Activation of microglial NmethylDaspartate receptors

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ORIGINAL ARTICLE

Activation of Microglial N-Methyl-DAspartate Receptors Triggers Inflammation and Neuronal Cell Death in the Developing and Mature Brain Angela M. Kaindl, MD, PhD,1,2,3,4,5 Vincent Degos, MD, PhD,1,2,3 St ephane Peineau, PhD,1,2,3,6 Elodie Gouadon, PhD,7,8,9 Vibol Chhor, MD,1,2,3 Gauthier Loron, MD,1,2,3 Tifenn Le Charpentier,1,2,3 Julien Josserand,1,2 Carine Ali, PhD,10,11 Denis Vivien, PhD,10,11 Graham L. Collingridge, PhD,6,12 Alain Lombet, PhD,5,6,7 Lina Issa,4,5 Fr ed erique Rene,13 Jean-Philippe Loeffler,13 Annemieke Kavelaars, PhD,14 Catherine Verney, PhD,1,2,3 Jean Mantz, MD, PhD,1,2,3,15 and Pierre Gressens, MD, PhD1,2,3,16 Objective: Activated microglia play a central role in the inflammatory and excitotoxic component of various acute and chronic neurological disorders. However, the mechanisms leading to their activation in the latter context are poorly understood, particularly the involvement of N-methyl-D-aspartate receptors (NMDARs), which are critical for excitotoxicity in neurons. We hypothesized that microglia express functional NMDARs and that their activation would trigger neuronal cell death in the brain by modulating inflammation. Methods and Results: We demonstrate that microglia express NMDARs in the murine and human central nervous system and that these receptors are functional in vitro. We show that NMDAR stimulation triggers microglia activation in vitro and secretion of factors that induce cell death of cortical neurons. These damaged neurons are further shown to activate microglial NMDARs and trigger a release of neurotoxic factors from microglia in vitro, indicating that microglia can signal back to neurons and possibly induce, aggravate, and/or maintain neurologic disease. Neuronal cell death was significantly reduced through pharmacological inhibition or genetically induced loss of function of the microglial NMDARs. We generated Nr1 LoxPþ/þ LysM Creþ/ mice lacking the NMDAR subunit NR1 in cells of the myeloid lineage. In this model, we further demonstrate that a loss of function of the essential NMDAR subunit NR1 protects from excitotoxic neuronal cell death in vivo and from traumatic brain injury. Interpretation: Our findings link inflammation and excitotoxicity in a potential vicious circle and indicate that an activation of the microglial NMDARs plays a pivotal role in neuronal cell death in the perinatal and adult brain. ANN NEUROL 2012;72:536–549

View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana.23626 Received Mar 18, 2011, and in revised form Mar 26, 2012. Accepted for publication Apr 6, 2012. Address correspondence to Dr Kaindl, Pediatric Neurology and Institute of Cell Biology and Neurobiology, Charit e–Universit€ atsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail: [email protected] or Dr Gressens, Inserm, U676, Hoˆpital Robert Debr e, 75019 Paris, France. E-mail: [email protected] e Hospital, Paris, France; 2Denis Diderot Faculty of Medicine, From the 1French Institute of Health and Medical Research U676, Robert Debr e–Universit€ atsmedizin Berlin, Berlin, Germany; University of Paris 7, Paris, France; 3PremUP, Paris, France; 4Department of Pediatric Neurology, Charit 5 Institute of Neurobiology and Cell Biology, Universit€ atsmedizin Berlin, Berlin, Germany; 6Medical Research Council Centre for Synaptic Plasticity, 7 Department of Anatomy, School of Medical Sciences, Bristol, United Kingdom; National Center for Scientific Research, UMR 8162, Le Plessis-Robinson, France; 8Marie Lannelongue Hospital, Le Plessis-Robinson, France; 9University of Paris-South, Le Plessis-Robinson, France; 10French Institute of Health and Medical Research U919, Caen, France; 11National Center for Scientific Research, Mixed Unit of Research 6232, Caen, France; 12Department of Brain and Cognitive Sciences, Seoul National University, Seoul, South Korea; 13French Institute of Health and Medical Research U692, Strasbourg, France; 14 University Medical Center Utrecht, Utrecht, the Netherlands; 15Anesthesiology and Intensive Care Service, Clichy, Beaujon Hospital, Public Assistance Hospitals of Paris, Paris, France; and 16Centre for the Developing Brain, Institute of Reproductive and Developmental Biology, Imperial College, Hammersmith Campus, London, United Kingdom. Additional supporting information can be found in the online version of this article.

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icroglia, the immune competent cells of the central nervous system (CNS), play a pivotal role in restoration of CNS integrity and in the pathogenesis of a large variety of acute and chronic neurological disorders affecting the infant and adult CNS.1,2 Amoeboid microglia originate from circulating monocytes that invade the brain during embryonic and early postnatal life.3–5 They ultimately transform into surveying microglia.6 The latter constantly screen the CNS and can be activated rapidly through various environmental changes.6 In the activation process, microglia change their phenotype to become amoeboid, proliferate, migrate to the site of damage, and secrete proinflammatory factors.1,7 There is, moreover, accumulating evidence that microglia have other physiologic roles beyond their defense-oriented action in the developing and the mature CNS.1,3,6,8 Recent findings indeed indicate neuron–microglia crosstalk and thereby raise the intriguing hypothesis that microglia can sense neuronal activity based on local neurotransmitter levels.8 Such intercellular interaction may also participate in the evolution of neurodegenerative disorders through glutamate excitotoxicity.8 Although excessive activation of the N-methyl-D-aspartate (NMDA) receptors (NMDARs; synonym GRIN or GLUN) is in general a key mechanism in excitotoxicity, the occurrence of these glutamate receptors in microglia has only been suggested recently.9–17 The mechanisms through which neurotoxic microglial activation is initiated remain poorly understood in the context of excitotoxicity.

