokine, was expressed by the brain microvascular endothelium. To test the role of CCR2 directly in mediating MCP-1 interactions with the brain microvasculature, we assessed MCP-1 binding activity in murine brain microvessels isolated from wild-type mice and from CCR2 (-/-) mice engineered to lack this receptor. Results demonstrate that MCP-1 binding is greatly attenuated in microvessels prepared from CCR2 (-/-) mice compared with wild-type controls. Moreover, microvessels from wild-type mice exhibited MCP-1-induced downmodulation in MCP-1 binding and a recovery of binding activity that was not dependent on de novo protein synthesis. Furthermore, MCP-1 was shown to be internalized within wild-type microvessels, but not within microvessels obtained from CCR2 (-/-) mice, additionally demonstrating that CCR2 is obligatory for MCP-1 endocytosis. Last, internalization of MCP-1, but not transferrin, was observed to be inhibited by disruption of caveolae. Internalized MCP-1 also colocalized at some sites with caveolin-1, a major protein of caveolae, implying that this chemokine is endocytosed, in part, via nonclathrin-coated vesicles. These results prompt consideration that MCP-1 signals may be relayed across the blood-brain barrier by highly specialized interactions of this chemokine with its cognate receptor, CCR2, along brain microvascular endothelial cells.

Qualitative and quantitative analysis of monocyte transendothelial migration by confocal microscopy and three-dimensional image reconstruction.

A novel method for qualitative and quantitative analysis of monocyte transendothelial migration is described. By labeling monocytes and endothelial cells with different fluorophores, and utilizing confocal microscopy and three-dimensional image reconstruction, transmigrating monocytes were resolved and quantified within a subendothelial collagen gel. Comparison of monocyte migration across endothelial monolayers derived from human brain microvessels versus umbilical veins revealed diapedesis across brain endothelium to be significantly delayed. Inclusion of astrocytes within the subendothelial collagen gel resulted in the formation of an array of astrocytic processes that simulated the glia limitans surrounding brain microvessels in situ, thus yielding a more physiologic paradigm of the blood-brain barrier. By virtue of its unique capacity to provide information on the total number of migrating cells, this analytic approach overcomes significant caveats associated with sampling only aspects of the migration process. The potential adaptability of this method to computer-assisted analysis further enhances its prospective use in high-throughput screening.

Monocyte:astrocyte interactions regulate MCP-1 expression in both cell types.

As astrocytes are a source of monocyte chemoattractant protein-1 (MCP-1) and lie in close apposition to brain microvessels, interactions between astrocytes and infiltrating monocytes might regulate production of this chemokine. To investigate this possibility, a monocyte:astrocyte co-culture model was utilized to assess the respective roles of these two cell types in regulating MCP-1 production. Results indicate that, while neither monocytes nor astrocytes alone produce detectable levels of MCP-1, co-culture of these two cell types results in time-dependent production of this chemokine. Such production requires de novo protein synthesis and is dependent on physical contact between monocytes and astrocytes, involving engagement of the cell-adhesion molecules ICAM-1 and VCAM-1. Additionally, interleukin 1-beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) are soluble mediators of this response. These findings imply that monocyte extravasation into the CNS may be critically regulated at the blood-brain barrier by specialized monocyte:astrocyte interactions.

Characterization of binding sites for chemokines MCP-1 and MIP-1alpha on human brain microvessels.

The presence of binding sites for the beta chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) has recently been identified on human brain microvessels. We extend these findings in this report to reveal that such sites exemplify characteristics of the recognized major receptors for MCP-1 and MIP-1alpha: CCR2, and CCR1 and CCR5, respectively. Specifically, labeled MCP-1 binding to isolated brain microvessels was inhibited by unlabeled MCP-1 and MCP-3, the latter another CCR2 ligand, but not by MIP-1alpha. Inhibition of labeled MIP-1alpha binding was achieved with unlabeled MIP-1alpha and RANTES, the latter a beta chemokine that binds to both CCR1 and CCR5, but not by MCP-1. Labeled MIP-1alpha binding was also antagonized by unlabeled MCP-3, which is also recognized by CCR1, and MIP-1beta, which is a ligand for CCR5. Labeled MCP-1 and MIP-1alpha were further observed to be internalized within the endothelial cells of brain microvessels, following their binding to the microvascular surface at 37 degrees C. Additionally, exposure of microvessels to unlabeled MCP-1 or MIP-1alpha was accompanied by the initial loss and subsequent recovery of surface binding sites for these chemokines, which occurred on a time scale consistent with ligand-induced endocytosis and recycling. These collective features bear striking similarity to those that characterize interactions of MCP-1 and MIP-1alpha with their receptors on leukocytes and underscore the concept of cognate chemokine receptors on brain microvascular endothelium.

