Amyloid-beta-induced occludin down-regulation and increased permeability in human brain endothelial cells is mediated by MAPK activation.

Vascular dysfunction is emerging as a key pathological hallmark in Alzheimer's disease (AD). A leaky blood-brain barrier (BBB) has been described in AD patient tissue and in vivo AD mouse models. Brain endothelial cells (BECs) are linked together by tight junctional (TJ) proteins, which are a key determinant in restricting the permeability of the BBB. The amyloid beta (Abeta) peptides of 1-40 and 1-42 amino acids are believed to be pivotal in AD pathogenesis. We therefore decided to investigate the effect of Abeta 1-40, the Abeta variant found at the highest concentration in human plasma, on the permeability of an immortalized human BEC line, hCMEC/D3. Abeta 1-40 induced a marked increase in hCMEC/D3 cell permeability to the paracellular tracer 70 kD FITC-dextran when compared with cells incubated with the scrambled Abeta 1-40 peptide. Increased permeability was associated with a specific decrease, both at the protein and mRNA level, in the TJ protein occludin, whereas claudin-5 and ZO-1 were unaffected. JNK and p38MAPK inhibition prevented both Abeta 1-40-mediated down-regulation of occludin and the increase in paracellular permeability in hCMEC/D3 cells. Our findings suggest that the JNK and p38MAPK pathways might represent attractive therapeutic targets for preventing BBB dysfunction in AD.

The expression of tissue-type plasminogen activator, matrix metalloproteases and endogenous inhibitors in the central nervous system in multiple sclerosis: comparison of stages in lesion evolution.

The expression of tissue-type plasminogen activator (t-PA) and a number of metalloproteases as well as plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of metalloproteases-1 (TIMP-1) was analyzed in the central nervous system (CNS) of normal control and multiple sclerosis (MS) cases by immunohistopathology. The expression of t-PA was detectable only in the blood vessel matrix in control white matter, but positive infiltrating mononuclear cells were also observed in MS white matter and primary lesions. In active plaques this pattern converted to strong positivity of foamy macrophages in areas of demyelination, declining in chronic lesions. In general PAI-1 expression paralleled that of t-PA. Gelatinase A and B were detected predominantly in astrocytes and microglia throughout normal control white matter, with additional positive mononuclear cells in perivascular cuffs in MS white matter. In the demyelinating lesion there is widespread prominent expression of gelatinase B in reactive astrocytes and macrophages, which persists in astrocytes in the chronic lesion. TIMP-1 was also present in the vessel matrix and in lesional macrophages. These observations on the coexpression of enzymes and inhibitors of the matrix degrading cascade in CNS tissue pinpoint t-PA, a rate-limiting enzyme, and gelatinase B as therapeutic targets in MS.

Regulation of eukaryotic initiation factor eIF2B: glycogen synthase kinase-3 phosphorylates a conserved serine which undergoes dephosphorylation in response to insulin.

Eukaryotic initiation factor eIF2B catalyses a key regulatory step in mRNA translation. eIF2B and total protein synthesis are acutely activated by insulin, and this requires phosphatidylinositol 3-kinase (PI 3-kinase). The epsilon-subunit of eIF2B is phosphorylated by glycogen synthase kinase-3 (GSK-3), which is inactivated by insulin in a PI 3-kinase-dependent manner. Here we identify the phosphorylation site in eIF2Bepsilon as Ser540 and show that treatment of eIF2B with GSK-3 inhibits its activity. Ser540 is phosphorylated in intact cells and undergoes dephosphorylation in response to insulin. This is blocked by PI 3-kinase inhibitors. Insulin-induced dephosphorylation of this inhibitory site in eIF2B seems likely to be important in the overall activation of translation by this hormone.

Detection of interleukin-1 and interleukin-6 in adult rat brain, following mechanical injury, by in vivo microdialysis: evidence of a role for microglia in cytokine production.

