Neurotoxic Mediators Research Articles

Hypoxia potentiates cytotoxicity of LPS-activated microglial BV2 cells in vitro by synergistic effects on cytokine and nitric oxide secretion

Results Activation of BV2 microglial cells by LPS exposure stimulated significant and persistent production of NO, IL-1b, IL-6, and TNF-a. Even after LPS removal, ongoing NO and cytokine secretion could be observed. While hypoxia alone mediated exclusively a significant, shortterm increase of IL-1b, oxygen deprivation enhanced LPS-induced secretion of NO, IL-1b, IL-6, and TNF-a significantly. Surprisingly, pre-stimulation of BV2 cells by hypoxia prior LPS exposure abolished microglial activation suppressing LPS-induced NO production. Hereby, cell-free supernatants derived from LPS-activated microglial cells exhibited a stronger cytotoxic effect in glial and neuronal cells than LPS exposition per se (P < 0.001). Again, hypoxia potentiated LPS-induced cytotoxicity. Conclusion Present data prove that i) the outcome of hypoxia is determined by the microglial activation status and that ii) LPS-induced soluble factors rather than LPS are mediators of microglial neurotoxicity under conditions of hypoxia in vitro. Activation of pro-inflammatory pathways may sensitize microglial cells to promote hypoxia-induced injury of the developing brain. Consequently, our findings may promote neuroprotective therapeutic strategies in the field of perinatal brain injury.

Mechanisms and potential therapeutic applications of microglial activation after brain injury.

As the resident immune cells of the central nervous system, microglia rapidly respond to brain insults, including stroke and traumatic brain injury. Microglial activation plays a major role in neuronal cell damage and death by releasing a variety of inflammatory and neurotoxic mediators. Their activation is an early response that may exacerbate brain injury and many other stressors, especially in the acute stages, but are also essential to brain recovery and repair. The full range of microglial activities is still not completely understood, but there is accumulating knowledge about their role following brain injury. We review recent progress related to the deleterious and beneficial effects of microglia in the setting of acute neurological insults and the current literature surrounding pharmacological interventions for intervention.

Neutralization of TNFSF10 ameliorates functional outcome in a murine model of Alzheimer's disease.

Alzheimer's disease is one of the most common causes of death worldwide, with poor treatment options. A tissue landmark of Alzheimer's disease is accumulation of the anomalous protein amyloid-β in specific brain areas. Whether inflammation is an effect of amyloid-β on the Alzheimer's disease brain, or rather it represents a cause for formation of amyloid plaques and intracellular tangles remains a subject of debate. TNFSF10, a proapoptotic cytokine of the TNF superfamily, is a mediator of amyloid-β neurotoxicity. Here, we demonstrate that blocking TNFSF10 by administration of a neutralizing monoclonal antibody could attenuate the amyloid-β-induced neurotoxicity in a triple transgenic mouse model of Alzheimer's disease (3xTg-AD). The effects of TNFSF10 neutralization on either cognitive parameters, as well as on the expression of TNFSF10, amyloid-β, inflammatory mediators and GFAP were studied in the hippocampus of 3xTg-AD mice. Treatment with the TNFSF10 neutralizing antibody resulted in dramatic improvement of cognitive parameters, as assessed by the Morris water maze test and the novel object recognition test. These results were correlated with decreased protein expression of TNFSF10, amyloid-β, inflammatory mediators and GFAP in the hippocampus. Finally, neutralization of TNFSF10 results in functional improvement and restrained immune/inflammatory response in the brain of 3xTg-AD mice in vivo. Thus, it is plausible to regard the TNFSF10 system as a potential target for efficacious treatment of amyloid-related disorders.

Platelet-activating factor mediates the cytotoxicity induced by W7FW14F apomyoglobin amyloid aggregates in neuroblastoma cells.