Engagement of the scavenger receptor is not responsible for beta-amyloid stimulation of monocytes to a neurocytopathic state.

Experiments were performed to determine if scavenger receptors (SRs) play a role in amyloid beta (Abeta) stimulation of peripheral blood monocyte (PBM) neurotoxicity. Results indicate that Abeta does not block binding of the SR ligand DiI-acetylated low density lipoprotein to PBM, nor does another SR ligand, fucoidin, inhibit Abeta-PBM binding. Moreover, neither of three SR ligands alone stimulates neurotoxicity in PBM, nor antagonizes the ability of Abeta to activate PBM to a neurocytopathic state. Such findings suggest that Abeta's action is not dependent upon engagement of the SR ligand binding domain and raise doubts about the role of SR in Abeta neurotoxicity.

Visualization of beta-amyloid peptide (Abeta) phagocytosis by human mononuclear phagocytes: dependency on Abeta aggregate size.

Previous studies from this laboratory have shown that human monocytes exposed to beta-amyloid peptide (Abeta) exert a graded neurotoxic response in an organotypic brain culture paradigm. Moreover, this toxicity can be reduced by compounds that inhibit cell motility and phagocytosis, suggesting that internalization of Abeta may be a requirement for neurotoxic action. To confirm that Abeta is indeed phagocytosed by monocytes and to further lay the groundwork for resolving the possible linkage between this process and neurotoxicity, we examined Abeta:monocyte interactions using immunocytochemistry and fluorescence histochemistry followed by confocal microscopy and three-dimensional image reconstruction. Internalization of Abeta was detected by 24 hr following exposure of monocytes to the purified peptide, and the relative efficacy of this process appeared to be influenced by the size of the Abeta aggregates. Specifically, smaller aggregates were observed to be more efficiently internalized, while larger Abeta masses tended to reside only on the monocyte surface, apparently bound to several monocytes at once. Both colchicine and cytochalasin D, cytoskeletal perturbants that block phagocytosis, caused Abeta to accumulate in deep pits within monocytes and inhibited complete envelopment by monocyte cytoplasm. These results suggest that monocytes can indeed phagocytose aggregates of Abeta and that this process may be critical in activating these cells to a neurocytopathic state. Accordingly, interference of Abeta phagocytosis by monocytes or monocyte-derived cells may be a novel target for therapeutic action.

Expression of binding sites for beta chemokines on human astrocytes.

Astrocytes are major sources of chemokines and are thus critical effectors of central nervous system (CNS) inflammation. However, it is as yet unclear whether these cells, like leukocytes, also possess receptors for chemokines (CCRs). To address this issue, we utilized a novel fluorescence approach to detect qualitatively and quantitatively binding sites for biotinylated derivatives of the beta chemokines monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) on cultured human fetal astrocytes. Both chemokines were found to bind to the surface of astrocytes in a specific and saturable manner and with the high-affinity typical of these chemokines' binding to leukocyte CCRs. Binding of labeled MCP-1 and of labeled MIP-1alpha was antagonized by the respective unlabeled homologue but not by the unlabeled heterologous chemokine. Binding of labeled MCP-1 was also inhibited by unlabeled MCP-3, both of which are ligands for CCR2. In a parallel manner, binding of labeled MIP-1alpha was first shown to be attenuated by unlabeled RANTES, both of which recognize CCR1 and CCR5, and then separately antagonized by MCP-3 and MIP-1beta, which bind to CCR1 and CCR5, respectively. Finally, binding of both labeled chemokines was observed to be modulated in response to astrocyte stimulation by proinflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), further indicating that these binding sites are subject to regulation and, thus, likely to be physiologically responsive. Collectively, these results indicate that binding sites exhibiting characteristics of chemokine receptors exist on human astrocytes. Such sites might function in the recruitment of both astrocytes and leukocytes to specified brain regions during physiological and pathophysiological processes.

