The NLRP3 inflammasome modulates glycolysis by increasing PFKFB3 in an IL-1ß-dependent manner in macrophages.

Inflammation and metabolism are intricately linked during inflammatory diseases in which activation of the nucleotide-binding domain-like receptors Family Pyrin Domain Containing 3 (NLRP3) inflammasome, an innate immune sensor, is critical. Several factors can activate the NLRP3 inflammasome, but the nature of the link between NLRP3 inflammasome activation and metabolism remains to be thoroughly explored. This study investigates whether the small molecule inhibitor of the NLRP3 inflammasome, MCC950, modulates the lipopolysaccharide (LPS) -and amyloid-ß (Aß)-induced metabolic phenotype and inflammatory signature in macrophages. LPS + Aß induced IL-1ß secretion, while pre-treatment with MCC950 inhibited this. LPS + Aß also upregulated IL-1ß mRNA and supernatant concentrations of TNFα, IL-6 and IL-10, however these changes were insensitive to MCC950, confirming that MCC950 specifically targets inflammasome activation in BMDMs. LPS + Aß increased glycolysis and the glycolytic enzyme, PFKFB3, and these effects were decreased by MCC950. These findings suggest that NLRP3 inflammasome activation may play a role in modulating glycolysis. To investigate this further, the effect of IL-1ß on glycolysis was assessed. IL-1ß stimulated glycolysis and PFKFB3, mimicking the effect of LPS + Aß and adding to the evidence that inflammasome activation impacts on metabolism. This contention was supported by the finding that the LPS + Aß-induced changes in glycolysis and PFKFB3 were attenuated in BMDMs from NLRP3-deficient and IL-1R1-deficient mice. Consistent with a key role for PFKFB3 is the finding that the PFKFB3 inhibitor, 3PO, attenuated the LPS + Aß-induced glycolysis. The data demonstrate that activation of the NLRP3 inflammasome, and the subsequent release of IL-1ß, play a key role in modulating glycolysis via PFKFB3. Reinstating metabolic homeostasis by targeting the NLRP3 inflammasome-PFKFB3 axis may provide a novel therapeutic target for treatment of acute and chronic disease.

Anti-TLR2 antibody triggers oxidative phosphorylation in microglia and increases phagocytosis of ß-amyloid.

BACKGROUND: Microglia are multifunctional cells that are primarily neuroprotective and a deficit in their functional integrity is likely to be a contributory factor in the deteriorating neuronal function that occurs with age and neurodegeneration. One aspect of microglial dysfunction is reduced phagocytosis, and this is believed to contribute to the accumulation of amyloid-ß (Aß) in Alzheimer's disease (AD). Therefore, improving phagocytosis should be beneficial in limiting the amyloidosis that characterises AD. METHODS: Here, we investigated whether an antibody that targets toll-like receptor (TLR)2 might attenuate the inflammatory and metabolic changes induced by lipopolysaccharide (LPS) and amyloid-ß. The impact on phagocytosis was assessed by immunohistochemistry. We evaluated the metabolic changes with the SeaHorse Extracellular Flux Analyser and studied the expression of key enzymes driving glycolysis by western blotting. For all experiments, statistical significance was determined by unpaired Student's t test and two-way analysis of variance (ANOVA). RESULTS: We have reported that, when exposed to an inflammatory stimulus, microglia switch their metabolism towards the metabolically- inefficient glycolysis; this potentially impacts on metabolically demanding functions like phagocytosis. Anti-TLR2 antibody increased phagocytosis of Aß in LPS + Aß-stimulated microglia and this was linked with the ability of the antibody to attenuate the LPS + Aß-triggered inflammasome activation. LPS + Aß increased glycolysis in microglia and increased the expression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB)3, an enzyme that plays a key role in driving glycolysis; these effects were inhibited when cells were incubated with the anti-TLR2 antibody. The data also show that antibody treatment increased oxidative metabolism. CONCLUSIONS: Thus, microglia with an inflammatory phenotype, specifically cells in which the inflammasome is activated, are glycolytic; this may compromise the metabolic efficiency of microglia and thereby provide an explanation for the reduced phagocytic function of the cells. We propose that, by restoring oxidative metabolism and reducing inflammasome activation in microglia, phagocytic function is also restored.

