Eric J. Downer

CD200 Ligand–Receptor Interaction Modulates Microglial Activation In Vivo and In Vitro: A Role for IL-4

Deficits in cognitive function are associated with neuroinflammatory changes, typified by activation of glial cells and an alteration of the pro- and anti-inflammatory cytokine balance in the brain. Although there is evidence to suggest that activation of microglia is regulated by interaction with other cell types in the brain, the mechanism(s) involved is poorly understood. Here, we provide evidence that interaction between CD200 and its receptor plays a role in modulating microglial activation under conditions of chronic and acute inflammation of the brain. We report that interleukin-4 (IL-4) plays a central role in modulating expression of CD200 and identify a mechanism by which IL-4 directly controls microglial cell activation. Our findings provide the first demonstration of a role for IL-4 in modulating CD200 expression and suggest a mechanism for regulation of microglial activation in the intact CNS under inflammatory conditions.

Fractalkine‐induced activation of the phosphatidylinositol‐3 kinase pathway attentuates microglial activation in vivo and in vitro

Several neurodegenerative disorders are associated with evidence of inflammation, one feature of which is increased activation of microglia, the most likely cellular source of inflammatory cytokines like interleukin‐1β. It is now recognized that interaction of microglia with other cells contributes to maintenance of microglia in a quiescent state and the complementary distribution of the chemokine, fractalkine (CX3CL1) on neurons and its receptor (CX3CR1) on microglia, suggests that this interaction may play a role in modulating microglial activation. Here we demonstrate that both soluble and membrane‐bound fractalkine attenuate lipopolysaccharide‐induced microglial activation in vitro. We also show that fractalkine expression is reduced in the brain of aged rats and this is accompanied by an age‐related increase in microglial activation. Treatment of aged rats with fractalkine attenuates the age‐related increase in microglial activation and the evidence indicates that fractalkine‐induced activation of the phosphatidylinositol‐3 kinase pathway is required to maintain microglia in a quiescent state both in vivo and in vitro.

The polyunsaturated fatty acids, EPA and DPA exert a protective effect in the hippocampus of the aged rat

Age is characterized by deficits in synaptic function identified by decreased performance of aged animals in spatial learning tasks and reduced ability of animals to sustain long term potentiation (LTP). Several cellular and molecular events are correlated with these deficits, many of which are indicative of age-related neuroinflammatory and oxidative cell stress. It is significant that agents which decrease microglial activation are commonly associated with restoration of function. We set out to examine whether the n-3 polyunsaturated fatty acid docosapentaenoic acid (DPA), which is a metabolite of eicosapentaenoic acid (EPA), could modulate the age-related increase in microglial activation and the associated increase in oxidative changes and therefore impact on synaptic function in aged rats. We demonstrate that docosapentaenoic acid possesses neurorestorative effects and is capable of downregulating microglial activation. The data show that it also decreases the coupled activation of sphingomyelinase and caspase 3, probably because of its ability to decrease age-related oxidative changes, and consequently attenuates the age-related decrease in spatial learning and long-term potentiation.

A role for c-Jun N-terminal kinase 1 (JNK1), but not JNK2, in the beta-amyloid-mediated stabilization of protein p53 and induction of the apoptotic cascade in cultured cortical neurons.

beta-Amyloid (A beta) peptide has been shown to induce neuronal apoptosis; however, the mechanisms underlying A beta-induced neuronal cell death remain to be fully elucidated. The stress-activated protein kinase, c-Jun N-terminal kinase (JNK), is activated in response to cellular stress and has been identified as a proximal mediator of cell death. In the present study, expression of active JNK was increased in the nucleus and cytoplasm of A beta-treated cells. Evaluation of the nature of the JNK isoforms activated by A beta revealed a transient increase in JNK1 activity that reached its peak at 1 h and a later activation (at 24 h) of JNK2. The tumour suppressor protein, p53, is a substrate for JNK and can serve as a signalling molecule in apoptosis. In cultured cortical neurons, we found that A beta increased p53 protein expression and phosphorylation of p53 at Ser(15). Thus it appears that A beta increases p53 expression via phosphorylation-mediated stabilization of the protein. Given the lack of availability of a JNK inhibitor that can distinguish between JNK1- and JNK2-mediated effects, we employed antisense technology to deplete cells of JNK1 or JNK2 selectively. Using this strategy, the respective roles of JNK1 and JNK2 on the A beta-mediated activation of the apoptotic cascade (i.e. p53 stabilization, caspase 3 activation and DNA fragmentation) were examined. The results obtained demonstrate a role for JNK1 in the A beta-induced stabilization of p53, activation of caspase 3 and DNA fragmentation. In contrast, depletion of JNK2 had no effect on the proclivity of A beta to activate capase 3 or induce DNA fragmentation. These results demonstrate a significant role for JNK1 in A beta-mediated induction of the apoptotic cascade in cultured cortical neurons.

