Activated microglia may be detrimental to neuronal survival in a number of neurodegenerative diseases. Thus, strategies that reduce microglial neurotoxicity may have therapeutic benefit. Stimulation of group II metabotropic glutamate (mGlu) receptors on rat primary microglia with the specific group II agonist 2S,2ЈR,3ЈR-2-(2Ј,3Ј-dicarboxy-cyclopropyl)glycine for 24 h induced microglial activation and resulted in a neurotoxic microglial phenotype. These effects were attributable to preferential mGlu2 stimulation, because N-acetyl-Laspartyl-L-glutamate, a specific mGlu3 agonist, did not induce microglial activation or neurotoxicity. Stimulation of microglial mGlu2 but not mGlu3 induced caspase-3 activation in cerebellar granule neurons in culture, using microglial-conditioned media as well as cocultures. Stimulation of microglial mGlu2 induced tumor necrosis factor-␣ (TNF␣) release, which contributed to microglial neurotoxicity mediated via neuronal TNF receptor 1 and caspase-3 activation. Stimulation of microglial group I or III mGlu receptors did not induce TNF␣ release. TNF␣ was only neurotoxic in the presence of microglia or microglial-conditioned medium. The toxicity of TNF␣ could be prevented by coexposure of neurons to conditioned medium from microglia stimulated by the specific group III agonist L-2-amino-4-phosphono-butyric acid. The neurotoxicity of TNF␣ derived from mGlu2-stimulated microglia was potentiated by microglial-derived Fas ligand (FasL), the death receptor ligand. FasL was constitutively expressed in microglia and shed after mGlu2 stimulation. Our data suggest that selective and inverse modulation of microglial mGlu2 and mGlu3 may prove a therapeutic target in neuroinflammatory diseases such as Alzheimer's disease and multiple sclerosis.

A reduction in microglial activation and subsequent neurotoxicity may prove critical for neuroprotection in neurodegenerative diseases. We examined the expression and functionality of group III metabotropic glutamate (mGlu) receptors on microglia. Rat microglia express mRNA and receptor protein for group III mGlu receptors mGlu4, mGlu6, and mGlu8 but not mGlu7. Activation of these receptors on microglia with the specific group III agonists (L)-2-amino-4-phosphono-butyric acid (L-AP-4) or (R,S)-phosphonophenylglycine (RS-PPG) inhibited forskolin-induced cAMP production, linking these receptors to the negative inhibition of adenylate cyclase. These agonists did not induce a fall in mitochondrial membrane potential or apoptosis in the microglia, suggesting that activation of these receptors is not in itself toxic to microglia. Fluorescence-activated cell sorting analysis revealed that activation of group III mGlu receptors induces a mild activation of the microglia, as evidence by their enhanced staining with ED1. However, this activation is not neurotoxic. Agonists of group III mGlu receptors reduced microglial reactivity when they were activated with lipopolysaccharide (LPS), chromogranin A (CGA) or amyloid ␤ peptide 25-35 (A␤25-35). Furthermore, L-AP-4 or RS-PPG treatment of microglia reduced their neurotoxicity after microglial stimulation with LPS or CGA but not A␤25-35. Similar results were obtained with microglial conditioned medium or in coculture, suggesting that the activation of microglial group III mGlu receptors may modulate the production of stable neurotoxins from the microglia. These results suggest that selective modulation of microglial group III mGlu receptors may provide a therapeutic target in neuroinflammatory diseases such as Alzheimer's disease.

Regulation of microglial reactivity and neurotoxicity is critical for neuroprotection in neurodegenerative diseases. Here we report that microglia possess functional group II metabotropic glutamate receptors, expressing mRNA and receptor protein for mGlu2 and mGlu3, negatively coupled to adenylate cyclase. Two different agonists of these receptors were able to induce a neurotoxic microglial phenotype which was attenuated by a specific antagonist. Chromogranin A, a secretory peptide expressed in amyloid plaques in Alzheimer's disease, activates microglia to a reactive neurotoxic phenotype. Chromogranin A-induced microglial activation and subsequent neurotoxicity may also involve an underlying stimulation of group II metabotropic glutamate receptors since their inhibition reduced chromogranin A-induced microglial reactivity and neurotoxicity. These results show that selective inhibition of microglial group II metabotropic glutamate receptors has a positive impact on neuronal survival, and may prove a therapeutic target in Alzheimer's disease.

Microglia, the resident macrophage of the brain, can release substances that aid neuronal development, differentiation and survival. We have investigated the effects of non-activated microglia on the survival of cultured rat cerebellar granule neurones. Microglial-conditioned medium, collected from primary rat microglial cultures, was used to treat 7-day-in-vitro neurones, and neuronal viability and proliferation was assessed following a further 1 or 7 days in culture. Microglialconditioned medium enhanced neuronal survival by up to 50% compared with untreated neurones and this effect was completely abated by pretreatment of the microglia with L-leucine methyl ester. The expression of the proliferation marker Ki-67 increased in neuronal cultures treated with microglial-conditioned medium suggesting enhanced proliferation of precursor neurones. Microglial-induced neuronal proliferation could be attenuated by specific inhibition of mitogen-activated protein kinase or phosphatidylinositol-3-kinase/Akt signalling pathways, and by selective fractionation and immunodepletion of the microglial-conditioned medium. Activation of the Notch pathway was enhanced as antibody against the Notch ligand, delta-1, prevented the microglial-induced neuronal proliferation. These results show that microglia release stable neurotrophic factors that can promote neuronal precursor cell proliferation.

