Lipopolysaccharide-activated SHP-1-deficient motheaten microglia release increased nitric oxide, TNF-alpha, and IL-1beta

Accumulating evidence suggests a deleterious role for activated microglia in facilitating neuronal death by producing neurocytotoxic substances during injury, infection, or neurodegenerative diseases. After cochlear ablation, abnormal microglial activation accompanied by increased neuronal loss within the auditory brainstem occurs in motheaten (me/me) mice deficient in the protein tyrosine phosphatase SHP-1. To determine whether abnormally activated microglia contribute to neuronal death in me/me mice, primary microglial cultures from me/me and wild-type mouse cortices were stimulated by the bacterial endotoxin lipopolysaccharide (LPS) to evaluate the secretion of the neurotoxic mediators nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta). Me/me microglia release significantly greater amounts of all three mediators compared with wild-type microglia. However, the increased release of these compounds in microglia lacking SHP-1 does not appear to occur through activation of extracellular signal-regulated kinase (ERK), p38 kinase subgroups of mitogen-activated protein (MAP) kinases, or increases in NF-kappaB-inducing kinase (NIK). These results suggest that abnormal microglial activation and release of neurotoxic compounds may potentiate neuronal death in deafferented cells and can thus potentiate neurodegeneration in the me/me brainstem. Our data also indicate that SHP-1 is engaged in signaling pathways in LPS-activated microglia, but not through regulation of the ERK and p38 MAP kinases.     

Blocked gap junctional coupling increases glutamate-induced neurotoxicity in neuron-astrocyte co-cultures

Gap junctional communication is likely one means by which neurons can endure glutamate cytotoxicity associated with CNS insults (i.e. ischemia). To examine this neuroprotective role of gap junctions, we employed gap junctional blockers to neuronal and astrocytic co-cultures during exposure to a high concentration of extracellular glutamate. Co-cultures were treated with the blocking agents carbenoxolone (CBX; 25 microM), 18alpha-glycyrrhetinic acid (AGA; 10 microM), vehicle or the inactive blocking analogue glycyrrhizic acid (GZA; 25 microM). Twenty-four hours following the insult, cell mortality was analyzed and quantified by the release of lactate dehydrogenase (LDH) into the media, the cells' inability to exclude propidium iodide, and terminal dUTP nick end labeling (TUNEL). Measurement of LDH release revealed that the glutamate insult was detrimental to the co-cultures when gap junctions were blocked with CBX and AGA. Based on propidium iodide and TUNEL labeling, the glutamate insult caused significant cell death compared to sham vehicle and mortality was amplified in the presence of CBX and AGA. Since blockers were not themselves toxic and did not affect astrocytic uptake of glutamate, it is likely that blocked gap junctions lead to the increased glutamate cytotoxicity. These findings support the hypothesis that gap junctions play a neuroprotective role against glutamate cytotoxicity.     

Effects of aging on neuroprotective and neurotoxic properties of microglia in neurodegenerative diseases

The inflammatory process in the brain, which is accompanied by changes in the levels of proinflammatory cytokines and neurotrophins, along with the presence of activated microglia, has recently gained much attention in neurodegenerative diseases. Activated microglia produce either neuroprotective or neurotoxic factors. Many reports indicate that activated microglia promote degeneration of dopamine (DA) neurons in Parkinson's disease (PD). On the other hand, there are several lines of evidence that microglia also have a neuroprotective function. Microglia activated with lipopolysaccharide in the nigrostriatum of neonatal mice protect DA neurons against the PD-producing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), whereas activated microglia in aged mice promote DA cell death by MPTP. These results suggest that the function of activated microglia may change in vivo from neuroprotective to neurotoxic during aging as neurodegeneration progresses in the PD brain.     

