F-spondin plays a critical role in murine neuroblastoma survival by maintaining IL-6 expression

F-spondin is associated with the regulation of axonal growth and the development of the nervous system. Its mechanism of action, however, is not clearly understood. In this study, we found that murine neuroblastoma Neuro-2a cells expressed a significant level of IL-6, but only trace amounts of IL-12, tumor necrosis factor α and nitric oxide. Knock-down of F-spondin mRNA in murine neuroblastoma NB41A3 and Neuro-2a cells using small interfering RNAs led to decreased IL-6 levels along with lower resistance to serum starvation and cytotoxic amyloid β_1–42 (Aβ_1–42) peptide. Restoring decline of F-spondin or IL-6 induced by F-spondin knock-down through adding exogenous F-spondin, IL-6 or over-expressing F-spondin reversed the cell death induced by Aβ_1–42 peptide or serum starvation. The decrease of IL-6 level was positively correlated with decrease of NF-κB and inhibition of p38 mitogen-activated protein kinase (MAPK). Over-expressing MEKK, a kinase activator of the p38 MAPK pathway, increased IL-6 production, restored the decrease of p38 induced by F-spondin knock-down, and rescued the cells from death caused by Aβ_1–42 peptide. Taken together, these results suggest that F-spondin may play a critical role in murine neuroblastoma survival under adverse conditions by maintaining IL-6 level via a MEKK/p38 MAPK/NF-κB-dependent pathway.     

Amyloid β Protein Primes Cultured Rat Microglial Cells for an Enhanced Phorbol 12-Myristate 13-Acetate-Induced Respiratory Burst Activity

Abstract: Activated microglia, often associated with neuritic amyloid plaques in the Alzheimer's disease brain, are likely to contribute to the progression of the disease process, e.g., by releasing neurotoxic reactive oxygen and/or nitrogen intermediates. In the present study, whether the amyloid β peptide (Aβ), the principal constituent of amyloid plaques, can stimulate microglial respiratory burst activity and/or microglial production of nitric oxide was examined. Using neonatal rat microglial cultures as a model, it was found that neither the spontaneous release of nitric oxide nor the lipopolysaccharide-induced production of nitric oxide was altered in cultures previously incubated with synthetic Aβ(1–40). for 24 h. In addition, no direct stimulatory effect of Aβ(1–40) on the respiratory burst activity was observed. Nevertheless, concomitant with an increase in the number of responsive cells, a profound priming of the phorbol 12-myristate 13-acetate-evoked production of superoxide anion was observed in Aβ(1–40)-treated cultures. Thus, both the maximal rate and the total phorbol 12-myristate 13-acetate-induced production of superoxide appeared to be statistically significantly higher as compared with untreated cultures. It is concluded that, as far as activation of the microglial respiratory burst is concerned, Aβ(1–40) may merely act as a priming rather than a triggering stimulus.     

Enhancement of β-Amyloid Peptide Aβ(1–40)-Mediated Neurotoxicity by Glutamine Synthetase

Abstract: The β-amyloid peptide (Aβ), a main constituent in both senile and diffuse plaques in Alzheimer's disease brains, was previously shown to be neurotoxic and to be able to interact with several macromolecular components of brain tissue. Previous investigations carried out in our laboratory demonstrated free radical species formation in aqueous solutions of Aβ(1–40) and its C-end fragment, Aβ(25–35). Toxic forms of Aβ rapidly inactivate the oxidation-sensitive cytosolic enzyme glutamine synthetase (GS). In this regard, we suggested and subsequently demonstrated that Aβ radicals can cause an oxidative damage of cell proteins and lipids resulting in disruption of membrane functions, enzyme inactivation, and cell death. Because GS can be a substrate for Aβ-derived oxidizing species, the present study was conducted to determine if GS could protect against Aβ neurotoxicity. In contrast to this initial hypothesis, we here report that GS significantly enhances the neurotoxic effects of Aβ(1–40). The Aβ-mediated inactivation of GS was found to be accompanied by the loss of immunoreactive GS and the significant increase of Aβ(1–40) neurotoxicity.     

p75 Neurotrophin receptor mediates neurotrophin activation of NF‐kappa B and induction of iNOS expression in P19 neurons