W7FW14F apomyoglobin (W7FW14F ApoMb) amyloid aggregates induce cytotoxicity in SH-SY5Y human neuroblastoma cells through a mechanism not fully elucidated. Amyloid neurotoxicity process involves calcium dyshomeostasis and reactive oxygen species (ROS) production. Another key mediator of the amyloid neurotoxicity is Platelet-Activating Factor (PAF), an inflammatory phospholipid implicated in neurodegenerative diseases. Here, with the aim at evaluating the possible involvement of PAF signaling in the W7FW14F ApoMb-induced cytotoxicity, we show that the presence of CV3899, a PAF receptor (PAF-R) antagonist, prevented the detrimental effect of W7FW14F ApoMb aggregates on SH-SY5Y cell viability. Noticeably, we found that the activation of PAF signaling, following treatment with W7FW14F ApoMb, involves a decreased expression of the PAF acetylhydroase II (PAF-AH II). Interestingly, the reduced PAF-AH II expression was associated with a decreased acetylhydrolase (AH) activity and to an increased sphingosine-transacetylase activity (TA(S)) with production of N-acetylsphingosine (C2-ceramide), a well known mediator of neuronal caspase-dependent apoptosis. These findings suggest that an altered PAF catabolism takes part to the molecular events leading to W7FW14F ApoMb amyloid aggregates-induced cell death.

Photoreceptor toxicity of subretinal Mononuclear Phagocytes

AbstractGrowing evidences indicates that inflammation play an important role in AMD. In particular, subretinal mononuclear phagocytes (MPs) accumulate in the vicinity of the atrophic lesion of GA patients and are thought to contribute to photoreceptor degeneration. The mechanism by which MP participates to neuronal cell loss is currently unknown and no treatment is available to date to delay degeneration. We have recently shown that subretinal MPs that originate from the blood circulation and accumulate in the subretinal space are particularly detrimental in animal models of subretinal inflammation. Defects in the CX3CR1/CX3CL1 axis have been associated in animals with cardinal features of AMD including photoreceptor loss. We here show that CX3CR1 deficiency resulted in an exacerbated neurotoxicity of subretinal MPs. we present evidences that CX3CR1‐/‐ deficiency lead to the differentiation of subretinal MPs into an exacerbated pro‐inflammatory profile. Inhibiting neurotoxic mediators produced by subretinaly differentiated monocytes efficiently reduces photoreceptor loss in vitro and in animal models where MP accumulates. Our result provides new rationales to protect retina from damaging age‐dependent subretinal inflammation.

Rapamycin Is Neuroprotective in a Rat Chronic Hypertensive Glaucoma Model

Glaucoma is a leading cause of irreversible blindness. Injury of retinal ganglion cells (RGCs) accounts for visual impairment of glaucoma. Here, we report rapamycin protects RGCs from death in experimental glaucoma model and the underlying mechanisms. Our results showed that treatment with rapamycin dramatically promote RGCs survival in a rat chronic ocular hypertension model. This protective action appears to be attributable to inhibition of neurotoxic mediators release and/or direct suppression of RGC apoptosis. In support of this mechanism, in vitro, rapamycin significantly inhibits the production of NO, TNF-α in BV2 microglials by modulating NF-κB signaling. In experimental animals, treatment with rapamycin also dramatically inhibited the activation of microglials. In primary RGCs, rapamycin was capable of direct suppression the apoptosis of primary RGCs induced by glutamate. Mechanistically, rapamycin-mediated suppression of RGCs apoptosis is by sparing phosphorylation of Akt at a site critical for maintenance of its survival-promoting activity in cell and animal model. These results demonstrate that rapamycin is neuroprotective in experimental glaucoma, possibly via decreasing neurotoxic releasing and suppressing directly apoptosis of RGCs.

Resveratrol counteracts lipopolysaccharide-mediated microglial inflammation by modulating a SOCS-1 dependent signaling pathway

Brain damage or exposure to inflammatory agents provokes the activation of microglia and secretion of pro-inflammatory and neurotoxic mediators responsible for neuronal loss. Several lines of evidence show that resveratrol, a natural non-flavonoid polyphenol, may exert a neuroprotective action in neurodegenerative diseases. Suppressor of cytokine signaling (SOCS) proteins are a family of eight members expressed by immune cells and the central nervous system (CNS) cells, that regulate immune processes within the CNS, including microglia activation. We demonstrate that resveratrol had anti-inflammatory effects in murine N13 microglial cells stimulated with lipopolysaccharide (LPS), through up-regulating SOCS-1 expression. Interestingly, in SOCS-1-silenced cells resveratrol failed to play a protective role after LPS treatment. Our data demonstrate that resveratrol can impair microglia activation by activating a SOCS-1 mediated signaling pathway.