LoxPþ/þ mice with LysM Creþ/þ mice to obtain Nr1 LoxPþ/þ LysM Creþ/ mice. Nr1 LoxPþ/þ LysM Cre/ were used as controls. Also, 2 human fetal and 1 adult control brain specimen with no detectable neurological abnormalities, as reported previously,3,18 were employed. As pathological cases, we used sections from 2 fetal brains with periventricular white matter injury, from the substantia nigra of a case Parkinson disease and from the spinal cord of a case with amyotrophic lateral sclerosis (ALS).

Primary Microglial and Neuron Cultures Primary glial cell cultures19,20 were prepared from the cortices of newborn (P0) and cultured neurons from the cerebral cortex of E14.5 Swiss mice,21 as described in detail in the Supplementary Materials and Methods.

Immunohistology, Immunocytochemistry, and Confocal Microscopy Brain sections or cells were incubated in primary antibodies (anti-NR1, anti-NR2B, anti-NR2D, tomato lectin, anti-CD68, anti-Iba1, anti–glial fibrillary acidic protein [GFAP], anti–neuronal nuclei [NeuN]) and the corresponding secondary antibodies as specified in Supplementary Table 1 and the Supplementary Materials and Methods.

Calcium Imaging Microglia were incubated in 2lm FURA 2-AM (Molecular Probes, Interchim, Montluc¸on, France) and intracellular Ca2þ changes in response to treatment with NMDA 300lm (Sigma, St Louis, MO) were monitored, and images were processed as described in the Supplementary Materials and Methods.22,23

Here, we demonstrate the presence of NMDARs on microglia in the immature and mature murine and human CNS. We further report that NMDAR activation of microglia causes an inflammatory response and triggers neocortical neuronal cell death. This neocortical damage was significantly reduced through pharmacological inhibition or a genetically induced loss of function of the NMDARs in microglia.

Whole-cell recordings were obtained at room temperature from the soma of visually identified microglia using patch electrodes (5–7MX) following bath application of NMDA 300lm and D-serine 20lm. Access resistance and membrane resistance were recorded online at 0.033Hz. Cells were continuously recorded at a holding potential of 70mV. Data were stored using the LTP Program.24,25

Materials and Methods

Quantification of Cell Survival

Detailed methods, especially regarding the generation and assessment of conditional knockout (KO) mice, human tissues, tissue processing, cell culture protocols, immunofluorescence staining, RNA and protein extraction, quantitative real time polymerase chain reaction (PCR), Western blotting, electrophysiological analyses, and in vitro and in vivo models are described in the Supplementary Materials and Methods.

Cell survival was quantified using the colorimetric CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI) according to the instructions of the manufacturer. Cell death was also assessed by evaluation of DAPIstained pyknotic nuclei following the treatment described above.26

Electrophysiological Recording

Animals and Human Tissues

Measurement of Reactive Oxygen Species Formation

Brains from 5-day-old (P5) and adult male Swiss mice and conditional KO mice and human brains were cryostat sectioned at a thickness of 10lm. Conditional microglial NR1 KO mice (Nr1 LoxPþ/þ LysM Creþ/) were generated by breeding Nr1

Reactive oxygen species (ROS) formation was measured using the nonfluorescent 20 ,70 -dichlorodihydrofluorescein diacetate (H2DCFDA; Invitrogen, Carlsbad, CA) as described in the Supplementary Materials and Methods.

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Luminex (Multiplex Immunoassay) The medium of NMDA- and/or MK801-treated microglia was harvested 12 hours after treatment initiation and further processed for Luminex multiple cytokine and chemokine analysis (mouse interleukin [IL]1a, IL1b, IL2-6, IL9, IL10, IL12 [p40], IL12 [p70], IL13, IL17, eotaxin, granulocyte colony-stimulating factor, granulocyte macrophage colony-stimulating factor [GMCSF], interferon [IFN]c, tumor necrosis factor [TNF]a, KC, monocyte chemotactic protein 1 [MCP1], macrophage inflammatory protein 1 [MIP1]a, MIP1b, and RANTES).27

Neuron Survival after Addition of Microglia Conditioned Medium Medium from Nr1 LoxPþ/þ LysM Creþ/ mice (KO) and wildtype (WT) mouse microglia following treatment with phosphatebuffered saline (PBS) 1; NMDA 300lm; NMDA 300lm and MK801 10lm; NMDA 300lm and etanercept 50lg/ml (Wyeth Pharmaceuticals, Madison, NJ); or lipopolysaccharide (LPS) 1lg/ ml was applied to days in vitro (DIV)5 neuronal cultures. Neuronal cell death was assessed by evaluation of DAPI-stained pyknotic nuclei following the treatment stated above.28

In Vitro Stroke Penumbra Model This model has been described previously in detail.29 Glutamate concentration was measured in the neuronal culture medium using the Amplex Red Glutamic Acid Kit (Invitrogen) according to the protocol supplied by the manufacturer.

In Vivo Excitotoxic Brain Lesion Model Excitotoxic brain lesions were induced and histological analysis was performed according to the well-characterized model through intracerebral injection of 10lg glutamate analogue ibotenate (5lg/ll, Sigma) on P10 and P56 mice, respectively, as described previously30–34. See Supplementary Materials and Methods for details.

In Vivo Traumatic Brain Injury Model Mice were subjected to a well-characterized and previously described model of mechanical head trauma on P7, and brains were analyzed histologically 1 and 5 days later35,36. See Supplementary Materials and Methods for details.