A novel anti-inflammatory role of NCAM-derived mimetic peptide, FGL.

Age-related cognitive deficits in hippocampus are correlated with neuroinflammatory changes, typified by increased pro-inflammatory cytokine production and microglial activation. We provide evidence that the neural cell adhesion molecule (NCAM)-derived mimetic peptide, FG loop (FGL), acts as a novel anti-inflammatory agent. Administration of FGL to aged rats attenuated the increased expression of markers of activated microglia, the increase in pro-inflammatory interleukin-1beta (IL-1beta) and the impairment in long-term potentiation (LTP). We report that the age-related increase in microglial activation was accompanied by decreased expression of neuronal CD200, and suggest that the proclivity of FGL to suppress microglial activation is due to its stimulatory effect on neuronal CD200. We demonstrate that FGL enhanced interleukin-4 (IL-4) release from glial cells and IL-4 in turn enhanced neuronal CD200 in vitro. We provide evidence that the increase in CD200 is reliant on IL-4-induced extracellular signal-regulated kinase (ERK) signal transduction. These findings provide the first evidence of a role for FGL as an anti-inflammatory agent and identify a mechanism by which FGL controls microglial activation.

Tetrahydrocannabinol-induced neurotoxicity depends on CB1 receptor-mediated c-Jun N-terminal kinase activation in cultured cortical neurons

Δ9‐Tetrahydrocannabinol (THC), the main psychoactive ingredient of marijuana, induces apoptosis in cultured cortical neurons. THC exerts its apoptotic effects in cortical neurons by binding to the CB1 cannabinoid receptor. The CB1 receptor has been shown to couple to the stress‐activated protein kinase, c‐Jun N‐terminal kinase (JNK). However, the involvement of specific JNK isoforms in the neurotoxic properties of THC remains to be established. The present study involved treatment of rat cultured cortical neurons with THC (0.005–50 μM), and combinations of THC with the CB1 receptor antagonist, AM 251 (10 μM) and pertussis toxin (PTX; 200 ng ml−1). Antisense oligonucleotides (AS) were used to deplete neurons of JNK1 and JNK2 in order to elucidate their respective roles in THC signalling. Here we report that THC induces the activation of JNK via the CB1 receptor and its associated G‐protein, Gi/o. Treatment of cultured cortical neurons with THC resulted in a differential timeframe of activation of the JNK1 and JNK2 isoforms. Use of specific JNK1 and JNK2 AS identified activation of caspase‐3 and DNA fragmentation as downstream consequences of JNK1 and JNK2 activation. The results from this study demonstrate that activation of the CB1 receptor induces JNK and caspase‐3 activation, an increase in Bax expression and DNA fragmentation. The data demonstrate that the activation of both JNK1 and JNK2 isoforms is central to the THC‐induced activation of the apoptotic pathway in cortical neurons.

Decreased neuronal CD200 expression in IL-4-deficient mice results in increased neuroinflammation in response to lipopolysaccharide

Maintenance of the balance between pro- and anti-inflammatory cytokines in the brain, which is affected by the activation state of microglia, is important for maintenance of neuronal function. Evidence has suggested that IL-4 plays an important neuromodulatory role and has the ability to decrease lipopolysaccharide-induced microglial activation and the production of IL-1beta. We have also demonstrated that CD200-CD200R interaction is involved in immune homeostasis in the brain. Here, we investigated the anti-inflammatory role of IL-4 and, using in vitro and in vivo analysis, established that the effect of lipopolysaccharide was more profound in IL-4(-/-), compared with wildtype, mice. Intraperitoneal injection of lipopolysaccharide exerted a greater inhibitory effect on exploratory behaviour in IL-4(-/-), compared with wildtype, mice and this was associated with evidence of microglial activation. We demonstrate that the increase in microglial activation is inversely related to CD200 expression. Furthermore, CD200 was decreased in neurons prepared from IL-4(-/-) mice, whereas stimulation with IL-4 enhanced CD200 expression. Importantly, neurons prepared from wildtype, but not from IL-4(-/-), mice attenuated the lipopolysaccharide-induced increase in pro-inflammatory cytokine production by glia. These findings suggest that the neuromodulatory effect of IL-4, and in particular its capacity to maintain microglia in a quiescent state, may result from its ability to upregulate CD200 expression on neurons.