Oxidative Stress Is Associated with Neuroinflammation in Animal Models of HIV-1 Tat Neurotoxicity.

HIV-1 trans-acting protein Tat, an essential protein for viral replication, is a key mediator of neurotoxicity. If Tat oxidant injury and neurotoxicity have been described, consequent neuroinflammation is less understood. Rat caudate-putamens (CPs) were challenged with Tat, with or without prior rSV40-delivered superoxide dismutase or glutathione peroxidase. Tat injection caused oxidative stress. Administration of Tat in the CP induced an increase in numbers of Iba-1- and CD68-positive cells, as well as an infiltration of astrocytes. We also tested the effect of more protracted Tat exposure on neuroinflammation using an experimental model of chronic Tat exposure. SV(Tat): a recombinant SV40-derived gene transfer vector was inoculated into the rat CP, leading to chronic expression of Tat, oxidative stress, and ongoing apoptosis, mainly located in neurons. Intra-CP SV(Tat) injection induced an increase in microglia and astrocytes, suggesting that protracted Tat production increased neuroinflammation. SV(SOD1) or SV(GPx1) significantly reduced neuroinflammation following Tat administration into the CP. Thus, Tat-induced oxidative stress, CNS injury, neuron loss and inflammation may be mitigated by antioxidant gene delivery.

Hydrogen sulfide attenuates hypoxia-induced neurotoxicity through inhibiting microglial activation

Endogenously produced hydrogen sulfide (H2S) may have multiple functions in the brain including potent anti-inflammatory effects. Activated microglia can secrete various pro-inflammatory cytokines and neurotoxic mediators, which may contribute to hypoxic injuries in the developing brain. The aim of this study is to investigate the potential role of H2S in altering hypoxia-induced neurotoxicity via its anti-inflammatory actions as examined in vitro and in vivo models. Using the BV-2 microglial cell line, we found that sodium hydrosulfide (NaHS), a H2S donor, significantly inhibited hypoxia-induced microglial activation and suppressed subsequent pro-inflammatory factor release. In addition, treating murine primary cortical neurons with conditioned medium (CM) from hypoxia-stimulated microglia induced neuronal apoptosis, an effect that was reversed by CM treated with NaHS. Further, NaHS inhibited phosphorylation of the p65 subunit of NF-κB, phosphorylation of ERK and p38 but not JNK MAPK in these hypoxia-induced microglia. When administered in vivo to neonatal mice subjected to hypoxia, NaHS was found to attenuate neuron death, an effect that was associated with suppressed microglial activation, pro-inflammatory cytokines and NO levels. Taken together, H2S exerts neuroprotection against hypoxia-induced neurotoxicity through its anti-inflammatory effect in microglia. This effect appears to be attributable to inhibition of iNOS, NF-κB, ERK and p38 MAPK signaling pathways. Our results suggest a potential therapeutic application of H2S releasing drugs in hypoxic brain damage treatment.

Protease-activated Receptor-1 (PAR-1) and Interleukin-1Beta Act Synergistically in Granzyme b Mediated Neurotoxicity (P1.159)

Protease-activated Receptor-1 (PAR-1) and Interleukin-1Beta Act Synergistically in Granzyme b Mediated Neurotoxicity (P1.159)

Reciprocal disruption of neuronal signaling and Aβ production mediated by extrasynaptic NMDA receptors: a downward spiral