Results Cortical Microglia Express NMDARs In Vitro and In Vivo To investigate the presence of the NMDARs in microglia, we first focused on the main NMDAR subunit NR1 (NMDAR1). NR1 mRNA was detected in cultured murine cortical microglia by real time PCR (Fig 1, Supplementary Fig 1, Supplementary Table 1; microglia culture purity 99%). Compared to the mRNA levels detected in cultured murine cortical neurons, gene expression levels in microglia were lower. The results obtained from analysis of the NR1 subunit protein levels by Western blot were consistent with those on the mRNA level, 538

demonstrating the presence of this NMDAR subunit in microglia and in neurons and suggesting that NMDARs are expressed at a lower level in microglia. In Western blots, the NR1 band intensity in microglia was about 42% of that seen in neurons. The presence of the NMDAR subunit NR1 was further assessed by immunocytological staining of cultured murine cortical microglia. We demonstrated in detail the specificity of the immunoreactions with antibodies directed against NMDAR1 by (1) blocking the NR1 antibody by addition of the corresponding peptide, (2) staining with the secondary antibodies only, (3) transfection experiments with a GFPtagged overexpression NR1 plasmid, and (4) immunostainings and Western blots of brains and cultured microglia from NR1 Cre-lox KO mice (Supplementary Methods, Fig 6, Supplementary Figs 2, 3, 7, and Supplementary Table 2). On mouse and human brain and spinal cord sections, ramified, intermediate to amoeboid microglia– macrophage phenotypes were detected with the microglia markers tomato lectin, Iba1, and CD68. In infant murine control brains at P5, most intermediate and amoeboid microglial cells were present in the white matter,3,37 whereas ramified microglia were evenly distributed in the gray matter. In the cortical white matter of these P5 mice, some of the intermediate–amoeboid microglia displayed NR1-immunoreactive dots on their cell bodies and short processes (see Fig 1E, Supplementary Fig 1). In adult murine brains at P56, the microglial phenotype was foremost ramified with intermediate microglia detected in the white matter. In these mice, again some intermediate microglial cells showed NR1 labeling in the cortical white matter. In parallel, sparse NR1-positive dots were also detected on intermediate microglia in the developing human frontal white matter (24–30 postovulatory weeks) with no detectable neuropathological abnormalities (data not shown). In control adult human brain sections, the microglial phenotype was foremost ramified with intermediate microglia detected in the white matter, and sparse NR1 labeling was detected on intermediate microglial cells (data not shown). However, numerous intermediate–amoeboid microglial cells with intense NR1 immunoreactivity were detected within lesions in pathological human brain and spinal cord sections, that is, in the white matter of human fetuses with periventricular leukomalacia, in the substantia nigra of adult brain with Parkinson’s disease and in the spinal cord of a patient with ALS (see Fig 1E, Supplementary Fig 4). In murine and human CNS sections, strong NR1 immunostaining was detected in neurons (data not shown). Control performed at the same time and in the same conditions as the specimen depicted in Figure 1 Volume 72, No. 4

FIGURE 1: Microglia express N-methyl-D-aspartate receptor (NMDAR) subunits in vitro and in vivo. (A) NMDAR subunit NR1, NR2A-D, NR3A mRNA expression levels assessed by real time polymerase chain reaction (n 5 4–5, each run in duplicates; unpaired 2-tailed t test, *p < 0.05, mean 6 standard error of the mean; see Supplementary Table 1 for primer sequences). (B) Purity of primary microglia cultures verified by immunostaining using cell type–specific antibodies against tomato lectin (microglia), glial fibrillary acidic protein (GFAP; astrocytes), and neuronal nuclei (NeuN; neurons), respectively (n 5 5). (C) NR1 protein levels analyzed by Western blot in cultured murine cortical microglia (days in vitro [DIV]15) and neuron (DIV10) samples (n 5 4). (D) Existence of NR1 revealed in DIV15 cultured murine cortical microglia by immunocytological staining (fluorescence microscopy; original magnification, 340). (E) NR1 immunopositivity was similarly detected on intermediate–amoeboid microglia in murine brain sections at postnatal day 5 (P5) and adult age (P56) as well as human postmortem brain sections from fetuses and adults with neurodegenerative diseases (periventricular leukomalacia, Parkinson disease) by immunohistological staining (tomato lectin [lectin], ionized calcium-binding adapter molecule 1 [Iba1], cluster of differentiation 68 [CD68], nuclei labeling with DAPI in blue; see Supplementary Table 2 for marker explanation). On confocal images, dotted membrane NR1 labeling was complementary to the tomato lectin labeling of microglia. Corpus callosum (mouse, P5), fimbria (mouse, P56), and cerebral cortex white matter (human) are shown. Fluorescence microscopy, original magnification, 363 (human, fetus, first row); otherwise confocal microscopy, original magnification, 363. Scale bar 5 10lm for adult mouse brain, otherwise 5lm. For controls see Figure 6C, Supplementary Figures 2, 3, and 7. GOI 5 glycerol-3-phosphate oxidase; HPRT 5 hypoxanthine-guanine phosphoribosyltransferase.