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) produces edema due to BBB disruption induced by MMP-9 activation in rat hippocampus.

The recreational drug of abuse, 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) disrupts blood-brain barrier (BBB) integrity in rats through an early P2X7 receptor-mediated event which induces MMP-9 activity. Increased BBB permeability often causes plasma proteins and water to access cerebral tissue leading to vasogenic edema formation. The current study was performed to examine the effect of a single neurotoxic dose of MDMA (12.5 mg/kg, i.p.) on in vivo edema development associated with changes in the expression of the perivascular astrocytic water channel, AQP4, as well as in the expression of the tight-junction (TJ) protein, claudin-5 and Evans Blue dye extravasation in the hippocampus of adult male Dark Agouti rats. We also evaluated the ability of the MMP-9 inhibitor, SB-3CT (25 mg/kg, i.p.), to prevent these changes in order to validate the involvement of MMP-9 activation in MDMA-induced BBB disruption. The results show that MDMA produces edema of short duration temporally associated with changes in AQP4 expression and a reduction in claudin-5 expression, changes which are prevented by SB-3CT. In addition, MDMA induces a short-term increase in both tPA activity and expression, a serine-protease which is involved in BBB disruption and upregulation of MMP-9 expression. In conclusion, this study provides evidence enough to conclude that MDMA induces edema of short duration due to BBB disruption mediated by MMP-9 activation.

Effects of repeated social defeat on adolescent mice on cocaine-induced CPP and self-administration in adulthood: integrity of the blood-brain barrier.

Social stress in adulthood enhances cocaine self-administration, an effect that has been related with an increase in extracellular signal-regulated kinase and p38α mitogen-activated protein kinase phosphorylation. A detrimental effect of cocaine on blood-brain barrier (BBB) integrity has also been reported. This study evaluates the effects of repeated social defeat (RSD) during adolescence on the reinforcing and motivational effects of cocaine in adult mice and the changes induced by RSD on BBB permeability. Cocaine self-administration, conditioned place preference and quantitative analysis of claudin-5, laminin, collagen-IV and IgG immunoreactivity took place 3 weeks after RSD. Mice socially defeated during adolescence developed conditioned place preference and exhibited reinstated preference with a non-effective dose of cocaine (1 mg/kg). RSD mice needed significantly more sessions than control animals for the preference induced by 25 mg/kg of cocaine to be extinguished. However, acquisition of cocaine self-administration (0.5 mg/kg per injection) was delayed in the RSD group. Mice exposed to RSD displayed significant changes in BBB structure in adulthood, with a marked reduction in expression of the tight junction protein claudin-5 and an increase in basal laminin degradation (reflected by a decrease in laminin and collagen-IV expression) in the nucleus accumbens and hippocampus. The detrimental effect induced by cocaine (25 mg/kg) on collagen-IV expression in the hippocampus was more pronounced in RSD mice. In summary, our findings suggest that stress and cocaine can increase the long-term vulnerability of the brain to subsequent environmental insults as a consequence of a sustained disruption of the BBB.

Disruption of blood-brain barrier integrity in postmortem alcoholic brain: preclinical evidence of TLR4 involvement from a binge-like drinking model.