It is becoming increasingly clear that aberrant neuronal activity can be the cause and the result of amyloid beta production. Synaptic activation facilitates non-amyloidogenic processing of amyloid precursor protein (APP) and cell survival, primarily through synaptic NMDA receptors (NMDARs) and perhaps specifically those containing GluN2A-subunits. In contrast, extrasynaptic and GluN2B-containing NMDARs promote beta-secretase cleavage of APP into amyloid-beta (Aβ). The opposing nature of these NMDAR populations is reflected in their control over cell survival and death pathways. Subtle changes in glutamate homeostasis may shift the balance between these pathways and could play a role in Alzheimer's disease (AD). Indeed, Aβ production, regional loss of brain connectivity and neurodegeneration correlate with neuronal activity in AD patients. From another perspective, Aβ oligomers (Aβo) alter neuronal signaling through several mechanisms involving NMDARs and intracellular calcium mishandling. While Aβo affect multiple receptors, GluN2B-NMDARs have emerged as primary mediators of altered synaptic plasticity and neurotoxicity. Memantine and its successor, NitroMemantine, are efficient at blocking or reversing the deleterious actions of Aβo largely due to their selectivity for extrasynaptic NMDARs. Recently, Aβo were shown to trigger astrocytic release of glutamate to the extrasynaptic space where it activates NMDARs to promote further Aβ production and synaptic depression. Combined with the reciprocal regulation between neuronal activity and Aβ production, extrasynaptic glutamate release adds to a maladaptive model and ultimately results in synaptotoxicity and neurodegeneration of AD. Extrasynaptic NMDAR antagonists remain as a promising therapeutic avenue by interfering with this cascade.

Association between anti-U1 ribonucleoprotein antibodies and inflammatory mediators in cerebrospinal fluid of patients with neuropsychiatric systemic lupus erythematosus

We investigated possible associations between neurotoxic inflammatory mediators (IMs) and anti-U1RNP antibodies (Abs) in cerebrospinal fluid (CSF) of neuropsychiatric systemic lupus erythematosus (NPSLE). Serum and CSF anti-U1RNP Abs were detected using an RNA-immunoprecipitation assay and CSF anti-U1RNP Ab levels were measured by ELISA. IFN-α, MCP-1 and IL-8 levels in CSF were determined by quantitative multiplex cytokine analysis. IM levels were compared among anti-U1RNP-positive and anti-U1RNP-negative NPSLE as well as other rheumatic disease controls (controls). Anti-U1RNP Abs were detected in serum (58%) and in CSF (18%) of 82 NPSLE patients. CSF MCP-1 levels were higher in NPSLE than in controls. CSF IFN-α level was higher in CSF anti-U1RNP Ab-positive than in -negative patients or controls. When limited to serum anti-U1RNP Ab-positive patients, however, levels of all three IMs in CSF were higher in CSF anti-U1RNP Ab-positive than in -negative patients. Anti-U1RNP Ab levels in CSF correlated with CSF MCP-1, but not IFN-α and IL-8 levels. CSF anti-U1RNP Ab positivity is associated with increased level of CSF IFN-α. MCP-1 levels correlated with CSF anti-U1RNP Ab levels, whereas the increased CSF MCP-1 was not specific to CSF anti-U1RNP Ab-positive NPSLE.

Up-regulation of ROS-Dependent Matrix Metalloproteinase-9 from High-Glucose-Challenged Astrocytes Contributes to the Neuronal Apoptosis

An elevated level of glucose has been found in the blood of hyperglycemia and diabetes patients associated with several central nervous system (CNS) complications. These disorders may be due to the up-regulation of many neurotoxic mediators by host cells triggered by high glucose (HG). Moreover, increased plasma levels of matrix metalloproteinases (MMPs), MMP-9 especially, have been observed in patients with brain injuries and may contribute to brain diseases. However, the HG level triggers the CNS pathological responses during the hyperglycemia and diabetes remain unclear. In this study, we use a transformed astroglial cell (RBA-1) as a model to investigate the signaling mechanisms of MMP-9 induction by HG and its effects on neuronal cells. First, the data by gelatin zymographic and Western blotting analyses demonstrated that HG-induced MMP-9 expression. Next, the data obtained with selective pharmacological inhibitors and small interfering RNAs showed that HG-induced MMP-9 expression via a c-Src-dependent transactivation of platelet-derived growth factor receptor and PI3K/Akt linking to NADPH oxidase 2-derived reactive oxygen species signal and activation of MAPKs. Subsequently, the transcriptional factor AP-1 was activated and thereby turned on transcription of MMP-9 gene. Ultimately, we found that HG-induced MMP-9 expression from astrocytes resulted in neuronal apoptosis. These results will provide new insights into the mechanisms and effects of the action of HG, supporting the hypothesis that HG may cause brain disorders in the development of diabetes and hyperglycemia-induced CNS complications such as neurodegenerative diseases.