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FIGURE 2: Microglial N-methyl-D-aspartate (NMDA) receptor activation triggers intracellular calcium increase and inward current response. (A) Pooled data of the recordings of fluorescence intensity of microglia during the application of 300lm NMDA (n 5 21 cells; shaded lines indicate point-by-point standard error of the mean [SEM]). Representative fluorescence microscopic images before and after application of 300lm NMDA are displayed. Fluorescence intensity is scaled from black (lowest) to pink (highest), and the scale bar represents 10lm. (B) Peak amplitude average of the fluorescence intensity of microglia following 300lm NMDA application. Cells presenting >5% fluorescence intensity increase from baseline following 300lm NMDA application are categorized as responding, the others as nonresponding (n of nonresponding microglia 5 164; n of responding microglia 5 21). Pretreatment of microglia with 30lm MK801 inhibited the NMDA-induced calcium response (n 5 123, 1-way analysis of variance, mean 6 SEM, ***p < 0.001). (C) For electrophysiological analyses, individual microglial cells were identified by their phenotype in 99% pure microglia cultures and patched. Application of NMDA 300lm and D-serine 20lm triggered an inward current in the absence of Mg21 in the extracellular solution. (D) Peak amplitude of current recorded in responding microglia following 300lm NMDA and 20lm D-serine application without (n 5 9) and in presence of 10lm MK801 (n 5 3). This current could be strongly reduced through coapplication of 10lm MK801. The calibration bars for the traces depict 5pA and 5 seconds (*p < 0.05, unpaired 2-tailed t test, mean 6 SEM).

verified the specificity of the NR1 immunolabeling (see Supplementary Fig 3). Functional NMDAR requires the presence of NR2/3 subunits. We confirmed the presence of these subunits in cultured microglia by real time PCR (see Fig 1A, Supplementary Table 1). Gene expression levels in microglia were significantly lower for all NR2/3 subunits except for NR2D when compared to neurons. NR2B and NR2D immunoreactivity was detected in cultured murine cortical microglia (Supplementary Fig 5) and in the white matter of murine brains and spinal cords (Supplementary Figs 4 and 6). NMDARs Are Functional in Cortical Microglia In Vitro To substantiate the existence of functional NMDARs in cortical microglia, we investigated whether the activation 540

of Ca2þ-permeable NMDARs triggers a local increase in cytosolic Ca2þ levels in cultured murine cortical microglia. Microglia were loaded with the Ca2þ indicator FURA 2-AM, and intracellular Ca2þ changes were monitored in response to 300lm of NMDA. NMDA triggered a transient increase in the fluorescence signal in microglia (peak amplitude 128.8 6 3.6% of baseline, n ¼ 21), indicating an increase of the intracellular Ca2þ concentration (Fig 2). This NMDA effect could be elicited in about 10% of analyzed microglia (n ¼ 21 in 185 recorded cells). To verify the specificity of the response for NMDARs, the same experiments were performed in the presence of MK801, a specific noncompetitive open channel blocker of NMDARs. Indeed, 10lm MK801 abolished the increase of the intracellular Ca2þ concentration induced by NMDA (n ¼ 123). Volume 72, No. 4

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Thapsigargin (0.5lm), a specific inhibitor of the endoplasmic and sarcoplasmic Ca2þ-adenosine triphosphatase, was applied as a positive control at the end of each experiment and effectively elicited an increase of the intracellular Ca2þ concentration (see Supplementary Materials and Methods). The presence of functional cortical microglial NMDARs was further studied through an electrophysiological investigation of individual microglial cells. Isolated cortical microglia were identified in primary cultures by their phase-bright small round- or rod-shaped cell bodies with no or few thick processes under the phase-contrast microscope. Individual cells were approached with a patch pipette to establish a whole cell recording mode. Because functional NMDAR channels display a voltage-dependent Mg2þ block at potentials below 40mV in neurons, we performed experiments in Mg2þ-free bath solution. Application of 300lm NMDA and 20lm D-serine triggered an inward current of 30 6

8pA in the absence of Mg2þ in the extracellular solution (n ¼ 9, holding potential of 70mV) in 5 to 10% of the recorded cells, which was partly inhibited by 10lm MK801 (n ¼ 3; see Fig 2C, D). These findings emphasize the presence of functional NMDARs in cortical microglia.

NMDAR Stimulation Activates Cortical Microglia and Thereby Contributes to Inflammatory Response In Vitro Activation of cortical microglia can be characterized by a change in their phenotype and oxidative activity and release of diffusible factors. Resting microglia displayed predominantly the phenotype of small round cells with sparse cytoplasm as observed in cultured cortical murine microglia (Fig 3). However, within 2 hours after treatment initiation with 300lm NMDA, several microglia adopted an activated phenotype characterized by a larger cell body with no processes; we observed some variability in this response (data not shown). This effect was still observed 22 hours later. We investigated, in parallel, the effects of LPS (endotoxin) as a positive control, because LPS is among the best-described strong stimuli of microglia activation. Treatment with 1lg/ml LPS elicited a similar phenotype change. FIGURE 3: N-Methyl-D-aspartate (NMDA) receptor stimulation activates cortical microglia in vitro. (A) Microglia phenotype in control condition (phosphate-buffered saline [PBS] 13) and 24 hours upon addition of (B) NMDA 300lm and (C) lipopolysaccharide (LPS) 1lg/ml (positive control; fluorescence microscopy, original magnification, 340; n 5 6 per condition). Immunocytological staining with an antibody directed against tomato lectin (lectin) and DAPI staining for nuclei are shown. (D) Twenty-four hours after NMDA 300lm treatment, the mean of area ratio (membrane/nucleus) is increased compared to control condition (control, 8.7 6 0.8; NMDA, 21.0 6 1.8; LPS, 20.9 6 1.2; 1-way analysis of variance [ANOVA]). (E) Reactive oxygen species accumulation following 24 hours treatment of microglia with PBS 13 (negative control), (F) NMDA 300lm, or (G) LPS 1lg/ml (positive control) evaluated within microglia loaded with the probe 20 ,70 -dichlorodihydrofluorescein diacetate (H2DCFDA) for 45 minutes. The nonfluorescent compound H2DCFDA turns into a fluorophore (20 ,70 -dichlorofluorescein) after reaction with oxidative species. (H) Microglia contained high levels of the fluorescent (oxidized) form of the probe when challenged with NMDA and LPS; fluorescence microscopy, original magnification, 320 (n 5 5–7). (I) inducible form of NO synthase (iNOS) mRNA levels were increased following 12 hours of NMDA 300lm treatment; this effect could be inhibited by a pretreatment with MK801 10lm (n 5 4). (J) Nitrite and nitrate levels were increased in the medium 8 hours after treatment initiation of cultured microglia with NMDA 300lm, MK801 10lm, and LPS 1lg/ml (positive control) when compared to the control situation (PBS 13; n 5 2–4; *p < 0.05 , **p < 0.01, ***p < 0.001; 1-way ANOVA unless otherwise noted, mean 6 SEM).