Scutellarin as a Potential Therapeutic Agent for Microglia-Mediated Neuroinflammation in Cerebral Ischemia

The cerebral ischemia is one of the most common diseases in the central nervous system that causes progressive disability or even death. In this connection, the inflammatory response mediated by the activated microglia is believed to play a central role in this pathogenesis. In the event of brain injury, activated microglia can clear the cellular debris and invading pathogens, release neurotrophic factors, etc., but in chronic activation microglia may cause neuronal death through the release of excessive inflammatory mediators. Therefore, suppression of microglial over-reaction and microglia-mediated neuroinflammation is deemed to be a therapeutic strategy of choice for cerebral ischemic damage. In the search for potential herbal extracts that are endowed with the property in suppressing the microglial activation and amelioration of neuroinflammation, attention has recently been drawn to scutellarin, a Chinese herbal extract. Here, we review the roles of activated microglia and the effects of scutellarin on activated microglia in pathological conditions especially in ischemic stroke. We have further extended the investigation with special reference to the effects of scutellarin on Notch signaling, one of the several signaling pathways known to be involved in microglial activation. Furthermore, in light of our recent experimental evidence that activated microglia can regulate astrogliosis, an interglial “cross-talk” that was amplified by scutellarin, it is suggested that in designing of a more effective therapeutic strategy for clinical management of cerebral ischemia both glial types should be considered collectively.     

高渗刺激引起培养的大鼠下丘脑星形胶质细胞和C6细胞合成并释放谷氨酸

目的 观察高渗刺激对培养的大鼠下丘脑星形胶质细胞或C6细胞合成、释放谷氨酸的影响.方法 获取一日龄SD大鼠下丘脑组织,进行星形胶质细胞培养、纯化和鉴定.培养细胞随机分五组:(1)等渗组:用新鲜等渗培养液置换原先的培养液,将细胞分成5个亚组,分别孵育1、3、5、10和15 min.(2)高渗组:用320 mosMNaCl高渗溶液置换原先的培养液,将细胞分成5个亚组,分别孵育1、3、5、10和15 min.(3)甘伯酸(carbenoxolone,CBX,一种缝隙连接阻断剂) 等渗组和(4)CBX 高渗组:用含有CBX(终浓度为100 mmol/L)的等渗培养液作用1 h,后分别用等渗或高渗培养液置换出原培养液,将细胞分成5个亚组,分别孵育1、3、5、10和15 min.(5)Ca2 高渗组:用含有Ca2 (1 000μmol/L)的等渗培养液作用1 h,后用高渗培养液置换出原培养液,将细胞分成5个亚组;分别孵育1、3、5、10和15 min.每个亚组5个培养皿.收集各组的细胞,进行抗谷氨酸和抗胶质原纤维酸性蛋白(Glial fibrillary acidic protein,GFAP)的双重免疫荧光染色,Confocal显微镜观察.用反相高效液相色谱荧光测定法测定细胞培养液中谷氨酸的含量.培养的C6细胞分为四组:等渗组,高渗组,CBX 等渗组和CBX 高渗组,用于流式细胞仪(flow cytometry,FCM)定量检测细胞内谷氨酸的含量.结果 星形胶质细胞内抗GFAP染色的荧光强度在五组间无明显差异.星彤胶质细胞内抗谷氨酸染色的荧光强度在等渗组中各时间点无明显变化,高渗组中在刺激1 min后表达增加,5 min达到高峰,15 min恢复到正常.CBX 高渗组的抗谷氨酸染色荧光强度明显高于CBX 等渗组和等渗组,一直维持到15 min不下降.培养基中的谷氨酸浓度在等渗组各时间点无明显变化,高渗组中谷氨酸浓度从5 min到15 min明显增加.Ca2 高渗组和CBX 高渗组中,培养基中谷氨酸浓度明显低于高渗组,各时间点之间没有明显变化.FCM测定的结果表明高渗或CBX 高渗刺激引起C6细胞内谷氨酸的水平明显上调.结论 高渗刺激可以激发培养的大鼠下丘脑星形胶质细胞合成、释放谷氨酸,CBX不能抑制其合成,但可阻断其释放.     