Voltage-Gated Potassium Channel KV1.5 Protects against MPP+ Mediated Neurotoxicity in PC12 Cells

Background: Parkinson’s disease (PD) is the second most common neurodegenerative disease and afflicts almost 1.8% of over 65-year-old group in the world. Epidemiological projections showed that the incidence of PD was increasing continuously each year, with a wider age range as well. A large number of studies indicated that voltage-gated potassium channel (Kv) played significant roles in cellular signaling in both excitable and non-excitable cells. What’s more, Kv was also ubiquitously expressed in neurons and participated in signaling pathway in neurons. Kv1.5 (encoded by KCNA5) is an important voltage-gated K+ channel, which is not only necessary for critical processes such as cell proliferation and apoptosis but ubiquitously expressed in neurons. Recent studies reported that PD clinical drugs could inhibit the expression of Kv1.5. To determine the mechanisms by which Kv1.5 protects against MPP+ mediated neurotoxicity in PC12 cells. Materials and Methods: Knockdown of Kv1.5 model was established with pSINsi-hU6- Kv1.5 treated by the RNAi method in PC12. MTT, and Western Blot were used to detect the influence of Kv1.5 on PC12 proliferation, and the effect of Kv1.5 on PC12 apoptosis after MPP+ treatment in vitro. Results: 1) Knockdown and overexpression of Kv1.5 participated in PC12 proliferation. Transiently over-expressed Kv1.5 could boost the survival rate of PC12, while transiently knockdown of Kv1.5 inhibited PC12 proliferation. 2) The effect of Kv1.5 on PC12 proliferation was through PI3K/Akt signaling pathway. Over-expressed Kv1.5 could induce the activation of Akt, and Bcl-2 expression in PC12; Knockdown of Kv1.5 in PC12 inhibited the activation of Akt, Bcl-2 expression, and promoted MAPK phosphorylation. 3) Over-expressed Kv1.5 could significantly prevent PC12 from apoptosis induced by MPP+ via activating Akt pathway and increasing Bcl-2 expression; Knockdown of Kv1.5 was more sensitive than its control counterpart when treated with MPP+ for 24 h. Conclusion: Kv1.5 could hinder MPP+ neurotoxicity to PC12 by PI3K/Akt signaling pathway.

Tau-Hsp70 Interaction at the Single-Molecule Level

As part of the protein quality control centre of the cell, many molecular chaperones tightly regulate amyloid proteins and seem to play an important role in neurodegeneration. However, the precise mechanism of this interplay and how its modification leads to disease remains elusive. Particularly oligomeric species of amyloid proteins have emerged as the most important mediators of neurotoxicity in neurodegenerative diseases. These amyloid species are exceedingly hard to characterize as they are transient, heterogeneous and rare relative to the monomeric and fibrillar states. Single-molecule fluorescence spectroscopy allows us to specifically characterise the oligomeric species and follow their structural development during fibril formation and dissociation. We employed this technique to characterise the oligomers evolving during the aggregation of a recombinant tau variant carrying the Δ280K mutation associated with some forms of frontotemporal dementia. Further, we studied the in vitro interaction of these oligomers with the chaperone Hsp70 which has previously been identified as a key regulator of tau turnover in the cell. We found that Hsp70 controls the oligomerisation and fibrillisation of tau interacting more strongly with oligomers of a certain size and structure. Our results provide insights into the molecular mechanism by which Hsp70 inhibits tau aggregation and targets oligomers for degradation.