Inflammatory cytokines and reactive oxygen species are reported to be involved in blood-brain barrier (BBB) disruption. Because there is evidence that ethanol (EtOH) induces release of free radicals, cytokines and inflammatory mediators we examined BBB integrity and matrix metalloproteinase (MMP) activity in postmortem human alcoholic brain and investigated the role of TLR4 signaling in BBB permeability in TLR4-knockout mice under a binge-like EtOH drinking protocol. Immunohistochemical studies showed reduced immunoreactivity of the basal lamina protein, collagen-IV and of the tight junction protein, claudin-5 in dorsolateral prefrontal cortex of alcoholics. There was also increased MMP-9 activity and expression of phosphorylated ERK1/2 and p-38. Greater number of CD45+ IR cells were observed associated with an enhanced neuroinflammatory response reflected by increased GFAP and Iba-1 immunostaining. To further explore effects of high EtOH consumption on BBB integrity we studied TLR4-knockout mice exposed to the drinking in the dark paradigm. Repetitive EtOH exposure in wild-type mice decreased hippocampal expression of laminin and collagen-IV and increased IgG immunoreactivity, indicating IgG extravasation. Western blot analysis also revealed increased MyD88 and p-ERK1/2 levels. None of these changes was observed in TLR4-knockout mice. Collectively, these findings indicate that chronic EtOH increases degradation of tight junctions and extracellular matrix in postmortem human brain and induces a neuroinflammatory response associated with activation of ERK1/2 and p-38 and greater MMP-9 activity. The EtOH-induced effects on BBB impairment are not evident in the hippocampus of TLR4-knockout mice, suggesting the involvement of TLR4 signaling in the underlying mechanism leading to BBB disruption in mice.

Inhibiting TLR2 activation attenuates amyloid accumulation and glial activation in a mouse model of Alzheimer's disease.

The effects of Toll-like receptor (TLR) activation in peripheral cells are well characterized but, although several TLRs are expressed on cells of the brain, the consequences of their activation on neuronal function remain to be fully investigated, particularly in the context of assessing their potential as therapeutic targets in neurodegenerative diseases. Several endogenous TLR ligands have been identified, many of which are soluble factors released from cells exposed to stressors. In addition, amyloid-ß (Aß) the main constituent of the amyloid plaques in Alzheimer's disease (AD), activates TLR2, although it has also been shown to bind to several other receptors. The objective of this study was to determine whether activation of TLR2 played a role in the developing inflammatory changes and Aß accumulation in a mouse model of AD. Wild type and transgenic mice that overexpress amyloid precursor protein and presenilin 1 (APP/PS1 mice) were treated with anti-TLR2 antibody for 7months from the age of 7-14months. We demonstrate that microglial and astroglial activation, as assessed by MHCII, CD68 and GFAP immunoreactivity was decreased in anti-TLR2 antibody-treated compared with control (IgG)-treated mice. This was associated with reduced Aß plaque burden and improved performance in spatial learning. The data suggest that continued TLR2 activation contributes to the developing neuroinflammation and pathology and may be provide a strategy for limiting the progression of AD.

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) disrupts blood-brain barrier integrity through a mechanism involving P2X7 receptors.

The recreational drug 3,4-methylenedioxymethamphetamine (MDMA; 'ecstasy') produces a neuro-inflammatory response in rats characterized by an increase in microglial activation and IL-1ß levels. The integrity of the blood-brain barrier (BBB) is important in preserving the homeostasis of the brain and has been shown to be affected by neuro-inflammatory processes. We aimed to study the effect of a single dose of MDMA on the activity of metalloproteinases (MMPs), expression of extracellular matrix proteins, BBB leakage and the role of the ionotropic purinergic receptor P2X7 (P2X7R) in the changes induced by the drug. Adult male Dark Agouti rats were treated with MDMA (10 mg/kg, i.p.) and killed at several time-points in order to evaluate MMP-9 and MMP-3 activity in the hippocampus and laminin and collagen-IV expression and IgG extravasation in the dentate gyrus. Microglial activation, P2X7R expression and localization were also determined in the dentate gyrus. Separate groups were treated with MDMA and the P2X7R antagonists Brilliant Blue G (BBG; 50 mg/kg, i.p.) or A-438079 (30 mg/kg, i.p.). MDMA increased MMP-3 and MMP-9 activity, reduced laminin and collagen-IV expression and increased IgG immunoreactivity. In addition, MDMA increased microglial activation and P2X7R immunoreactivity in these cells. BBG suppressed the increase in MMP-9 and MMP-3 activity, prevented basal lamina degradation and IgG extravasation into the brain parenchyma. A-438079 also prevented the MDMA-induced reduction in laminin and collagen-IV immunoreactivity. These results indicate that MDMA alters BBB permeability through an early P2X7R-mediated event, which in turn leads to enhancement of MMP-9 and MMP-3 activity and degradation of extracellular matrix.

