Blockade of glutamate release from microglia attenuates experimental autoimmune encephalomyelitis in mice.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating and neurodegenerative disease of the central nervous system. Despite a variety of anti-inflammatory or immunomodulation drugs including interferon-beta are effective to reduce relapse risk, most patients have progressive neurological deterioration due to axonal degeneration. Accumulation of activated microglia is a pathological hallmark of active MS lesion. Microglia can act as not only antigen-presenting cells but also effector cells to damage other cells in the central nervous system. Especially, glutamate released by activated microglia induces excito-neurotoxicity and may contribute to neurodegeneration in MS. Gap junction is a major cell-to-cell channel and is composed of paired hemichannels on coupled cells. Recent studies showed that cells release various small molecules (including ions, ATP, and amino acids) from unpaired hemichannel of gap junction that is openly exposed to the extracellular space. We have previously revealed that activated microglia produce glutamate via glutaminase and release it through hemichannels of gap junctions. Thus, in this study, we examined whether the glutaminase inhibitor and the gap junction blocker relieved experimental autoimmune encephalomyelitis (EAE) that is an animal model of MS. Here we show that the gap junction blocker carbenoxolone (CBX) and the glutaminase inhibitor 6-diazo-5-oxo-L-norleucine (DON) decreased glutamate release from activated microglia and rescued neuronal death in a dose-dependent manner in vitro. In EAE mice, treatment with CBX or DON also attenuated EAE clinical symptoms. Thus, blockade of glutamate release from activated microglia with CBX or DON may be an effective therapeutic strategy against neurodegeneration in MS.

Macrophage-induced neurotoxicity is mediated by glutamate and attenuated by glutaminase inhibitors and gap junction inhibitors.

We have shown previously, that the most neurotoxic factor from activated microglia is glutamate that is produced by glutaminase utilizing extracellular glutamine as a substrate. Drugs that inhibit glutaminase or gap junction through which the glutamate is released were effective in reducing neurotoxic activity of microglia. In this study, to elucidate whether or not a similar mechanism is operating in macrophages infiltrating into the central nervous system during inflammatory, demyelinating, and ischemic brain diseases, we examined the neurotoxicity induced by macrophages, in comparison with microglia in vitro. LPS- or TNF-alpha-stimulated macrophage-conditioned media induced robust neurotoxicity, which was completely inhibited by the NMDA receptor antagonist MK801. Both the glutaminase inhibitor 6-diazo-5-oxo-l-norleucine (DON), and the gap junction inhibitor carbenoxolone (CBX), effectively suppressed glutamate production and subsequent neurotoxicity by activated macrophages. These results revealed that macrophages produce glutamate via glutaminase from extracelluar glutamine, and release it through gap junctions. This study demonstrated that a similar machinery is operating in macrophages as well, and DON and CBX that prevent microglia-mediated neurotoxicity should be effective for preventing macrophage-mediated neurotoxicity. Thus, these drugs may be effective therapeutic reagents for inflammatory, demyelinating, and ischemic brain diseases.

Excitatory amino acid transporter expression by astrocytes is neuroprotective against microglial excitotoxicity.

Glutamate-induced excitotoxicity is considered as a major cause of neurodegenerative disease. Excitatory amino acid transporters (EAATs) on glial cells are responsible for the homeostasis of extracellular glutamate in the central nervous system which may contribute to the prevention of excitotoxic neurodegeneration. However, the differential EAAT expression in astrocytes and microglia is not fully understood. In this study, we compared the expression of EAATs in astrocytes and microglia, and we assessed the neuroprotective and neurotoxic function of astrocytes and microglia by a co-culture system. RT-PCR analyses detected that astrocytes expressed each EAAT (EAAT1-5) whereas microglia did not express EAAT4. Western blot analyses demonstrated that astrocytes express a much larger amount of membrane-localized EAATs than microglia. Astrocytes prevented excito-neurotoxicity by the reduction of exogenous glutamate whereas microglia did not. Conversely, activated microglia released an excess of glutamate that induced excitotoxic neuronal death. Astrocytes rescued neurons from microglial glutamate-induced death in a ratio-dependent manner. Inhibition of EAATs abolished glutamate uptake and the neuroprotective effect of astrocytes, but it did not alter any microglial neurotoxic or neuroprotective effects. These results revealed that astrocytic EAATs can counteract microglial glutamate-induced neuronal death whereas microglial EAATs are inconsequential to neurotoxicity and neuroprotection.

The direct and indirect effects of serofendic acid on neuroprotection.

Serofendic acid is a novel neuroprotective factor isolated from fetal calf serum. To elucidate the mechanisms how serofendic acid exerts neuroprotection, we examined its effects on glutamate-induced excito-toxicity in mouse cortical neurons. The effects of serofendic acid on inflammatory cytokine and neurotrophin production by glial cells were also examined to evaluate the indirect neuroprotection. Serofendic acid significantly and dose dependently increased survival of mouse cortical neurons after 10 muM N-methyl-D-asparate (NMDA) exposure. However, it did not affect production of inflammatory cytokines and neurotrophins by microglia as assessed by reverse transciption polymerase chain reaction (RT-PCR) for mRNA expression and ELISA for protein levels, though it suppressed tumor necrosis factor (TNF)-alpha production by astrocytes. Thus, serofendic acid works directly on neurons to protect against glutamate toxicity. Suppression of TNF-alpha production by astoryctes may also synergistically exert neuroprotective functions of serofendic acid. Serofendic acid may be of use for the future therapeutic strategy against ischemic and degenerative neurological disorders.

Production of IL-27 and other IL-12 family cytokines by microglia and their subpopulations.

Production of IL-27 and other IL-12 family cytokines by murine microglia were examined using RT-PCR, real-time RT-PCR and Western blot analysis. We show for the first time that murine microglia produce IL-27 in response to lipopolysaccharide (LPS) and/or interferon-gamma. Primary microglia, but not their cell lines, also induce IL-12 and IL-23 upon above stimulation. Therefore, microglia may play a critical role initiating Th1 responses via producing IL-12 family cytokines in the brain.

Role of histaminergic neurons in development of epileptic seizures in EL mice.

The EL mouse is an animal model for hereditary temporal lobe epilepsy. When the mice receive weekly vestibular stimulation, e.g., 30 "tosses", 10-15 cm vertically, they start to convulse after 1-2 weeks. The aim of this study was to evaluate the role of the histaminergic neurons in the regulation of seizure development in the EL mice. The obtained results indicated that administration of either histidine, a substrate for histamine synthesis, or metoprine (2,4-diamino-5-(3,4-dichlorophnyl)-6-methyl-pyrimidine), an inhibitor of histamine N-methyltransferase (HNMT), retarded the onset of seizure episodes in the mice. The co-administration of histidine and metoprine caused a more marked delay in it. The histamine levels in the brain significantly increased in response to any of these treatments. The intraperitoneal injection of diphenhydramine, a H1-antagonist accelerated the initiation of seizure episodes in the mice, whereas thioperamide, a H3-antagonist caused a delay in the response. There were significant increases in the brain histamine levels upon injection of any of these drugs with concomitant rises in the activity of the histidine decarboxylase (HDC). These results, taken together, suggest that the histaminergic neurons play crucial roles in the development of seizures in the EL mice. They inhibit convulsion in a H1-dependent fashion, while the neurons enhance it in a H3-receptor-mediated way.