Chapter 16 - Human herpesvirus 6 and the nervous system

Human herpesvirus (HHV)-6 is a β-herpesvirus that infects most infants by 2 years of age and persists in a variety of host cells after primary infection, with intermittent reactivation typically during periods of immunosuppression. HHV-6 has two closely related species, HHV-6A and B, which are both neurotropic and can be detected in up to 85% of brain samples at autopsy. This pleiotropic virus has been implicated in many central nervous system (CNS) diseases, including febrile seizures (FS), epilepsy due to medial temporal lobe sclerosis, multiple sclerosis, encephalomyelitis, progressive multifocal leukoencephalopathy, chronic fatigue syndrome, and cognitive dysfunction. The significance of HHV-6 infection is often controversial due to the challenge implicit in attributing disease to a commensal virus of the brain. Studies have used a variety of unstandardized techniques on an array of sample sources to detect HHV-6, making direct comparisons problematic. In addition, many detection methods do not distinguish between latent and active infection. However, there is accumulating evidence implicating HHV-6 as a cause of CNS pathology, especially in FS and encephalitis. Treatment options are limited, fraught with side-effects, and poorly studied. Whether HHV-6 is a commensal pathogen of the brain, marker of immune dysregulation, trigger of autoimmunity, or directly neurotoxic mediator of CNS disease will remain uncertain for many CNS diseases until more refined studies are performed.

TAM Receptors Affect Adult Brain Neurogenesis by Negative Regulation of Microglial Cell Activation

TAM tyrosine kinases play multiple functional roles, including regulation of the target genes important in homeostatic regulation of cytokine receptors or TLR-mediated signal transduction pathways. In this study, we show that TAM receptors affect adult hippocampal neurogenesis and loss of TAM receptors impairs hippocampal neurogenesis, largely attributed to exaggerated inflammatory responses by microglia characterized by increased MAPK and NF-κB activation and elevated production of proinflammatory cytokines that are detrimental to neuron stem cell proliferation and neuronal differentiation. Injection of LPS causes even more severe inhibition of BrdU incorporation in the Tyro3(-/-)Axl(-/-)Mertk(-/-) triple-knockout (TKO) brains, consistent with the LPS-elicited enhanced expression of proinflammatory mediators, for example, IL-1β, IL-6, TNF-α, and inducible NO synthase, and this effect is antagonized by coinjection of the anti-inflammatory drug indomethacin in wild-type but not TKO brains. Conditioned medium from TKO microglia cultures inhibits neuron stem cell proliferation and neuronal differentiation. IL-6 knockout in Axl(-/-)Mertk(-/-) double-knockout mice overcomes the inflammatory inhibition of neurogenesis, suggesting that IL-6 is a major downstream neurotoxic mediator under homeostatic regulation by TAM receptors in microglia. Additionally, autonomous trophic function of the TAM receptors on the proliferating neuronal progenitors may also promote progenitor differentiation into immature neurons.

Licorice-derived dehydroglyasperin C increases MKP-1 expression and suppresses inflammation-mediated neurodegeneration

Recent studies have demonstrated that microglial hyperactivation-mediated neuroinflammation is involved in the pathogenesis of several neurodegenerative diseases. Thus, inhibiting microglial production of the neurotoxic mediator tumor necrosis factor-α (TNF-α) is considered a promising strategy to protect against neurodegeneration. Here, we investigated the inhibitory effect of licorice-derived dehydroglyasperin C (DGC) on lipopolysaccharide (LPS)-induced TNF-α production and inflammation-mediated neurodegeneration. We found that DGC pre-treatment attenuated TNF-α production in response to LPS stimulation of BV-2 microglia. DGC pre-treatment attenuated LPS-induced inhibitor of κB-α (IκB-α) and p65 phosphorylation and decreased the DNA binding activity of nuclear factor-κB (NF-κB). DGC pre-treatment also inhibited LPS-mediated phosphorylation of p38 mitogen-activated protein kinases (MAPKs) and extracellular signal-regulated kinase (ERK). Interestingly, DGC treatment of BV-2 microglia significantly increased MAPK phosphatase 1 (MKP-1) mRNA and protein expression, which is a phosphatase of p38 MAPK and ERK, suggesting that the DGC-mediated increase in MKP-1 expression might inhibit LPS-induced MAPKs and NF-κB activation and further TNF-α production. We also found that LPS-mediated microglial neurotoxicity can be attenuated by DGC. The addition of conditioned media (CM) from DGC- and LPS-treated microglia to neurons helped maintain healthy cell body and neurite morphology and increased the number of microtubule-associated protein 2-positive cells and the level of synaptophysin compared to treatment with CM from LPS-treated microglia. Taken together, these data suggest that DGC isolated from licorice may inhibit microglia hyperactivation by increasing MKP-1 expression and acting as a potent anti-neurodegenerative agent.