Visualization of chemokine binding sites on human brain microvessels.

The chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) aid in directing leukocytes to specific locales within the brain and spinal cord during central nervous system inflammation. However, it remains unclear how these chemokines exert their actions across a vascular barrier, raising speculation that interaction with endothelial cells might be required. Therefore, experiments were performed to determine whether binding domains for these chemokines exist along the outer surface of brain microvessels, a feature that could potentially relay chemokine signals from brain to blood. Using a biotinylated chemokine binding assay with confocal microscopy and three-dimensional image reconstruction, spatially resolved binding sites for MCP-1 and MIP-alpha around human brain microvessels were revealed for the first time. Binding of labeled MCP-1 and MIP-1alpha could be inhibited by unlabeled homologous but not heterologous chemokine, and was independent of the presence of heparan sulfate, laminin, or collagen in the subendothelial matrix. This is the first evidence of specific and separate binding domains for MCP-1 and MIP-1alpha on the parenchymal surface of microvessels, and highlights the prospect that specific interactions of chemokines with microvascular elements influence the extent and course of central nervous system inflammation.

Macrophages/microglial cells in human central nervous system during development: an immunohistochemical study.

The development of microglia and macrophages was studied in 14 human embryos and fetuses ranging in age from 4.5-13.5 gestational weeks (g.w.), using lectins, Ricinus communis agglutinin-1 [RCA-1], and Lycopersicon esculentum, tomato lectin (TL), which recognize macrophages and microglia, and antibodies for the macrophage antigen CD68. Lectin-positive (+) cells were observed at 4.5 g.w., the youngest age examined. They were detected in the leptomeninges around the neural tube, and only rarely were observed in the CNS parenchyma. At 5.5 g.w., lectin+ cells were present throughout the CNS parenchyma, and a portion of these cells could also be labeled with antibody to CD68. In subsequent weeks, both types of cells, lectin+ and CD68+/lectin+ cells co-existed and progressively developed typical microglial morphology. In addition, in double label experiments, an antibody that labels CD14 antigen present on monocytes, hematogenous precursors of tissue macrophages, did not label either lectin+ or CD68+/lectin+ cells in CNS parenchyma. Additional immunocytochemical studies with appropriate markers excluded the possibility that any of the cells described here were either astrocytes, oligodendrocytes, endothelial cells or neurons. Our finding that one class of cells can be labeled early only with lectins, while another can be labeled with both lectins and CD68 macrophage antibody, may reflect a different origin of microglia in the early embryonic CNS compared to the fetal stages. This subdivision appears to be maintained in the adult brains as well.

Central nervous system endothelium in neuroinflammatory, neuroinfectious, and neurodegenerative disease.

Accumulating evidence points toward a significant role for the microvascular endothelium in the pathogenesis of several neurologic conditions. This review highlights those biochemical, anatomical, and physiological features of the endothelium thought to be dysfunctional in these disease states, and elaborates on novel treatment modalities that target the endothelium.

Suppression of A beta-induced monocyte neurotoxicity by antiinflammatory compounds.

Previous work from this laboratory has demonstrated that prior exposure of peripheral blood monocytes (PBM) to aggregated beta-amyloid peptide (A beta), the major protein comprising the amyloid plaques characteristically present in the brain of Alzheimer disease (AD)-afflicted individuals, activates these cells to a neurotoxic state when co-cultured with brain tissue. In this report we extend these findings to further show that such A beta-induced PBM neurotoxicity can be inhibited by three differentially-acting antiinflammatory drugs, indomethacin, dexamethasone, and colchicine, which are typically used clinically to treat peripheral inflammatory disease. In addition, evidence is presented that these toxic effects are initiated, in large part, by soluble factors released from A beta-stimulated PBM. Our results suggest a rationale for antiinflammatory therapy in the treatment of AD.