The ribotoxin deoxynivalenol affects the viability and functions of glial cells

Glial cells are responsible for maintaining brain homeostasis. Modification of the viability and functions of glial cells, including astrocytes and microglia, are associated with neuronal death and neurological diseases. Many toxins (heavy metals, pesticides, bacterial or viral toxins) are known to impact on brain cell viability and functions. Although recent publications suggest a potential link between environmental exposure of humans to mycotoxins and neurological diseases, data regarding the effects of fungal toxins on brain cells are scarce. In the present study, we looked at the impact of deoxynivalenol (DON), a fungal ribotoxin, on glial cells from animal and human origin. We found that DON decreased the viability of glial cells with a higher toxicity against microglial cells compared with astrocytes. In addition to cellular toxicity, DON affected key functions of glial cells. Thus, DON caused a biphasic effect on the neuroinflammatory response of microglia to lipopolysaccharide (LPS), while sublethal doses of DON increased the LPS-induced secretion of TNF-α and nitric oxide, toxic doses inhibited it. In addition to affecting microglial functions, sublethal doses of DON also suppressed the uptake of L-glutamate by astrocytes. This inhibition was associated with a modification of the expression of the glutamate transporters at the plasma membrane. Our results suggest that environmental ribotoxins such as DON could, at low doses, cause modifications of brain homeostasis and possibly participate in the etiology of neurological diseases in which alterations of the glia are involved.     

Attenuation of proliferation in oligodendrocyte precursor cells by activated microglia

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. © 2010 Wiley‐Liss, Inc.     

Activated microglia initiate motor neuron injury by a nitric oxide and glutamate-mediated mechanism

Recent studies suggest that motor neuron (MN) death may be non-cell autonomous, with cell injury mediated by interactions involving non-neuronal cells, such as microglia and astrocytes. To help define these interactions, we used primary MN cultures to investigate the effects of microglia activated by lipopolysaccharide or IgG immune complexes from patients with amyotrophic lateral sclerosis. Following activation, microglia induced MN injury, which was prevented by a microglial iNOS inhibitor as well as by catalase or glutathione. Glutamate was also required since inhibition of the MN AMPA/kainate receptor by CNQX prevented the toxic effects of activated microglia. Peroxynitrite and glutamate were synergistic in producing MN injury. Their toxic effects were also blocked by CNQX and prevented by calcium removal from the media. The addition of astrocytes to cocultures of MN and activated microglia prevented MN injury by removing glutamate from the media. The protective effects could be reversed by inhibiting astrocytic glutamate transport with dihydrokainic acid or pretreating astrocytes with H2O2. Astrocytic glutamate uptake was also decreased by activated microglia or by added peroxynitrite. These data suggest that free radicals released from activated microglia may initiate MN injury by increasing the susceptibility of the MN AMPA/kainate receptor to the toxic effects of glutamate.     

Molecular effects of activated BV-2 microglia by mitochondrial toxin 1-methyl-4-phenylpyridinium

Microglia plays an important role in inflammation-mediated neurodegeneration. Compelling evidence supports the hypothesis that microglial activation contributes to the pathogenesis of various neurodegenerative diseases. However, little is known about the molecular outcome of activated microglia. In this report, we investigate the molecular consequences of MPP(+) toxin-induced activated BV-2 microglia. Intoxication of specific mitochondrial toxin methyl-4-phenylpyridinium iodide ion (MPP(+)) to BV-2 cells induced significant mitochondrial dysfunction and increased the reactive oxygen species generation, caspase-3 activation, and poly ADP ribose polymerase proteolysis. Further, MAC-1 immunostaining in the midbrain of mice revealed a decrease in activated microglia at day 4 after intoxication with MPP(+). From this study, it was confirmed that BV-2 microglia respond to the mitochondrial toxin MPP(+) which may lead to apoptotic cell death. Understanding of the mechanistic basis of apoptotic elimination of activated microglia may help to develop new strategies for the treatment of neurodegenerative diseases.     

