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Published as doi: 10.1096/fj.07-099499.
 (The FASEB Journal. 2008;22:1797-1806.)
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Interferon-{gamma} directly induces neurotoxicity through a neuron specific, calcium-permeable complex of IFN-{gamma} receptor and AMPA GluR1 receptor

Tetsuya Mizuno*,1,2, Guiqin Zhang*,1, Hideyuki Takeuchi*, Jun Kawanokuchi*, Jinyan Wang*, Yoshifumi Sonobe*, Shijie Jin*, Naoki Takada{dagger}, Yukio Komatsu{dagger} and Akio Suzumura*

* Department of Neuroimmunology and

{dagger} Department of Visual Neuroscience, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan

2Correspondence: Department of Neuroimmunology, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601 Japan. E-mail: tmizuno{at}riem.nagoya-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interferon-{gamma} (IFN-{gamma}) is a proinflammatory cytokine that plays a pivotal role in pathology of diseases in the central nervous system (CNS), such as multiple sclerosis. However, the direct effect of IFN-{gamma} on neuronal cells has yet to be elucidated. We show here that IFN-{gamma} directly induces neuronal dysfunction, which appears as dendritic bead formation in mouse cortical neurons and enhances glutamate neurotoxicity mediated via alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptors but not N-methyl-D-aspartate receptors. In the CNS, IFN-{gamma} receptor forms a unique, neuron-specific, calcium-permeable receptor complex with AMPA receptor subunit GluR1. Through this receptor complex, IFN-{gamma} phosphorylates GluR1 at serine 845 position by JAK1·2/STAT1 pathway, increases Ca2+ influx and following nitric oxide production, and subsequently decreases ATP production, leading to the dendritic bead formation. These findings provide novel mechanisms of neuronal excitotoxicity, which may occur in both inflammatory and neurodegenerative diseases in the CNS.—Mizuno, T., Zhang, G., Takeuchi, H., Kawanokuchi, J., Wang, J., Sonobe, Y., Jin, S., Takada, N., Komatsu, Y., Suzumura, A. Interferon-{gamma} directly induces neurotoxicity through a neuron specific, calcium- permeable complex of IFN-{gamma} receptor and AMPA GluR1 receptor.


Key Words: proinflammatory cytokine • neuronal dysfunction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
INTERFERON (IFN) -{gamma} IS a pleiotropic cytokine released mainly by T-lymphocytes and natural killer cells. IFN-{gamma} is considered to play a major role in the pathogenesis of multiple sclerosis (MS) (1) , which is an inflammatory demyelinating and neurodegenerative disease in the central nervous system (CNS). Autoimmune inflammatory reaction to myelin components and axonal degeneration occur in MS, and neurodegeneration is the pathological correlate of the irreversible neurological impairment (1 2 3) . IFN-{gamma} enhances the production of tumor necrosis factor (TNF) -{alpha}, nitric oxide (NO), and superoxide by surrounding microglia in MS lesions (4 5 6 7) and exacerbates the microglial reaction to axonal degeneration (8) . Thus, the neurotoxic effects of IFN-{gamma} appear to be mediated mainly by activation of microglia. In contrast, the direct effects of IFN-{gamma} on neuronal cells are controversial and poorly understood. Although IFN-{gamma} reportedly promotes neurite outgrowth and survival of neuronal cells in embryonic cortical and hippocampal cultures (9 , 10) , another study has shown that IFN-{gamma} induces retrograde dendritic retraction and inhibits synapse formation (11) .

Excito-neurotoxicity is associated with the pathogenesis of various neuroinflammatory and neurodegenerative diseases. Glutamate excitotoxicity through alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA)/kainate receptors contributes to the mechanism of neuronal injury in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and an antagonist of these receptors NBQX is a candidate of effective therapy for MS (12) . N-methyl-D-aspartate (NMDA) receptor-mediated glutamate excitotoxicity has been implicated in β-amyloid-induced neuronal death, and an NMDA-receptor antagonist is therapeutically effective in Alzheimer’s disease (13) . In amyotrophic lateral sclerosis (ALS), persistently elevated Ca2+ influx via AMPA receptors causes degeneration of motor neurons. A defect in editing the messenger RNA encoding the GluR2 subunit of AMPA receptors has been observed in spinal motor neurons from patients with ALS (14) . Therefore, various types of glutamate receptors may be involved in the pathogenesis of neuronal degeneration.