A study on the effect of JNK inhibitor, SP600125, on the disruption of blood-brain barrier induced by methamphetamine.

Methamphetamine (METH) is a widely consumed drug with high abuse potential. Studies in animals have shown that the drug produces dopaminergic neurotoxicity following both single high-dose and repeated low-dose administration. In addition, METH produces an increase in matrix metalloproteinase expression and loss of BBB integrity. We have examined the effect of repeated low-dose METH on MMP-9/2 expression and activity and laminin expression and the role of MMPs and JNK 1/2 phosphorylation on the changes induced by the drug in BBB integrity. Mice were given METH (4 mg/kg, i.p., three times separated by 3 h) and killed at different times after the last dose. Striatal MMP-9/2 activity was determined by zymography and expression of MMPs, laminin and phosphorylated JNK 1/2 was determined by western blot. BBB integrity was determined by IgG immunoreactivity. SP600125 and BB-94 were used to inhibit JNK and MMPs respectively. METH increased striatal MMP-9 expression and activity, IgG immunoreactivity and p-JNK 1/2 expression and decreased laminin expression. Increased IgG immunoreactivity colocalized with areas of greater MMP-9 activity. JNK inhibition prevented METH-induced changes in MMP-9 activity, laminin degradation and BBB leakage. BB-94 also prevented laminin degradation and BBB leakage. The decrease in BBB integrity induced by METH is mediated by the JNK pathway which activates MMP-9 causing degradation of laminin and BBB leakage.

Apolipoprotein-E controls adenosine triphosphate-binding cassette transporters ABCB1 and ABCC1 on cerebral microvessels after methamphetamine intoxication.

BACKGROUND AND PURPOSE: Methamphetamine is a powerful addictive, which has been associated with ischemic stroke and brain hemorrhage in humans. Whether and how methamphetamine influences the expression of tight junctions and adenosine triphosphate-binding cassette transporters, which have previously been shown to be regulated by apolipoprotein-E (ApoE) under conditions of brain ischemia, was unknown. METHODS: C57BL/6J mice received intraperitoneal injections of methamphetamine (3 times 4 mg/kg separated by 3 hours) either alone or in combination with the ApoE receptor-2 inhibitor receptor-associated protein (40 µg/kg) or the inducible nitric oxide synthase inhibitor 1400W (5 mg/kg). Animals were euthanized 3 or 24 hours after methamphetamine exposure. Tissue responses were evaluated with Western blots, immunoprecipitation, and immunohistochemistry using total brain and cerebral microvessel extracts. RESULTS: Methamphetamine induced a transient activation of stress kinases c-Jun N-terminal kinase 1/2 and p38 in the brain parenchyma and increased intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression on cerebral microvessels without inducing loss of tight junction proteins and without inducing IgG extravasation. Methamphetamine transiently increased the expression of the luminal adenosine triphosphate-binding cassette transporter ABCB1 on cerebral microvessels and reduced the expression of the abluminal transporter ABCC1. Elevated expression of ApoE was noted in the brain parenchyma by methamphetamine, activating ApoE receptor-2 on brain capillaries, deactivating c-Jun N-terminal kinase 1/2 and c-Jun, and regulating ABCB1 and ABCC1 expression. Indeed, ApoE receptor-2 and inducible nitric oxide synthase inhibition prevented the ABCB1 and ABCC1 expression changes. CONCLUSIONS: Acute exposure to methamphetamine at doses comparable to those consumed in drug addiction does not induce tight junction breakdown but differentially regulates adenosine triphosphate-binding cassette transporters through the ApoE/ApoE receptor-2/c-Jun N-terminal kinase 1/2 pathway.