96. Anti-depressants and kynurenine pathway inhibition: A therapeutic strategy for neuroprotection in vitro?

The kynurenine pathway (KP) is the main pathway by which tryptophan is metabolized. Its rate limiting enzyme indolamine 2, 3-dioxygenase (IDO) catalyses the production of kynurenine, which in turn is metabolised by either kynurenine 3-hydroxylase (KMO) which leads to the production of quinolinic acid (neurotoxic), or by kynurenine aminotransferase (KAT II) which yields kynurenic acid (neuroprotective). These enzymes are localised in microglia and astrocytes respectively. The present studies demonstrated that IFN- γ induces expression of IDO, KMO and kynase along with the production of neurotoxic molecules from BV-2 microglia. This was determined using a cell culture system whereby microglia were activated with IFN- γ and this conditioned medium was then placed on primary cortical neurons. Inhibition of the kynurenine pathway with 1-(D)-methyl-tryptophan (IDO inhibitor) and Ro 61-8048 (KMO inhibitor) completely attenuated this microglial-mediated neurotoxicity, as did the NMDA antagonist MK-801. Furthermore the anti-depressant fluoxetine partially attenuated this neurotoxicity and IFN- γ induced increases in IDO and kynase mRNA expression. Inhibition of classical neurotoxic factors released from microglia i.e. iNOS and the enzyme NADPH oxidase had no effect on this microglial mediated neurotoxicity, and neither did the anti-inflammatory agent dexamethasone. These results propose IFN- γ activated microglia induce neurotoxicity by activating the KP, and this can be attenuated using fluoxetine, suggesting a possible role for anti-depressants in ameliorating neuronal damage and in influencing the KP balance.

Microglial P2Y12 Deficiency/Inhibition Protects against Brain Ischemia

ObjectiveMicroglia are among the first immune cells to respond to ischemic insults. Triggering of this inflammatory response may involve the microglial purinergic GPCR, P2Y12, activation via extracellular release of nucleotides from injured cells. It is also the inhibitory target of the widely used antiplatelet drug, clopidogrel. Thus, inhibiting this GPCR in microglia should inhibit microglial mediated neurotoxicity following ischemic brain injury.MethodsExperimental cerebral ischemia was induced, in vitro with oxygen-glucose deprivation (OGD), or in vivo via bilateral common carotid artery occlusion (BCCAO). Genetic knock-down in vitro via siRNA, or in vivo P2Y12 transgenic mice (P2Y12−/− or P2Y12+/−), or in vivo treatment with clopidogrel, were used to manipulate the receptor. Neuron death, microglial activation, and microglial migration were assessed.ResultsThe addition of microglia to neuron-astrocyte cultures increases neurotoxicity following OGD, which is mitigated by microglial P2Y12 deficiency (P<0.05). Wildtype microglia form clusters around these neurons following injury, which is also prevented in P2Y12 deficient microglia (P<0.01). P2Y12 knock-out microglia migrated less than WT controls in response to OGD-conditioned neuronal supernatant. P2Y12 (+/−) or clopidogrel treated mice subjected to global cerebral ischemia suffered less neuronal injury (P<0.01, P<0.001) compared to wild-type littermates or placebo treated controls. There were also fewer microglia surrounding areas of injury, and less activation of the pro-inflammatory transcription factor, nuclear factor Kappa B (NFkB).InterpretationP2Y12 participates in ischemia related inflammation by mediating microglial migration and potentiation of neurotoxicity. These data also suggest an additional anti-inflammatory, neuroprotective benefit of clopidogrel.