P19 embryonic carcinoma cells can be differentiated into neurons that form synaptic connections and that produce a variety of neurotransmitters. Results of RT‐PCR indicate that P19 neurons express several neurotrophin receptors (p75^NTR, trkB, and trkC, but not trkA) but they do not express any of the four neurotrophins. Consistent with the presence of trkB but not trkA, BDNF causes rapid phosphorylation of MAP kinases ERK1 and ERK2, but NGF does not. Neurotrophins induce translocation of NF‐κB into the nucleus. All four neurotrophins induce activation of NF‐κB in a biphasic manner. This effect is apparently mediated by p75^NTR, because an inhibitor of trk receptors, K252a, does not inhibit activation of NF‐κB. Instead, K252a itself promotes activation of NF‐κB and this effect is additive with the effect of neurotrophins. Inhibition of reactive oxygen intermediates with PDTC completely abolishes basal activity of NF‐κB and strongly inhibits activation of NF‐κB by neurotrophins, indicating an important role of reactive oxygen intermediates in the pathway by which neurotrophins activate NF‐κB. NF‐κB is known to promote expression of the iNOS gene. We found that all four neurotrophins increased iNOS mRNA levels, resulting in increased accumulation of iNOS protein. In contrast, none of the neurotrophins stimulated nNOS mRNA or protein synthesis. PDTC abolishes constitutive and neurotrophin‐induced expression of iNOS mRNA and protein and abolishes constitutive expression of nNOS mRNA, suggesting that reactive oxygen intermediates promote expression of nNOS. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 191–203, 2003     

Probiotic Lactobacillus reuteri promotes TNF-induced apoptosis in human myeloid leukemia-derived cells by modulation of NF-kappaB and MAPK signalling

The molecular mechanisms of pro-apoptotic effects of human-derived Lactobacillus reuteri ATCC PTA 6475 were investigated in this study. L. reuteri secretes factors that potentiate apoptosis in myeloid leukemia-derived cells induced by tumour necrosis factor (TNF), as indicated by intracellular esterase activity, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling assays and poly (ADP-ribose) polymerase cleavage. L. reuteri downregulated nuclear factor-κB (NF-κB)-dependent gene products that mediate cell proliferation (Cox-2, cyclin D1) and cell survival (Bcl-2, Bcl-xL). L. reuteri suppressed TNF-induced NF-κB activation, including NF-κB-dependent reporter gene expression in a dose-and time-dependent manner. L. reuteri stabilized degradation of IκBα and inhibited nuclear translocation of p65 (RelA). Although phosphorylation of IκBα was not affected, subsequent polyubiquitination necessary for regulated IκBα degradation was abrogated by L. reuteri . In addition, L. reuteri promoted apoptosis by enhancing mitogen-activated protein kinase (MAPK) activities including c-Jun N-terminal kinase and p38 MAPK. In contrast, L. reuteri suppressed extracellular signal-regulated kinases 1/2 in TNF-activated myeloid cells. L. reuteri may regulate cell proliferation by promoting apoptosis of activated immune cells via inhibition of IκBα ubiquitination and enhancing pro-apoptotic MAPK signalling. An improved understanding of L. reuteri- mediated effects on apoptotic signalling pathways may facilitate development of future probiotics-based regimens for prevention of colorectal cancer and inflammatory bowel disease.     

Aggregation of β-Amyloid Peptide Is Promoted by Membrane Phospholipid Metabolites Elevated in Alzheimer's Disease Brain

Abstract: Increased amounts of β-amyloid (Aβ) peptide deposits are found in Alzheimer's disease brain. These amyloid deposits have been implicated in the pathophysiology of this common dementing illness. Aβ peptides have been shown to be toxic to neurons in cell culture, and this toxicity is critically dependent on the aggregation of the peptide into cross-β-pleated sheet fibrils. Also, in vivo and postmortem NMR studies have shown changes in certain brain membrane phospholipid metabolites in normal aging and more extensive alterations in patients with Alzheimer's disease. The finding that membrane phospholipids affect the aggregation of Aβ suggests that the abnormalities in membrane metabolism found in Alzheimer's disease could affect the deposition of Aβ in vivo. Therefore, we examined the effect of membrane phospholipid metabolites that are altered in Alzheimer's disease brain on the aggregation of Aβ(1–40) using a light scattering method. Certain metabolites (glycerophosphocholine, glycerophosphoethanolamine, and α-glycerophosphate) augment the aggregation of Aβ. Other membrane phospholipid metabolites (phosphocholine, phosphoethanolamine, and inositol-1-phosphate) have no effect. We conclude that increased membrane phospholipid metabolite concentrations may play a role in the deposition of Aβ seen in normal aging and the even greater deposition of Aβ observed in Alzheimer's disease.     