Iron and immunity are closely linked: firstly by the fact that many of the genes/proteins involved in iron homoeostasis play a vital role in controlling iron fluxes such that bacteria are prevented from utilising iron for growth; secondly, cells of the innate immune system, monocytes, macrophages, microglia and lymphocytes, are able to combat bacterial insults by carefully controlling their iron fluxes, which are mediated by hepcidin and ferroportin. In addition, lymphocytes play an important role in adaptive immunity. Thirdly, a variety of effector molecules, e.g. toll-like receptors, NF-κB, hypoxia factor-1, haem oxygenase, will orchestrate the inflammatory response by mobilising a variety of cytokines, neurotrophic factors, chemokines, and reactive oxygen and nitrogen species. Pathologies, where iron loading and depletion occur, may adversely affect the ability of the cell to respond to the bacterial insult.

Marked abnormalities of the magnetic resonance intensity of N‐acetylaspartate (NAA) have been reported in patients with various neurological disorders, but the neurochemical consequences of these alterations are difficult to assess because the function of NAA remains speculative. The purpose of this study was to examine whether NAA plays a role in protecting neurons against osmotic stress. Intracerebral microdialysis was used to expose a small region of the rat dorsolateral striatum to an increasingly hyposmotic environment and to measure resulting changes in NAA extracellular concentrations. NAA changes in the extracellular fluid (ECF) were compared with those of the amino acids, in particular, taurine, known to be involved in brain osmoregulation. Stepped increases in cellular hydration produced by hyposmotic perfusion media induced a marked increase in ECF NAA, reflecting a redistribution of NAA from intra‐to extracellular space. Parallel experiments showed that, of all the extracellular amino acids measured, only taurine markedly increased with hyposmolar perfusion medium, indicating that the ECF NAA increase associated with hyposmotic stress was a specific response and not passive leakage out of the cells. As NAA is predominantly neuronal, it may contribute to the protection of neurons against swelling (i.e., regulatory volume decrease). In conditions with impaired blood‐brain barrier and cytotoxic oedema, efflux of intracellular NAA subsequent to sustained cellular swelling might lead to a reduction in total brain NAA detectable by magnetic resonance spectroscopy. Alternatively, redistribution of NAA from intra‐to extracellular space implies changes in its chemical environment that may alter its magnetic resonance visibility.

Activated microglia can influence the survival of neural cells through the release of cytotoxic factors. Here, we investigated the interaction between Toll-like receptor 4 (TLR4)-activated microglia and oligodendrocytes or their precursor cells (OPC). Primary rat or N9 microglial cells were activated by exposure to TLR4-specifc lipopolysaccharide (LPS), resulting in mitogen-activated protein kinase activation, increased CD68 and inducible nitric oxide synthase expression, and release of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6). Microglial conditioned medium (MGCM) from LPS-activated microglia attenuated primary OPC proliferation without inducing cell death. The microglial-induced inhibition of OPC proliferation was reversed by stimulating group III metabotropic glutamate receptors in microglia with the agonist L-AP4. In contrast to OPC, LPS-activated MGCM enhanced the survival of mature oligodendrocytes. Further investigation suggested that TNF and IL-6 released from TLR4-activated microglia might contribute to the effect of MGCM on OPC proliferation, insofar as TNF depletion of LPS-activated MGCM reduced the inhibition of OPC proliferation, and direct addition of TNF or IL-6 attenuated or increased proliferation, respectively. OPC themselves were also found to express proteins involved in TLR4 signalling, including TLR4, MyD88, and MAL. Although LPS stimulation of OPC did not induce proinflammatory cytokine release or affect their survival, it did trigger JNK phosphorylation, suggesting that TLR4 signalling in these cells is active. These findings suggest that OPC survival may be influenced not only by factors released from endotoxin-activated microglia but also through a direct response to endotoxins. This may have consequences for myelination under conditions in which microglial activation and cerebral infection are both implicated. , Inc.

Maple syrup urine disease (MSUD) is an inborn error of metabolism caused by a deficiency in branched chain ␣-keto acid dehydrogenase that can result in neurodegenerative sequelae in human infants. In the present study, increased concentrations of MSUD metabolites, in particular ␣-keto isocaproic acid, specifically induced apoptosis in glial and neuronal cells in culture. Apoptosis was associated with a reduction in cell respiration but without impairment of respiratory chain function, without early changes in mitochondrial membrane potential and without cytochrome c release into the cytosol. Significantly, ␣-keto isocaproic acid also triggered neuronal apoptosis in vivo after intracerebral injection into the developing rat brain. These findings suggest that MSUD neurodegeneration may result, at least in part, from an accumulation of branched chain amino acids and their ␣-keto acid derivatives that trigger apoptosis through a cytochrome c-independent pathway.