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Microglia activation through LPS treatment is associated with increased oxidative stress.38 We hypothesized that an activation of microglia via NMDAR similarly promotes an increase of cellular ROS and generation of nitric oxide (NO). We used the fluorogenic ROS probe H2DCFDA to detect the microglial oxidative activity. When challenged with LPS, high levels of the fluorescent (oxidized) form of the probe were detected in microglia (see Fig 3). A strong increase in oxidized probe levels was also elicited by a treatment with 300lm NMDA. In microglia, NO is generated by the inducible form of NO synthase (iNOS), and microglia activation as well as a wide variety of neurological diseases are associated with an induction of microglial iNOS and the generation of NO.39 Both the iNOS mRNA expression level (see Supplementary Table 1) and the NO production increased following NMDA, and these effects could be inhibited by MK801. Activated microglia send signals to other cells through the secretion of diffusible factors such as cytokines and chemokines and thereby regulate the inflammatory response.38 Release of pro- and antiinflammatory cytokines and chemokines by microglia was determined 12 hours after treatment initiation with PBS (control); NMDA 300lm; NMDA 300lm and MK801 10lm; and MK801 10lm, respectively. Quantification of cytokine and chemokine levels in the culture medium of microglia activated by NMDA revealed a significant increase of IL1a, IL1b, IL3, IL4, IL5, IL9, IL10, IL12 (p70), IL13, IL17, eotaxin, GMCSF, MCP1, and TNFa levels (Fig 4). Pretreatment of microglia with MK801 strongly reduced the respective increase of IL1a, IL10, eotaxin, MCP1, and TNFa levels, demonstrating that NMDA receptor stimulation accounts for these observed NMDA-induced effects. For IL1b, IL3, IL4, IL5, IL9, IL12, IL13, IL17, GMCSF, and IFNc, a reduction trend through MK801 was detected after NMDA treatment. Together, these data confirm that microglial NMDAR activation elicits a massive release and accumulation of pro- and anti-inflammatory cytokines and chemokines in vitro. No significant effect of MK801 treatment (without NMDA) compared to the control condition was detected (data not shown). Cortical Microglia Activated via Their NMDARs Are Neurotoxic In Vitro To investigate whether factors secreted into the medium by NMDA-activated microglia cause cell death of cortical neurons, we first assessed the effect of microglia-conditioned medium on cortical neuron survival in vitro. Microglia cell cultures were treated with PBS (control); NMDA 300lm; NMDA 300lm and MK801 10lm; and NMDA 300lm and etanercept 50lg/ml (TNFa in542

hibitor), respectively. We focused on TNFa, as this key inflammatory mediator40 is highly secreted in our model following NMDA treatment (see Fig 4), and potent specific inhibitors are available. To minimize effects of residual drugs and microglial cells in the conditioned medium added to neuron cultures, the microglia medium was exchanged for fresh medium 1 hour after treatment, recovered 11 hours later, and cleared of contaminating microglial cells by filtration. The exposure of neurons to conditioned medium from NMDA-treated microglia increased significantly the rate of neurons undergoing cell death (Fig 5A). This effect could be strongly diminished by pretreatment of microglia with MK801, demonstrating that microglial NMDAR stimulation accounts for at least part of the observed neurotoxic effect of NMDAactivated microglia. The neurotoxic effect of NMDARactivated microglia is in part mediated by TNFa production, because pretreatment with the TNFa inhibitor etanercept 50lg/ml reduced neuronal cell death upon addition of microglia-conditioned medium. Damaged Cortical Neurons Can Activate Microglia via Their NMDARs and Render Them Neurotoxic In Vitro, Thereby Possibly Inducing a Vicious Circle We further examined the role of cortical microglial NMDARs in an in vitro model of ischemic stroke penumbra described in detail previously.29 After stroke, glutamate released by damaged neurons in the infarct core can diffuse to nearby cells, and this can lead to an activation of microglia.41 After focal ischemia, microglia are thought to orchestrate damage in the penumbra.42 Because microglia activation and propagating neuronal death are implicated in the development of the stroke penumbra, this experiment was designed to test whether damaged/dying cortical neurons can activate microglia via NMDARs and whether the neurotoxic effect of activated microglia can be reduced through blockage of microglial NMDARs (see Fig 5). Cortical neuron cultures were subjected to oxygen–glucose deprivation (OGD) or normal culture conditions (control) for 45 minutes on DIV10 and cultured a further 24 hours after treatment initiation. This protocol was chosen because the inflammatory response, representing the expanding penumbra following an ischemic infarct, develops over many hours. Thereafter, MK801-pretreated (4 hours treatment) and control microglia plated in neuron medium on inserts with 1lm diameter pores were exposed to OGD-stressed or control cortical neurons, respectively. To avoid carryover of MK801 used to treat microglia, the microglia-bearing inserts were thoroughly washed before placing them on neurons. Microglia and neurons Volume 72, No. 4