Role of lipids in killing mycobacteria by macrophages: evidence for NF-κB-dependent and -independent killing induced by different lipids

We have shown that several lipids can modulate the macrophage innate immune response against mycobacteria and enhance their killing. Since NF-κB is required for mycobacterial killing, we tested the ability of lipids to activate NF-κB in uninfected macrophages and those infected with mycobacteria. In uninfected cells, sphingomyelin (SM), phosphatidylinositol-4-phosphate (PIP) and arachidonic acid (AA) enhanced NF-κB activation and the cell surface expression of CD69, a macrophage activation marker regulated by NF-κB. Sphingosine (Sph), sphingosine-1-phosphate (S1P), diacylglycerol (DAG), eicosapentanoic acid (EPA) and phosphatidyl choline (PC) failed to activate either NF-κB or CD69. Ceramide (Cer) activated CD69 expression without activating NF-κB. In Mycobacterium smegmatis- infected cells, NF-κB was transiently activated in a manner that was enhanced by SM, PIP and AA. In contrast Mycobacterium avium mostly repressed NF-κB activation and only SM and AA could induce its partial activation. While lipids that activate NF-κB in uninfected cells tend to kill mycobacteria in macrophages Sph and S1P failed to activate NF-κB under most conditions but nevertheless enhanced killing of M. smegmatis , M. avium and M. tuberculosis H37Rv. Our results argue that both NF-κB-dependent and -independent mechanisms are involved in macrophage killing of mycobacteria and that both mechanisms can be enhanced by selected lipids.     

Proinflammatory cytokine interferon-γ increases induction of indoleamine 2,3-dioxygenase in monocytic cells primed with amyloid β peptide 1–42: implications for the pathogenesis of Alzheimer's disease

Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme of the kynurenine pathway of tryptophan metabolism, ultimately leading to production of the excitotoxin quinolinic acid (QUIN) by monocytic cells. In the Tg2576 mouse model of Alzheimer's disease, systemic inflammation induced by lipopolysaccharide leads to an increase in IDO expression and QUIN production in microglia surrounding amyloid plaques. We examined whether the IDO over-expression in microglia could be mediated by brain proinflammatory cytokines induced during the peripheral inflammation using THP-1 cells and peripheral blood mononuclear cells (PBMC) as models for microglia. THP-1 cells pre-treated with 5–25 μM amyloid β peptide (Aβ) (1–42) but not with Aβ (1–40) or Aβ (25–35) became an activated state as indicated by their morphological changes and enhanced adhesiveness. IDO expression was only slightly increased in the reactive cells but strongly enhanced following treatment with proinflammatory cytokine interferon-γ (IFN-γ) but not with interleukin-1β, tumor necrosis factor-α, or interleukin-6 at 100 U/mL. The concomitant addition of Aβ (1–42) with IFN-γ was totally ineffective, indicating that Aβ pre-treatment is prerequisite for a high IDO expression. The priming effect of Aβ (1–42) for the IDO induction was also observed for PBMC. These findings suggest that IFN-γ induces IDO over-expression in the primed microglia surrounding amyloid plaques.     

Effects of HNE-modification induced by Aβ on neprilysin expression and activity in SH-SY5Y cells

The cerebral accumulation of β-amyloid (Aβ) is a consistent feature of and likely contributor to the development of Alzheimer's disease. In addition to dysregulated production, increasing experimental evidence suggests reduced catabolism also plays an important role in Aβ accumulation. We have previously shown that neprilysin (NEP), the major protease which cleaves Aβ in vivo , is modified by 4-hydroxy-nonenal (HNE) adducts in the brain of Alzheimer's disease patients. To determine if these changes affected Aβ, SH-SY5Y cells were treated with HNE or Aβ, and then NEP mRNA, protein levels, HNE adducted NEP, NEP activity and secreted Aβ levels were determined. Intracellular NEP developed HNE adducts after 24 h of HNE treatment as determined by immunoprecipitation, immunoblotting, and double immunofluorescence staining. HNE-modified NEP showed decreased catalytic activity, which was associated with elevations in Aβ1–40 in SH-SY5Y and H4 APP695wt cells. Incubation of cells with Aβ1–42 also induced HNE adduction of NEP. In an apparent compensatory response, Aβ-treated cells showed increased NEP mRNA and protein expression. Despite elevations in NEP protein, the activity was significantly lower compared with the NEP protein level. This study demonstrates that NEP can be inactivated by HNE-adduction, which is associated with, at least partly, reduced Aβ cleavage and enhanced Aβ accumulation.     

Alzheimer's β-Amyloid Peptide 1–42 Induces a Phagocytic Response in Murine Microglia

Abstract: β-Amyloid (Aβ) peptides are a key component of the senile plaques that characterize Alzheimer's disease. Cytokine-producing microglia have been shown to be intimately associated with amyloid deposits and have also been implicated as scavengers responsible for clearing Aβ deposits. However, little is known about the initial activation of these microglia or the effect of Aβ on phagocytosis. Murine BV-2 microglia were used to assess the effect of synthetic Aβ 1–42 on phagocytosis by quantifying uptake of fluorescent microspheres, acetylated low-density lipoproteins, and zymosan particles by flow cytometry. Aβ 1–42 stimulated microglial phagocytosis in a time- and dose-dependent manner. Aβ fibrils produced the greatest potentiation, and once activated, phagocytosis remained elevated after removal of Aβ from the cultures. Aβ-stimulated phagocytosis could be blocked if proteoglycans were first complexed to Aβ fibrils. These data suggest that Aβ fibrils act as an immune signal to stimulate microglial phagocytosis and that extracellular matrix molecules may modify Aβ function.     