In vivo levels of interleukin-1 (IL-1) and IL-6, present in the interstitial spaces of brain, have been repeatedly monitored up to 7 days after insertion of a microdialysis probe, designed to induce mechanical trauma to the brain. IL-1 is barely detectable immediately after implantation but over a 24-48 h period a 15-fold increase is seen. In contrast IL-6 levels at day 0 are high, increasing slightly (10%) by day 1 but decreasing to 40% by day 2. The temporal pattern of IL-6 recovery in the cerebrospinal fluid was similar to that in the dialysate but the levels were significantly lower and may reflect diffusion from the site of the probe lesion. Cellular sources of these cytokines include macrophages and neutrophils, which have infiltrated the lesion and microglia resident in the brain, which can be identified at the lesion site within 24 h of probe implantation. The astrocytic response to injury, evidenced by increased glial fibrillary acidic protein staining occurs much later, by day 7, and is unlikely to be responsible for IL-1 and IL-6 production found at 24-48 h. Since upon isolation and stimulation of microglia in vitro with lipopolysaccharide IL-1 and IL-6 can be measured in the supernatant, it would appear that they have the capacity to produce cytokines in vivo. Localised synthesis of cytokines at sites of brain injury by microglia would further stimulate microglia in an autocrine manner and also propagate the astrocytic reaction.

Inhibitory effect of interferon-gamma on LPS-induced interleukin 1 beta production by isolated adult rat brain microglia.

The effect of interferon-gamma (IFN-gamma) on lipopolysaccharide (LPS)-induced cytokine production has been examined in adult rat brain microglia. Following treatment of isolated cells in vitro with LPS, interleukin-1 (IL-1) and interleukin-6 (IL-6) levels in culture supernatants, determined by bioassay, were increased in a dose-dependent manner. IL-6 was particularly sensitive to LPS-stimulation. Using in situ hybridisation techniques, the induction by LPS of IL-1 and IL-6 mRNA in cultured microglia has been demonstrated. When IFN-gamma was included, LPS-induced production of both IL-1 and IL-6 by microglia was significantly reduced and this effect was particularly marked in the case of IL-1. Comparisons with peritoneal macrophages demonstrate that whilst both cell types produce IL-1 and IL-6 in response to LPS, IFN-gamma had no significant effect on this response in peritoneal macrophages. This suggests potential control mechanisms which may be effective in down-regulating cytokine production by microglia in vivo.

Exploring disparities in acute myocardial infarction events between Aboriginal and non-Aboriginal Australians: roles of age, gender, geography and area-level disadvantage.

We investigated disparities in rates of acute myocardial infarction (AMI) between Aboriginal and non-Aboriginal people in the 199 Statistical Local Areas (SLAs) in New South Wales, Australia. Using routinely collected and linked hospital and mortality data from 2002 to 2007, we developed multilevel Poisson regression models to estimate the relative rates of first AMI events in the study period accounting for area of residence. Rates of AMI in Aboriginal people were more than two times that in non-Aboriginal people, with the disparity greatest in more disadvantaged and remote areas. AMI rates in Aboriginal people varied significantly by SLA, as did the Aboriginal to non-Aboriginal rate ratio. We identified almost 30 priority areas for universal and targeted preventive interventions that had both high rates of AMI for Aboriginal people and large disparities in rates.

Regulation of Fc receptor and major histocompatibility complex antigen expression on isolated rat microglia by tumour necrosis factor, interleukin-1 and lipopolysaccharide: effects on interferon-gamma induced activation.

Isolated rat brain microglia display enhanced expression of Fc receptors on treatment with interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) and lipopolysaccharide (LPS), whereas major histocompatibility complex (MHC) antigen expression is enhanced only by IFN-gamma. Although TNF and LPS individually have no effect on MHC expression by microglia, they both antagonize IFN-gamma-induced expression. The enhanced expression of Fc receptors observed in the presence of IFN-gamma, TNF or LPS is significantly inhibited by the combination of IFN-gamma with either LPS or TNF. IL-1 alpha has little effect on IFN-gamma-induced MHC or Fc receptor expression by microglia. Peritoneal macrophages behave similarly to microglia, with the notable exception that IL-1 alpha enhances IFN-gamma-induced FcR expression. These observations suggest that the functional activity of microglia during inflammation or demyelination in the central nervous system can be influenced by the changing profile of cytokines present during lesion development.

Modulation of interferon-gamma-induced major histocompatibility complex class II and Fc receptor expression on isolated microglia by transforming growth factor-beta 1, interleukin-4, noradrenaline and glucocorticoids.