Myeloperoxidase: Bridging the gap in neurodegeneration

Neurodegenerative conditions present a group of complex disease pathologies mostly due to unknown aetiology resulting in neuronal death and permanent neurological disability. Any undesirable stress to the brain, disrupts homeostatic balance, through a remarkable convergence of pathophysiological changes and immune dysregulation. The crosstalk between inflammatory and oxidative mechanisms results in the release of neurotoxic mediators apparently spearheaded by myeloperoxidase derived from activated microglia, astrocytes, neurons as well as peripheral inflammatory cells. These isolated entities combinedly have the potential to flare up and contribute significantly to neuropathology and disease progression. Recent, clinicopathological evidence support the association of myeloperoxidase and its cytotoxic product, hypochlorous acid in a plethora of neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, Amyotrophic lateral sclerosis, Multiple sclerosis, Stroke, Epilepsy etc. But the biochemical and mechanistic insights into myeloperoxidase mediated neuroinflammation and neuronal death is still an uncharted territory. The current review outlines the emerging recognition of myeloperoxidase in neurodegeneration, which may offer novel therapeutic and diagnostic targets for neurodegenerative disorders.     

Neurodegeneration in Excitotoxicity, Global Cerebral Ischemia, and Target Deprivation: A Perspective on the Contributions of Apoptosis and Necrosis

In the human brain and spinal cord, neurons degenerate after acute insults (e.g., stroke, cardiac arrest, trauma) and during progressive, adult-onset diseases [e.g., amyotrophic lateral sclerosis, Alzheimer’s disease]. Glutamate receptor-mediated excitotoxicity has been implicated in all of these neurological conditions. Nevertheless, effective approaches to prevent or limit neuronal damage in these disorders remain elusive, primarily because of an incomplete understanding of the mechanisms of neuronal death in in vivo settings. Therefore, animal models of neurodegeneration are crucial for improving our understanding of the mechanisms of neuronal death. In this review, we evaluate experimental data on the general characteristics of cell death and, in particular, neuronal death in the central nervous system (CNS) following injury. We focus on the ongoing controversy of the contributions of apoptosis and necrosis in neurodegeneration and summarize new data from this laboratory on the classification of neuronal death using a variety of animal models of neurodegeneration in the immature or adult brain following excitotoxic injury, global cerebral ischemia, and axotomy/target deprivation. In these different models of brain injury, we determined whether the process of neuronal death has uniformly similar morphological characteristics or whether the features of neurodegeneration induced by different insults are distinct. We classified neurodegeneration in each of these models with respect to whether it resembles apoptosis, necrosis, or an intermediate form of cell death falling along an apoptosis-necrosis continuum. We found that N-methyl-d-aspartate (NMDA) receptor- and non-NMDA receptor-mediated excitotoxic injury results in neurodegeneration along an apoptosis-necrosis continuum, in which neuronal death (appearing as apoptotic, necrotic, or intermediate between the two extremes) is influenced by the degree of brain maturity and the subtype of glutamate receptor that is stimulated. Global cerebral ischemia produces neuronal death that has commonalities with excitotoxicity and target deprivation. Degeneration of selectively vulnerable populations of neurons after ischemia is morphologically nonapoptotic and is indistinguishable from NMDA receptor-mediated excitotoxic death of mature neurons. However, prominent apoptotic cell death occurs following global ischemia in neuronal groups that are interconnected with selectively vulnerable populations of neurons and also in nonneuronal cells. This apoptotic neuronal death is similar to some forms of retrograde neuronal apoptosis that occur following target deprivation. We conclude that cell death in the CNS following injury can coexist as apoptosis, necrosis, and hybrid forms along an apoptosis–necrosis continuum. These different forms of cell death have varying contributions to the neuropathology resulting from excitotoxicity, cerebral ischemia, and target deprivation/axotomy. Degeneration of different populations of cells (neurons and nonneuronal cells) may be mediated by distinct or common causal mechanisms that can temporally overlap and perhaps differ mechanistically in the rate of progression of cell death.     