Interleukin-12 P40 induces the expression of TNF-α in microglia and macrophages

Recently, it has been found that overproduction of IL-12 can be dangerous to the host as it is involved in the pathogenesis of a number of autoimmune inflammatory diseases such as multiple sclerosis. It is composed of two different subunits – p40 and p35. Expression of p40 mRNA but not that of p35 mRNA in excessive amount in the CNS of patients with Multiple Sclerosis (MS) suggests that IL-12 p40 may have a role in the pathogenesis of the disease. The present study was undertaken to explore the role of p40 in the expression of TNF-α in microglia. Interestingly, we have found that IL-12 p70, p402 (the p40 homodimer) and p40 (the p40 monomer) dose-dependently induced the production of TNF-α in BV-2 microglial cells. This induction of TNF-α production was accompanied by an induction of TNF-α mRNA. In addition to BV-2 glial cells, p70, p402 and p40 also induced the production of TNF-α in mouse primary microglia and peritoneal macrophages. Since the activation of both NF-κB and C/EBPb is important for the expression of TNF-α in microglial cells, we investigated the effect of p40 on the activation of NF-κB as well as C/EBPb. Activation of NF-κB as well as C/EBPb by p40 and inhibition of p40-induced expression of TNF-α by Dp65, a dominant-negative mutant of p65, and DC/EBPb, a dominant-negative mutant of C/EBPb, suggests that p40 induces the expression of TNF-α through the activation of NF-κB and C/EBPb. This study delineates a novel role of IL-12 p40 in inducing the expression of TNF-α in microglial cells which may participate in the pathogenesis of neuroinflammatory diseases.
Acknowledgements:   This study was supported by NIH grants (NS39940 and AG19487).     

Mitochondrial Free Radical Signal in Ceramide-Dependent Apoptosis: A Putative Mechanism for Neuronal Death in Parkinson''s Disease

Abstract: We investigated the potential role of different proteases in the death of cultured rat hippocampal pyramidal neurons induced by β-amyloid(Aβ) (25–35). Both Aβ(25–35)- and staurosporine-induced death of these neurons appeared to involve apoptosis, as indicated using Hoechst 33342 and terminal dUDP nick end labeling staining, whereas NMDA-induced death appeared more complex. Two irreversible inhibitors of the interleukin-1β converting enzyme (ICE) and related proteases, Z-Val-Ala-Asp-CH₂F and acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone, blocked neuronal death produced by Aβ(25–35), staurosporine, and NMDA to differing extents. Furthermore, MDL 28,170, a selective inhibitor of the calcium-regulated protease calpain, also inhibited death induced by all agents. Aβ(25–35) and staurosporine stimulated the breakdown of the protein spectrin, a calpain substrate. Spectrin breakdown was inhibited by MDL 28,170 but not by ICE inhibitors. Leupeptin was only effective in preventing NMDA-induced death. These results support the role of apoptosis in neuronal death due to Aβ(25–35) treatment and also suggest a role for calcium-regulated proteases in this process.     

Role of GSK-3beta activation and alpha7 nAChRs in Abeta(1-42)-induced tau phosphorylation in PC12 cells

β-amyloid peptide 1–42 (Aβ_1–42) and hyperphosphorylated tau are associated with neurodegeneration in Alzheimer's disease. Emerging evidence indicates that Aβ_1–42 can potentiate hyperphosphorylation of tau in cell lines and in transgenic mice, but the underlying mechanism(s) remains unclear. In this study, Aβ_1–42-induced tau phosphorylation was investigated in differentiated PC12 cells. Treatment of cells with Aβ_1–42 increased phosphorylation of tau at serine-202 as detected by AT8 antibody. This Aβ_1–42-induced tau phosphorylation paralleled phosphorylation of glycogen synthase kinase-3β (GSK-3β) at tyrosine-216 (GSK-3β-pY216), which was partially inhibited by the GSK-3β inhibitor, CHIR98023. Aβ_1–42-induced tau phosphorylation and increase in GSK-3β-pY216 phosphorylation were also partially attenuated by α7 nicotinic acetylcholine receptor (α7 nAChR) selective ligands including agonist A-582941 and antagonists methyllycaconitine and α-bungarotoxin. The α7 nAChR agonist and the GSK-3β inhibitor had no additive effect. These observations suggest that α7 nAChR modulation can influence Aβ_1–42-induced tau phosphorylation, possibly involving GSK-3β. This study provides evidence of nAChR mechanisms underlying Aβ_1–42 toxicity and tau phosphorylation, which, if translated in vivo , could provide additional basis for the utility of α7 nAChR ligands in the treatment of Alzheimer's disease.