Δ9-Tetrahydrocannabinol induces the apoptotic pathway in cultured cortical neurones via activation of the CB1 receptor

Δ9-Tetrahydrocannabinol, the principal psychoactive component of marijuana, exerts a variety of effects on the CNS, including impaired cognitive function and neurobehavioural deficits. The mechanisms underlying these neuronal responses to tetrahydrocannabinol are unclear but may involve alterations in neuronal viability. Tetrahydrocannabinol has been shown to influence neuronal survival but the role of the cannabinoid receptors in the regulation of neuronal viability has not been fully clarified. In this study we demonstrate that tetrahydrocan- nabinol promotes the release of cytochrome c, activates caspase-3, promotes cleavage of the DNA repair enzyme poly-ADP ribose polymerase and induces DNA fragmentation in cultured cortical neurones. These effects of tetrahydrocannabinol were completely abrogated by the CB1 receptor antagonist AM-251. The findings of this study demonstrate that tetrahydrocannabinol induces apoptosis in cortical neurones in a manner involving the CB1 subtype of cannabinoid receptor.

Cannabinoids and Innate Immunity: Taking a Toll on Neuroinflammation

The biologically active components of cannabis have therapeutic potential in neuroinflammatory disorders due to their anti-inflammatory propensity. Cannabinoids influence immune function in both the peripheral and the central nervous system (CNS), and the components of the cannabinoid system, the cannabinoid receptors and their endogenous ligands (endocannabinoids), have been detected on immune cells as well as in brain glia. Neuroinflammation is the complex innate immune response of neural tissue to control infection and eliminate pathogens, and Toll-like receptors (TLRs), a major family of pattern recognition receptors (PRRs) that mediate innate immunity, have emerged as players in the neuroinflammatory processes underpinning various CNS diseases. This review will highlight evidence that cannabinoids interact with the immune system by impacting TLR-mediated signaling events, which may provide cues for devising novel therapeutic approaches for cannabinoid ligands.

A synthetic NCAM‐derived mimetic peptide, FGL, exerts anti‐inflammatory properties via IGF‐1 and interferon‐γ modulation

Microglial cell activity increases in the rat hippocampus during normal brain aging. The neural cell adhesion molecule (NCAM)‐derived mimetic peptide, FG loop (FGL), acts as an anti‐inflammatory agent in the hippocampus of the aged rat, promoting CD200 ligand expression while attenuating glial cell activation and subsequent pro‐inflammatory cytokine production. The aim of the current study was to determine if FGL corrects the age‐related imbalance in hippocampal levels of insulin‐like growth factor‐1 (IGF‐1) and pro‐inflammatory interferon‐γ (IFNγ), and subsequently attenuates the glial reactivity associated with aging. Administration of FGL reversed the age‐related decline in IGF‐1 in hippocampus, while abrogating the age‐related increase in IFNγ. FGL robustly promotes IGF‐1 release from primary neurons and IGF‐1 is pivotal in FGL induction of neuronal Akt phosphorylation and subsequent CD200 ligand expression in vitro. In addition, FGL abrogates both age‐ and IFNγ‐induced increases in markers of glial cell activation, including major histocompatibility complex class II (MHCII) and CD40. Finally, the proclivity of FGL to attenuate IFNγ‐induced glial cell activation in vitro is IGF‐1‐dependent. Overall, these findings suggest that FGL, by correcting the age‐related imbalance in hippocampal levels of IGF‐1 and IFNγ, attenuates glial cell activation associated with aging. These findings also highlight a novel mechanism by which FGL can impact on neuronal CD200 ligand expression and subsequently on glial cell activation status.