Protective effects of carbenoxolone are associated with attenuation of oxidative stress in ischemic brain injury

Accumulating evidence has suggested that the gap junction plays an important role in the determination of cerebral ischemia, but the underlying mechanisms remain to be elucidated. In this study, we assessed the effect of a gap-junction blocker, carbenoxolone (CBX), on ischemia/reperfusion-induced brain injury and the possible mechanisms. By using the transient cerebral ischemia model induced by occlusion of the middle cerebral artery for 30 min followed by reperfusion for 24 h, we found that pre-administration of CBX (25 mg/kg, intracerebroventricular injection, 30 min before cerebral ischemic surgery) diminished the infarction size in rats. And this was associated with a decrease of reactive oxygen species generation and inhibition of the activation of astrocytes and microglia. In PC12 cells, H₂O₂ treatment induced more coupling and apoptosis, while CBX partly inhibited the opening of gap junctions and improved the cell viability. These results suggest that cerebral ischemia enhances the opening of gap junctions. Blocking the gap junction with CBX may attenuate the brain injury after cerebral ischemia/reperfusion by partially contributing to amelioration of the oxidative stress and apoptosis.     

CCL11 enhances excitotoxic neuronal death by producing reactive oxygen species in microglia

The chemokine CCL11 (also known as eotaxin‐1) is a potent eosinophil chemoattractant that mediates allergic diseases such as asthma, atopic dermatitis, and inflammatory bowel diseases. Previous studies demonstrated that concentrations of CCL11 are elevated in the sera and cerebrospinal fluids (CSF) of patients with neuroinflammatory disorders, including multiple sclerosis. Moreover, the levels of CCL11 in plasma and CSF increase with age, and CCL11 suppresses adult neurogenesis in the central nervous system (CNS), resulting in memory impairment. However, the precise source and function of CCL11 in the CNS are not fully understood. In this study, we found that activated astrocytes release CCL11, whereas microglia predominantly express the CCL11 receptor. CCL11 significantly promoted the migration of microglia, and induced microglial production of reactive oxygen species by upregulating nicotinamide adenine dinucleotide phosphate‐oxidase 1 (NOX1), thereby promoting excitotoxic neuronal death. These effects were reversed by inhibition of NOX1. Our findings suggest that CCL11 released from activated astrocytes triggers oxidative stress via microglial NOX1 activation and potentiates glutamate‐mediated neurotoxicity, which may be involved in the pathogenesis of various neurological disorders. GLIA 2015;63:2274–2284     

Neuroinflammation, Oxidative Stress and the Pathogenesis of Parkinson's Disease

Neuroinflammatory processes play a significant role in the pathogenesis of Parkinson’s disease (PD). Epidemiologic, animal, human, and therapeutic studies all support the presence of a neuroinflammatory cascade in disease. This is highlighted by the neurotoxic potential of microglia. In steady-state, microglia serve to protect the nervous system by acting as debris scavengers, killers of microbial pathogens, and regulators of innate and adaptive immune responses. In neurodegenerative diseases, activated microglia affect neuronal injury and death through production of glutamate, pro-inflammatory factors, reactive oxygen species, quinolinic acid among others and by mobilization of adaptive immune responses and cell chemotaxis leading to transendothelial migration of immunocytes across the blood–brain barrier and perpetuation of neural damage. As disease progresses, inflammatory secretions engage neighboring glial cells, including astrocytes and endothelial cells, resulting in a vicious cycle of autocrine and paracrine amplification of inflammation perpetuating tissue injury. Such pathogenic processes contribute to neurodegeneration in PD. Research from others and our own laboratories seek to harness such inflammatory processes with the singular goal of developing therapeutic interventions that positively affect the tempo and progression of human disease.     