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FIGURE 4: Cytokines and chemokine induction through N-methyl-D-aspartate (NMDA) receptor activation in microglia. Interleukin (IL)1a, IL1b, IL3, IL4, IL5, IL9, IL10, IL12 (p70), IL13, IL17, eotaxin, granulocyte macrophage colony-stimulating factor (GMCSF), interferon (IFN)c, monocyte chemotactic protein 1 (MCP1), and tumor necrosis factor (TNF)a levels 12 hours after treatment initiation with phosphate-buffered saline (control); NMDA 300lm; and NMDA 300lm and MK801 10lm (n 5 3–4; 1way analysis of variance [ANOVA], and Bonferroni multiple comparison test, **p < 0.01; values represent mean 6 standard error of the mean [SEM]). Pretreatment of microglia with MK801 reduced the NMDA-induced increase of IL1a, IL1b, IL3, IL10, IL13, MCP1, and TNFa levels, demonstrating that NMDA receptor stimulation accounts for these observed NMDA-induced effects; for IL4, IL5, IL9, IL12, and IL17 there was a reduction trend through MK801 (n 5 3-4; 1-way ANOVA, Bonferroni multiple comparison test, #p < 0.05; values represent mean 6 SEM). ND 5 not done.

were allowed to chemically communicate with each other for 4 hours. We confirmed that neuronal glutamate release following OGD-treatment was not affected by the pretreatment status of microglia (glutamate mean 1.3lm vs 1.5lm in medium of OGD-stressed neurons following communication with MK801-treated vs nontreated microglia, p < 0.05). Microglia-bearing inserts were then October 2012

thoroughly washed again with neuron medium before being placed on healthy neurons not exposed to OGD. These later neurons were fixed 24 hours later and stained with DAPI, and cell death was assessed. Cell death was observed in about 20% of cortical neurons when they were exposed to untreated microglia pre-exposed to healthy neurons. When untreated microglia were pre543

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FIGURE 5: N-Methyl-D-aspartate (NMDA) treatment renders microglia toxic toward cortical neurons in vitro. (A) Neuronal cell death increased after exposure to 300lm NMDA-conditioned microglia medium; the effect was significantly reduced by microglia pretreatment with MK801 10lm or etanercept 50lg/ml (TNFa inhibitor; n 5 7–10, unpaired 2-tailed t test). (B) In the model illustrated in C, microglia were activated by damaged neurons via their NMDA receptors and subsequently mediated neuronal damage; this effect was significantly reduced by blockage of microglial NMDA receptors with MK801 10lm (n 5 4–5). (C) Experimental paradigm of modified in vitro penumbra model.29 Step 1: 10 days in vitro neurons were subjected to oxygen–glucose deprivation (OGD; 45 minutes) or not (controls) and cultured for a further 24 hours (model of expanding penumbra following an ischemic infract); microglia were treated with MK801 10lm or phosphate-buffered saline (PBS; controls) for 4 hours. Step 2: microglia were grown on inserts with a porous membrane as a bottom and were exposed to OGD-stressed or control neurons for 24 hours, allowing for chemical communication between microglia and neurons. Step 3: microglia-bearing inserts were placed on fresh, healthy neurons; neurons were fixed 24 hours later; cell death was assessed in DAPI-stained cells.

exposed to OGD-stressed cortical neurons, the target neuron cell death was increased to about 50%, and pretreatment of microglia with MK801 significantly reduced the neurotoxic potential of OGD-pretreated microglia. We thereby demonstrate that OGD-stressed cortical neurons activated the neurotoxic functions of microglia via microglial NMDARs and demonstrated neuroprotection through inhibition of these NMDARs. Our results highlight that the NMDARs are a target for microglia activation in a model of ischemic stroke. Loss of Function of the Microglial NMDARs Protects from Gray Matter Damage In Vitro and In Vivo To examine whether a loss of microglial NMDAR function is protective in vitro and in vivo, we analyzed the effect of brain damage models. For this, we generated mice carrying a conditional microglia-targeted KO of 544

their NMDAR1. Such Nr1 LoxPþ/þ LysM Creþ/ mice lack the NMDAR1 subunit NR1 specifically in cells of the myeloid lineage (microglia, macrophages, monocytes; Fig 6A–C, Supplementary Fig 7). In these mice, a strong reduction of NR1 mRNA and protein levels could be detected in microglia but not in astrocytes or neurons of conditional KO mice, and microglia did not stain positively for the NR1 subunit in Nr1 LoxPþ/þ LysM Creþ/ mice. Nr1 LoxPþ/þ LysM Creþ/ mice grew and bred normally, showed no gross clinical neurological deficits (normal brain, body weight, movement) or brain malformations at P5 and P56, and had normal blood counts as well as bone marrow phenotypes at P56 (Supplementary Fig 7C–F). To investigate whether a conditional KO of NR1 in microglia inhibits the release of pro- and anti-inflammatory cytokines and chemokines by microglia described above, we quantified corresponding protein levels in the culture medium of microglia Volume 72, No. 4