A rapid and sensitive radioimmunoassay for the detection of human cytomegalovirus binding and infection of human fibroblasts.

A rapid and sensitive radioimmunoassay for the quantitation of HCMV binding and infection of human fibroblasts (HFF) was developed. The protocol involves the use of a monoclonal antibody (27-156) reactive with HCMV gB (alpha-gB), followed by an 125I-labeled second antibody to mouse IgG. Antibody to gB bound specifically to HFF inoculated with HCMV when compared to sham inoculated cells or cells inoculated with HSV (strain KOS). Antibody to gB also bound to HFF infected with HCMV 48 h prior to assay. The binding of antibody to HFF inoculated with HCMV was found to be dependent on antibody concentration and to demonstrate saturable kinetics. Moreover, antibody binding was directly dependent on the concentration of the virus inoculum, using either conventional viral preparations or gradient purified HCMV. The binding of antibody to HFF inoculated with HCMV at 4 degrees C was found to be dependent on antibody concentration and to demonstrate saturable kinetics. Displacement of HCMV binding to HFF with the proteoglycan heparin sulfate could be detected, thus allowing for competitive binding studies. This binding assay allows for the relative quantitation of HCMV binding to cells and will be useful for examining the early events of cell-viral interactions.

Neurocytopathic effects of beta-amyloid-stimulated monocytes: a potential mechanism for central nervous system damage in Alzheimer disease.

Growing evidence indicates that cells of the mononuclear phagocyte lineage, which includes peripheral blood monocytes (PBM) and tissue macrophages, participate in a variety of neurodestructive events and may play a pivotal role in neurodegenerative conditions such as Alzheimer disease. The present study sought to determine whether exposure of PBM to beta-amyloid peptide (A beta), the major protein of the amyloid fibrils that accumulate in the brain in Alzheimer disease, could induce cytopathic activity in these cells upon their subsequent incubation with neural tissue. PBM were incubated with A beta for 3 days, centrifuged and washed to remove traces of cell-free A beta, and then applied to organotypic cultures of rat brain for varying periods of time. By using a cell-viability assay to quantitate neurocytopathic effect, an increase in the ratio of dead to live cells was detected in cultures containing A beta-stimulated PBM versus control PBM (stimulated with either bovine serum albumin or reverse A beta peptide) as early as 3 days after coculture. The ratio of dead to live cells increased further by 10 days of coculture. By 30 days of coculture, the dead to live cell ratio remained elevated, and the intensity of neurocytopathic effect was such that large areas of brain mass dissociated from the cultures. These results indicate that stimulation of PBM with A beta significantly heightens their neurocytopathic activity and highlight the possibility that inflammatory reactions in the brain play a role in the neurodegeneration that accompanies Alzheimer disease.

Isolation and culture of human brain microvessel endothelial cells for the study of blood-brain barrier properties in vitro.

A simplified protocol for isolating brain microvessel endothelial cells (BMEC) from human cortex and culturing them on a thick collagen plug is described. This method results in the establishment of monolayers of BMEC that retain numerous properties indicative of the blood-brain barrier (BBB) phenotype, such as elevated transendothelial electrical resistance, attenuated paracellular flux of sucrose, peripheral actin filament distribution and asymmetric localization of the efflux peptide, P-glycoprotein, to the apical (luminal) BMEC surface. The novel 3-dimensional nature of this model system renders it ideally suitable for assaying such varied aspects of BBB physiology as solute transport, pathogen penetrance, leukocyte infiltration and tumor metastasis into the brain. Moreover, the fact that the system is derived from human brain allows for the study of pathogenetic mechanisms that may only be operative in humans.

Growth of brain microvessel endothelial cells on collagen gels: applications to the study of blood-brain barrier physiology and CNS inflammation.