Nitric oxide from inflammatory-activated glia synergizes with hypoxia to induce neuronal death

Inflammatory-activated glia are seen in numerous central nervous system (CNS) pathologies and can kill nearby neurons through the release of cytotoxic mediators. Glia, when activated, can express the inducible isoform of nitric oxide synthase (iNOS) producing high levels of nitric oxide (NO), which can kill neurons in certain conditions. We show, however, that inflammatory activation of glia in a mature culture of cerebellar granule neurons and glia causes little or no neuronal death under normal (21%) oxygen conditions. Similarly, hypoxia (2% oxygen) or low levels of an NO donor (100 microM DETA/NO) caused little or no neuronal death in nonactivated cultures. If inflammatory activation of glia or addition of NO donor was combined with hypoxia, however, extensive neuronal death occurred. Death in both cases was prevented by the N-methyl-D-aspartate (NMDA) receptor blocker MK-801, implying that death was mediated by the glutamate receptor. Low levels of NO were found to increase the apparent K(M) of cellular oxygen consumption for oxygen, probably due to NO-induced inhibition of mitochondrial respiration, in competition with oxygen, at cytochrome oxidase. Necrotic death, induced by hypoxia plus DETA/NO, was increased further by deoxyglucose, an inhibitor of glycolysis, suggesting that necrosis was mediated by energy depletion. Hypoxia was found to be a potent stimulator of microglia proliferation, but this proliferation was not significant in inflammatory-activated cultures. These results suggest that low levels of NO can induce neuronal death under hypoxic conditions, mediated by glutamate after NO inhibition of respiration in competition with oxygen. Brain inflammation can thus sensitize to hypoxia-induced death, which may be important in pathologies such as stroke, neurodegeneration, and brain aging.     

Unconjugated bilirubin activates and damages microglia

Microglia are the resident immune cells of the brain and are the principal source of cytokines produced during central nervous system inflammation. We have previously shown that increased levels of unconjugated bilirubin (UCB), which can be detrimental to the central nervous system during neonatal life, induce the secretion of inflammatory cytokines and glutamate by astrocytes. Nevertheless, the effect of UCB on microglia has never been investigated. Hence, the main goal of the present study was to evaluate whether UCB leads to microglial activation and to the release of the cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6. Additionally, we investigated the effects of UCB on glutamate efflux and cell death. The results showed that UCB induces morphological changes characteristic of activated microglia and the release of high levels of TNF-alpha, IL-1beta, and IL-6 in a concentration-dependent manner. In addition, UCB triggered extracellular accumulation of glutamate and an increased cell death by apoptosis and necrosis. These results demonstrate, for the first time, that UCB is toxic to microglial cells and point to microglia as an important target of UCB in the central nervous system. Moreover, they suggest that UCB-induced cytokine production, by mediating cell injury, can further contribute to exacerbate neurototoxicity. Interestingly, microglia cells are much more responsive to UCB than astrocytes. Collectively, these data indicate that microglia may play an important role in the pathogenesis of encephalopathy during severe hyperbilirubinemia.     

The Gap-junction inhibitor Carbenoxolone suppresses the differentiation of Th17 cells through inhibition of IL-23 expression in antigen presenting cells

Carbenoxolone (CBX) is a widely used gap-junction inhibitor. We have previously shown that treatment with CBX significantly delayed the onset of experimental autoimmune encephalomyelitis (EAE). However, the mechanism by which CBX delays the onset of EAE remains to be elucidated. Here, we show that CBX specifically inhibits the production of IL-23 by dendritic cells (DCs) and microglia in vitro. CBX treatment significantly reduced the population of Th17 cells in EAE mice. Furthermore, CBX downregulated the expression of IL-23 p19 via increased production of protein phosphatase 2A (PP2A). Thus, CBX may be an effective therapeutic strategy against Th17-mediated autoimmune diseases, such as multiple sclerosis.     