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FIGURE 6: Conditional microglia NR1 knockout (KO) mice are protected from N-methyl-D-aspartate receptor (NMDAR)-induced neocortical injury. (A) Conditional microglial NR1 KO mice were generated by breeding Nr1 LoxP1/1 mice (loxP sites in intron between exons 10 and 11 and with a neomycin-resistance gene downstream of the last NMDAR1 gene exon) with LysM Cre1/1 mice that express the Cre recombinase from the endogenous M lysozyme locus common to the myeloid lineage (see Supplementary Materials and Methods, Supplementary Fig 7 for details). Polymerase chain reaction for genotyping was performed with LoxP primers that generated a 280bp (mutant) and 180bp (wild type [WT]) product and with Cre primers that generated a 350bp (WT) and a 700bp and 1.7kb (LysM Cre recombinase) product. (B) NR1 mRNA levels were significantly lower in cultured murine cortical microglia (days in vitro [DIV]15) from KO when compared to WT mice; these levels did not show any difference when astrocytes (DIV15) or neurons (DIV10) were assessed (n 5 3–5, 1-way analysis of variance [ANOVA], ***p < 0.001; mean 6 standard error of the mean [SEM]). (C) NR1 protein levels analyzed by Western blot in cultured murine cortical microglia (DIV15), in the respective frontal cortex before brain dissection (birth to 1 day old), and in cultured cortical neurons (DIV10). (D) Cell death of cortical neurons is significantly reduced when conditioned medium was derived from conditional NR1 KO mouse microglia (n 5 11, unpaired 2-tailed t test). (E) In the model illustrated in Figure 5C, control microglia were activated by damaged neurons via their NMDARs and subsequently mediated neuronal damage; this effect was significantly reduced when microglia from conditional microglia-targeted NMDAR subunit NR1 KO mice were used. One-way ANOVA; values represent mean 6 SEM; **p < 0.01 (n 5 6). (F) In an in vivo excitotoxic brain damage model, a cortical lesion (L) was induced by intracerebral injection of 10lg ibotenate. (G) Cortical gray matter lesion size was assessed 5 days later (n 5 18) for 5-day-old (P5) mice and for P56 adult mice (n 5 9; unpaired 2-tailed t test). Conditional NR1 KO mice (Nr1 LoxP1/1 LysM Cre1/2) lacking to a large extent the NMDAR subunit NR1 in cells of the myeloid lineage (ie, in microglia) showed significantly reduced cortical gray matter lesion size as compared to their WT littermates. One-way ANOVA; values represent mean 6 SEM; ***p < 0.001. (H) In an in vivo traumatic brain injury (TBI) model, the ventricle size as a measure for the extent of the lesion was significantly smaller 1 and 5 days following TBI in the conditional Nr1 LoxP1/1 LysM Cre1/2 KO mice when compared to WT mice. *p < 0.05. (I) At the same time, there were fewer Iba1-positive cells near the lesion site. Two-tailed t test; values represent mean 6 SEM; **p < 0.01. HPRT 5 hypoxanthine–guanine phosphoribosyltransferase; OGD 5 oxygen–glucose deprivation.

12 hours after treatment initiation with PBS (negative control), NMDA 300lm, and LPS 1lg/ml (positive control), respectively. For all but IL9, there was no change in the concentration of these secreted factors when microglia from conditional KO mice were treated with NMDA in comparison to PBS (Supplementary Fig 8). To further determine whether this is also associated with a reduction in overall neurotoxic factors secreted into the medium by NMDAR-activated microglia, we assessed October 2012

the effect of microglia-conditioned medium on cortical neuron survival in vitro. Microglia cell cultures from WT or KO mice were treated with PBS (control) or NMDA 300lm, respectively. The exposure of cortical neurons to conditioned medium from NMDA-treated microglia from WT mice increased significantly the rate of cortical neurons undergoing cell death. This effect was strongly reduced when microglia from KO mice were treated in a similar way, demonstrating that microglial NMDAR 545

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stimulation accounts for at least part of the observed neurotoxic effect of NMDA-activated microglia. In line with this, neuronal cell death was significantly reduced through genetically induced loss of function of the microglial NMDARs in the in vitro stroke penumbra model described above. In a further experiment, we determined the lesion size in conditional KO (Nr1 LoxPþ/þ LysM Creþ/) and WT animals 1 day and 5 days after intracerebral ibotenate injection of infant mice on P5 and adult mice on P56. In this well-characterized model of excitotoxic brain damage, ibotenate activates NMDARs and metabotropic glutamate receptors and induces cortical necrosis and white matter cysts.32,33,43 Following ibotenate injection, infant mice developed cortical gray matter (see Fig 6F,G) and periventricular white matter lesions (see Supplementary Fig 7G). The cortical lesion was typically characterized by dramatic neuronal loss in all neocortical layers and an almost complete disappearance of neuronal cell bodies along the axis of the ibotenate injection.11 Conditional Nr1 LoxPþ/þ LysM Creþ/ mice lacking NMDAR1 specifically in cells of the myeloid lineage (ie, in microglia) showed a significantly reduced gray and white matter lesion size as compared to their WT littermates, at both P6 (data not shown) and P10 (Fig 6F,G; Supplementary Fig 7G), respectively. Similarly, adult mice injected with ibotenate typically showed gray matter damage that was strongly reduced in conditional Nr1 LoxPþ/þ LysM Creþ/ KO mice. To further examine whether a loss of microglial NMDAR function is protective in vivo, we analyzed the effect in a well-characterized traumatic brain injury (TBI) model in conditional Nr1 LoxPþ/þ LysM Creþ/ KO mice and their WT littermates. In this TBI model, excitotoxic lesion are triggered with a rapid evolution at the impact site, and only approximately 6 hours postTBI a delayed neurodegenerative response becomes apparent at the impact site but also in many other brain regions distant from the site of impact.35,36 Consistent with our previous results, increased rates of cell death were observed in brains of P7 mice 24 hours following trauma (data not shown). As previously reported, cell death occurred predominantly in bilateral cortices, thalamic nuclei, hippocampal dentate gyrus, subiculum, and striatum (data not shown). The calculated lesion size was significantly smaller 1 and 5 days following TBI in the conditional Nr1 LoxPþ/þ LysM Creþ/ KO mice when compared to WT mice (see Fig 6H). At the same time, there were fewer Iba1-positive cells near the lesion site (see Fig 6I). 546