Interleukin-4 (IL-4) enhances Fc receptor (FcR) expression on isolated rat brain microglia and peritoneal macrophages but has little effect on major histocompatibility complex (MHC) class II antigen expression. In contrast transforming growth factor-beta 1 (TGF-beta 1) causes a reduction in expression of MHC class II on macrophages and of FcR on both cell types. Both microglia and peritoneal macrophages demonstrate enhanced expression of FcR and MHC class II on treatment with interferon-gamma (IFN-gamma). The effect of IL-4 or TGF-beta 1 in combination with IFN-gamma, added either sequentially or simultaneously, has been investigated. TGF-beta 1 down-regulates IFN-gamma-induced effects in both microglia and macrophages when present before or during the activation stage. In combination, IL-4 and IFN-gamma can be additive or antagonistic, depending on their concentrations and the sequence in which cells are exposed to the cytokines. Non-cytokine mediators present during stimulation, such as noradrenaline, dexamethasone and corticosterone, are also potent inhibitors of IFN-gamma-induced activation of microglia and macrophages.

Characterization of the mammalian initiation factor eIF2B complex as a GDP dissociation stimulator protein.

Initiation factor eIF2B mediates a key regulatory step in the initiation of mRNA translation, i.e. the regeneration of active eIF2.GTP complexes. It is composed of five subunits, alpha-epsilon. The largest of these (epsilon) displays catalytic activity in the absence of the others. The catalytic mechanism of eIF2B and the functions of the other subunits remain to be clarified. Here we show that, when present at similar concentrations to eIF2, mammalian eIF2B can mediate release of eIF2-bound GDP even in the absence of free nucleotide, indicating that it acts as a GDP dissociation stimulator protein. Consistent with this, addition of GDP to purified eIF2.eIF2B complexes causes them to dissociate. The alternative sequential mechanism would require that eIF2Bepsilon itself bind GTP. However, we show that it is the beta-subunit of eIF2B that interacts with GTP. This indicates that binding of GTP to eIF2B is not an essential element of its mechanism. eIF2B preparations that lack the alpha-subunit display reduced activity compared with the holocomplex. Supplementation of such preparations with recombinant eIF2Balpha markedly enhances activity, indicating that eIF2Balpha is required for full activity of mammalian eIF2B.

Myelin basic protein content of aggregating rat brain cell cultures treated with cytokines and/or demyelinating antibody: effects of macrophage enrichment.

The demyelinative potential of the cytokines interleukin-1 alpha (IL-1 alpha), interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) has been investigated in myelinating aggregate brain cell cultures. Treatment of myelinated cultures with these cytokines resulted in a reduction in myelin basic protein (MBP) content. This effect was additively increased by anti-myelin/oligodendrocyte glycoprotein (alpha-MOG) in the presence of complement. Qualitative immunocytochemistry demonstrated that peritoneal macrophages, added to the fetal telencephalon cell suspensions at the start of the culture period, successfully integrated into aggregate cultures. Supplementing the macrophage component of the cultures in this fashion resulted in increased accumulation of MBP. The effect of IFN-gamma on MBP content of cultures was not affected by the presence of macrophages in increased numbers.

Myelination and remyelination of aggregate rat brain cell cultures enriched with macrophages.

We reported previously that accumulation of myelin basic protein (MBP) in foetal brain aggregate cultures is enhanced by supplementation with peritoneal macrophages. The present study demonstrates that the rate of MBP accumulation in macrophage-enriched cultures continues to increase over time unaccompanied by a matching increase in the oligodendrocyte marker cyclic nucleotide phosphodiesterase, while that of control cultures reaches a plateau. These observations are supported by electron microscopic evidence of cumulative numbers of myelinated axons in the aggregates over time and by enhanced expression of myelin protein genes in macrophage-enriched relative to control cultures. Aggregates demyelinate following short-term exposure to cytokines and antimyelin oligodendrocyte glycoprotein antibody, and MBP synthesis resumes following removal of demyelinating agents. Supplementation of cultures with macrophages influences the degree of myelin breakdown and remyelination, drawing attention to the role that macrophage-derived growth factors may play in myelinogenesis and myelin repair in inflammatory demyelinating disease.