Novel mGluR5 Positive Allosteric Modulator Improves Functional Recovery,Attenuates Neurodegeneration,and Alters Microglial Polarization after Experimental Traumatic Brain Injury

Traumatic brain injury (TBI) causes microglial activation and related neurotoxicity that contributes to chronic neurodegeneration and loss of neurological function. Selective activation of metabotropic glutamate receptor 5 (mGluR5) by the orthosteric agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), is neuroprotective in experimental models of TBI, and has potent anti-inflammatory effects in vitro. However, the therapeutic potential of CHPG is limited due to its relatively weak potency and brain permeability. Highly potent, selective and brain penetrant mGluR5 positive allosteric modulators (PAMs) have been developed and show promise as therapeutic agents. We evaluated the therapeutic potential of a novel mGluR5 PAM, VU0360172, after controlled cortical impact (CCI) in mice. Vehicle, VU0360172, or VU0360172 plus mGluR5 antagonist (MTEP), were administered systemically to CCI mice at 3 h post-injury; lesion volume, hippocampal neurodegeneration, microglial activation, and functional recovery were assessed through 28 days post-injury. Anti-inflammatory effects of VU0360172 were also examined in vitro using BV2 and primary microglia. VU0360172 treatment significantly reduced the lesion, attenuated hippocampal neurodegeneration, and improved motor function recovery after CCI. Effects were mediated by mGluR5 as co-administration of MTEP blocked the protective effects of VU0360172. VU0360172 significantly reduced CD68 and NOX2 expression in activated microglia in the cortex at 28 days post-injury, and also suppressed pro-inflammatory signaling pathways in BV2 and primary microglia. In addition, VU0360172 treatment shifted the balance between M1/M2 microglial activation states towards an M2 pro-repair phenotype. This study demonstrates that VU0360172 confers neuroprotection after experimental TBI, and suggests that mGluR5 PAMs may be promising therapeutic agents for head injury.

Electronic supplementary material

The online version of this article (doi:10.1007/s13311-014-0298-6) contains supplementary material, which is available to authorized users.     

Novel mGluR5 Positive Allosteric Modulator Improves Functional Recovery,Attenuates Neurodegeneration,and Alters Microglial Polarization after Experimental Traumatic Brain Injury

Traumatic brain injury (TBI) causes microglial activation and related neurotoxicity that contributes to chronic neurodegeneration and loss of neurological function. Selective activation of metabotropic glutamate receptor 5 (mGluR5) by the orthosteric agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), is neuroprotective in experimental models of TBI, and has potent anti-inflammatory effects in vitro. However, the therapeutic potential of CHPG is limited due to its relatively weak potency and brain permeability. Highly potent, selective and brain penetrant mGluR5 positive allosteric modulators (PAMs) have been developed and show promise as therapeutic agents. We evaluated the therapeutic potential of a novel mGluR5 PAM, VU0360172, after controlled cortical impact (CCI) in mice. Vehicle, VU0360172, or VU0360172 plus mGluR5 antagonist (MTEP), were administered systemically to CCI mice at 3 h post-injury; lesion volume, hippocampal neurodegeneration, microglial activation, and functional recovery were assessed through 28 days post-injury. Anti-inflammatory effects of VU0360172 were also examined in vitro using BV2 and primary microglia. VU0360172 treatment significantly reduced the lesion, attenuated hippocampal neurodegeneration, and improved motor function recovery after CCI. Effects were mediated by mGluR5 as co-administration of MTEP blocked the protective effects of VU0360172. VU0360172 significantly reduced CD68 and NOX2 expression in activated microglia in the cortex at 28 days post-injury, and also suppressed pro-inflammatory signaling pathways in BV2 and primary microglia. In addition, VU0360172 treatment shifted the balance between M1/M2 microglial activation states towards an M2 pro-repair phenotype. This study demonstrates that VU0360172 confers neuroprotection after experimental TBI, and suggests that mGluR5 PAMs may be promising therapeutic agents for head injury.