The endocannabinoid system modulates a transient TNF pathway that induces neural stem cell proliferation.

Evidence is emerging that the tumour necrosis factor (TNF-alpha) is a potent signal that induces neural stem cell proliferation and migration. We show that NSC self-renewal is controlled by bi-directional cross-talk between the endocannabinoid system and the TNF signalling pathway. By blocking endogenous TNF-alpha activity, we demonstrate that the TNF system is critical for the proliferation of NSC. Furthermore, we show that pharmacological blockade of the CB1/CB2 cannabinoid receptors dramatically suppresses TNF-alpha-induced NSC proliferation. Interestingly, we found that CB1 or CB2 agonists induce NSC proliferation coupled to a significant increase in both TACE/ADAM 17 and TNF-alpha levels. Overall these data suggest a novel mode of action for the endocannabinoid system in NSC proliferation that is coupled to TNF signalling and that may be of therapeutic interest in the emerging field of brain repair.

Cannabinoids modulate Olig2 and polysialylated neural cell adhesion molecule expression in the subventricular zone of post-natal rats through cannabinoid receptor 1 and cannabinoid receptor 2.

The subventricular zone (SVZ) is a source of post-natal glial precursors that can migrate to the overlying white matter, where they may differentiate into oligodendrocytes. We showed that, in the post-natal SVZ ependymocytes, radial glia and astrocyte-like cells express cannabinoid receptor 1 (CB1), whereas cannabinoid receptor 2 (CB2) is found in cells expressing the polysialylated neural cell adhesion molecule. To study CB1 and CB2 function, post-natal rats were exposed to selective CB1 or CB2 agonists (arachidonyl-2-chloroethylamide and JWH-056, respectively) for 15 days. Accordingly, we found that CB1 activation increases the number of Olig2-positive cells in the dorsolateral SVZ, whereas CB2 activation increases polysialylated neural cell adhesion molecule expression in this region. As intense myelination occurs during the first weeks of post-natal development, we examined how modulating these factors affected the expression of myelin basic protein. Pharmacological administration of agonists and antagonists of CB1 and CB2 showed that the activation of both receptors is needed to augment the expression of myelin basic protein in the subcortical white matter.

CB2 cannabinoid receptors promote mouse neural stem cell proliferation.

Neurospheres are clonal cellular aggregates of neural stem/precursor cells that grow in culture as free-floating clusters. Activation of CB1 cannabinoid receptors, which are expressed by these cells, promotes proliferation. In the present study we investigated the expression of CB2 cannabinoid receptors and the effect of exogenous cannabinoids on neural stem/precursor cell proliferation. Neurospheres containing nestin-positive and sn-1 diacylglycerol lipase alpha-positive cells expressed both CB1 and CB2 receptors, which were maintained through several passages. Application of the non-selective cannabinoid agonist (HU-210, 0.5 microM) stimulated bromodeoxyuridine incorporation and neurosphere formation. This action involved both CB1 and CB2 receptors as neurosphere formation was stimulated by either selective CB1 [arachidonyl-2'chloroethylamide/(all Z)-N-(2-cycloethyl)-5,8,11,14-eicosatetraenamide (ACEA), 200 nM and 1 microM] or CB2 (JWH-056, 0.5 microM) agonists. In addition, CB1 or CB2 antagonists (1 microM SR-141716A and SR-144528, respectively) blocked basal proliferation, suggesting that endogenous cannabinoids are implicated in neurosphere proliferation. In addition, cannabinoid agonist-stimulated proliferation was reduced by the Akt translocation inhibitor BML-257 (12.5 microM), suggesting a role for phosphoinositide-3 kinase signalling. Together, our results suggest that cannabinoids stimulate proliferation of neural stem/precursor cells acting on both CB1 and CB2 cannabinoid receptors through a phosphoinositide-3 kinase/Akt pathway.