Discussion We have confirmed that NMDARs exist on microglia in the immature and the mature CNS of mice and humans, and that these are functional. Moreover, we have demonstrated in animal models that the microglial NMDARs contribute significantly to cortical damage. In the CNS, bidirectional communication between neighboring neurons and microglia through NMDARmediated neurotransmitter signaling may influence neuronal function and, conversely, control microglia activation by conveying neuronal activity to microglia. Such neurotransmitter-dependent mechanisms have been described previously in the context of non-NMDA glutamate receptors; cytokines released by microglia may modulate glutamatergic transmission by regulating glutamate receptors and transporters,44–47 and neurotransmitter treatment changes LPS-induced release of various proinflammatory factors such as NO, IL-1b, TNFa, IL6, and chemokines.48–51 Because microglia lack well-defined synaptic structures, their NMDARs may be activated by diffusion of neurotransmitters outside of the synapses, their spreading within the extracellular space, and their subsequent binding to extrasynaptic receptors, according to the volume transmission model.52 Typical neuronal NMDARs are believed to be capable of maximal activation fully operational at membranes polarized around 40mV due to their specific Mg2þ-dependent voltage-dependent block.53–55 Contrary to neuronal or nonmicroglia glial cells, which display a resting potential of 60 to 80mV, resting and activated microglia have a membrane potential of about 40mV56,57 and 45 mV,57–59 respectively, and thus sustain optimal conditions for NMDAR activation. We detected a unique NMDAR subunit expression in microglia with a high level of the NR2D subunit that is distinct from that found in neurons, suggesting that NMDAR subunit composition might be different. This renders it likely that microglial NMDAR properties differ from their neuronal counterparts; further analysis devoted to microglial NMDAR characterization will be needed to address this point. The pathophysiologic relevance of microglial NMDARs is particularly high, as they confer sensitivity to excitotoxic cortical injury that is likely to be significant for a large variety of neurological diseases (Fig 7). Excitotoxicity has been related to acute and chronic neurological disorders such as perinatal brain damage, epilepsy, ischemic stroke, TBI, and progressive neurodegenerative disorders.60 We found that NMDAR stimulation is a mechanism for microglia activation in vitro and in vivo. Factors released by microglia as a Volume 72, No. 4

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FIGURE 7: Microglial N-methyl-D-aspartate receptors (NMDARs) mediate neuronal injury. The schematic drawing indicates the potential contribution of microglial NMDARs to neurodegeneration within the central nervous system. Various insults trigger neuronal cell death and thereby the secretion of factors such as glutamate that can activate microglia via their NMDAR. Microglia can become overactivated and cause neuronal cell death through the secretion of neurotoxic factors such as interleukins, tumor necrosis factor a, reactive oxygen species, and nitric oxide. This can result in a vicious circle.

consequence of NMDAR activation proved to be neurotoxic in vitro and in vivo, indicating that microglia can signal back to neurons and possibly induce, aggravate, and/or maintain neurologic disease. To further test our hypothesis that an inhibition of the microglial NMDAR protects from damage to the cerebral cortex in vivo, we exposed conditional Nr1 LoxPþ/þ LysM Creþ/ mice, lacking NMDAR1 in microglia, and WT littermates to excitotoxic brain damage through intracerebral ibotenate injections and, in a further experiment, subjected such mice to TBI. Through this approach, we were able to demonstrate that both infant (P5) and adult (P56) conditional microglia NR1 KO mice are significantly protected from an excitotoxic ibotenate insult and from TBI. In a preliminary study using human brain tissue, we detected NR1-positive intermediate microglia in the developing frontal white matter (24–30 postovulatory weeks) and in adult cortical sections, both free of neuropathological abnormalities (data not shown). Moreover, numerous intermediate–amoeboid microglial cells with intense NR1 immunoreactivity were detected October 2012

within lesions in pathological human brain and spinal cord sections from patients with neurodegenerative diseases (fetuses with periventricular leukomalacia, adults with Parkinson disease or ALS). Thus, the microglial NMDARs and/ or downstream pathways may be promising targets for antiexcitotoxic and/or anti-inflammatory therapy aimed at targeting different acute and/or chronic diseases of the CNS.

Acknowledgments This work was supported by Inserm, Universite Paris 7, Leducq Foundation, Sixth Framework Program of the European Commission (contract No. LSHM-CT-2006036534/neobrain), PremUP, Institut pour la Recherche sur la Moelle epinie`re et l’Encephale, Fondation des Gueules Cassees, Fondation Motrice, ELA Foundation, Fondation Grace de Monaco, Sonnenfeld Stiftung, German Research Foundation (SFB665), Berliner Krebsgesellschaft e.V., Societe Franc¸aise d’Anesthesie Reanimation, MRC (UK), and APHP (Contrat d’Interface to P.G.). 547

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We thank M. Mallat, P. Rustin, V. Lelievre, O. Fenneteau, M. Eveillard, H. Adle-Biassette, M. Teeuwen, C. Zabel, J. Stuwe, S. Sigaut, A.-M. Bodiou, and N. Hofmann for helpful discussions and support; and BrainNet Europe for brain and spinal cord samples.

Authorship

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Potential Conflicts of Interest A.K.: employment, University Medical Center Utrecht; grants/grants pending, EU, NIH.

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