Brain microvessel endothelial cells (BMEC) exhibit the tendency to migrate through 3.0-vm pore semipermeable inserts and establish monolayers on both apical and basal filter surfaces. This can potentially lead to complications in accurately assessing a wide variety of physiologic parameters uniquely associated with these cells. To avoid this problem, we have explored growing BMEC on Transwell filters coated with hydrated collagen gels. BMEC seeded on such gels grow as a monolayer until confluency, but do not invade the subendothelial collagen matrix or the underlying support filter. Furthermore, BMEC grown in this manner exhibit biochemical, morphologic, and electrophysiologic properties reflective of the endothelial cells that comprise the blood-brain barrier in vivo. Although the collagen gel acts as an impenetrable barrier to BMEC, and thus ensures the growth of only a single layer of cells, it nevertheless can be infiltrated by monocytes that have been stimulated by a chemotaxin to undergo diapedesis. Thus, growing BMEC on collagen gel-coated Transwells has broad applications for the in vitro study of both blood-brain barrier physiology as well as the mechanisms underlying central nervous system inflammation.

Beta-actin mRNA-binding proteins associated with the cytoskeletal framework.

Association of mRNA with the cytoskeletal framework (CSK) is thought to play a strategic role in the placement of mRNA in the cytoplasm. However, the molecular determinants underlying mRNA/CSK association are completely unknown. To begin addressing this issue, we have employed a binding assay to identify proteins of the CSK compartment of NIH 3T3 cells that bind in-vitro-transcribed 32P-labelled beta-actin mRNA with high affinity. Three proteins, of approximate molecular masses 27, 50 and 97 kDa, were observed to exhibit strong binding. Binding to these proteins took place at physiological salt concentration and withstood washing in 0.5 M salt. Furthermore, binding was unaffected by heparin but was inhibited by unlabelled beta-actin mRNA. Treatment of isolated CSKs with the microfilament-severing agent DNase I abolished all beta-actin mRNA-binding activities, thus suggesting a possible association of beta-actin mRNA with the microfilament network in situ. Removal of the 3' untranslated region (UTR) significantly reduced beta-actin mRNA binding to all three CSK proteins but removal of the 5' UTR mainly affected binding to the 97-kDa species and that to a lesser extent. beta-Tubulin mRNA bound to the same three CSK proteins as did beta-actin mRNA, but with considerably less avidity. In contrast, vimentin mRNA strongly recognized these CSK proteins, and further bound to a group of smaller proteins (< 29 kDa). As beta-actin mRNA, beta-tubulin mRNA and vimentin mRNA have been observed to occupy separate cytoplasmic locales, the proteins detected here may be operative both in binding mRNAs to the CSK in situ, as well as in localizing mRNA in the cytoplasm.

mRNA association with the cytoskeletal framework likely represents a physiological binding event.

A multitude of studies has indicated that the vast majority of mRNA and polyribosomes is associated with the detergent-resistant cytoskeletal framework (CSK). However, the nature and purpose of this association remain unclear. To begin unraveling the factors which may mediate this phenomenon, we examined the extent of association of four mRNAs (tubulin, vimentin, actin, and histone mRNA) with the CSKs of NIH 3T3 cells over a wide range of salt concentrations. Results indicate that the vast majority (greater than 90%) of each of these mRNAs remains associated with the CSK after detergent extraction of cells in low ionic strength buffer (25 mM NaCl). This association is manifest under conditions that cause the complete depolymerization of microtubules but that leave microfilaments and intermediate filaments intact. Even after extensive washing in buffer of approximately physiological ionic strength (150 mM NaCl), 75-85% of these mRNAs still remain associated with the CSK. However, at least 50% of each of these mRNAs can be eluted from the CSK by washing with buffer containing 250 mM NaCl. Not all the mRNAs, though, display the same elution profile. This suggests that different binding sites and/or different binding affinities may exist for different mRNAs. Surprisingly, close to 50% of the polyribosome population remains bound to the CSK despite washing in as much as 1.0 M NaCl. These adherent polyribosomes appear to be of the same size as those that are eluted, allaying the possibility that they are retained by the CSK simply due to size exclusion. Collectively, these data strongly imply that mRNAs are neither weakly adsorbed to the CSK nor physically trapped within the meshwork of cytoskeletal filaments.(ABSTRACT TRUNCATED AT 250 WORDS)