Here we report for the first time that IFN-{gamma} directly induces neuronal damage in mouse cortical neurons through a unique neuron-specific complex containing the IFN-{gamma} receptor (IFNGR) and AMPA receptor GluR1, moreover, IFN-{gamma} phosphorylates GluR1 at the serine 845 position by using JAK1·2/STAT1 pathway. We also demonstrate that IFN-{gamma} synergistically enhances glutamate neurotoxicity mediated by AMPA receptors but not NMDA receptors. The present study provides novel mechanisms of neuronal excitotoxicity, which may occur in both inflammatory and neurodegenerative diseases in the CNS.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Primary neuronal cultures
The protocols for animal experiments were approved by the Animal Experiment Committee of Nagoya University. Primary neuronal cultures were prepared from the neocortices of embryonic day 17 (E17) C57BL/6 mouse embryos as described previously (15 , 16) . Briefly, cortical fragments were dissociated into single cells in dissociation solution (Sumitomo Bakelite, Akita, Japan) and resuspended in Nerve Culture Medium (Sumitomo Bakelite). Neurons were plated onto 12 mm polyethyleneimine (PEI) -coated glass coverslips (Asahi Techno Glass Corp., Chiba, Japan) in 24-well multidishes at a density of 5 x 104 cells per well, or poly-L-Lysine-coated plastic 96-well plates (BD Biosciences, San Jose, CA, USA) at a density of 2 x 104 cells per well. The purity of the cultures was more than 95% as determined by NeuN-specific immunostaining as described previously (16) .

Organotypic slice cultures
Organotypic slice cultures were prepared from newborn C57BL/6 mice. The whole brain was dissected in cold Hanks’ balanced solution, and cortical slices were sectioned and cultured using organotypic culture techniques as described previously (17 , 18) . In brief, coronal slices (thickness 300–400 µm) of each hemisphere at rostro-caudal levels containing the parietal cortex were cut and cultured in serum-free Nerve Culture Medium (Sumitomo Bakelite) on culture membranes (Millicell-CM PICMORG50; Millipore, Bedford, MA, USA). Cultures were incubated for 5 days at 37°C in a 95% air and 5% CO2 atmosphere. The medium was exchanged on day 2 in vitro.

MTS assay
To assess the effects of IFN-{gamma} on neurons, cell viability was analyzed using 3- (4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetra-zolium (MTS) assay kit from Promega (Madison, WI, USA) as described previously (19) . Purified neuronal cells (2x104 cells per well) were plated on 96-well culture plates and incubated at 37°C in a humidified atmosphere containing 5% CO2. Then 100 ng/ml IFN-{gamma} (R&D Systems, Minneapolis, MN, USA) was added to cultures for up to 72 h on day 13 in vitro. At the end of the treatment period, 20 µl of CellTiter 96 AQueous One Solution Reagent containing MTS was added to each well, and the cells were incubated for 2 h at 37°C. Absorbance at 490 nm was measured in a multiple plate reader (Thermo Scientific, Milford, MA, USA). Assays were performed in five independent experiments.

Measurement of intracellular ATP levels
To measure intracellular ATP levels, we used the ApoSENSOR Cell Viability Assay Kit (BioVision, Mountain View, CA, USA) as described previously (20) . ATP concentration was calculated as a percentage of control. Assays were performed at each time point (0, 12, 24, 48, and 72 h) in five independent experiments.

Measurement of intracellular calcium
Ca2+ influx was measured by means of Fura-2-based fluorometry with a Wallac 1420 ARVOMX (PerkinElmer Japan, Yokohama, Japan). Mouse cortical neuronal cultures were loaded with 5 µM Fura-2-AM (Invitrogen, Carlsbad, CA, USA) for 1 h in 37°C, then rinsed twice with Hanks’ balanced salt solution (HBSS) and incubated with HBSS including 1.8 mM CaCl2 for 30 min in 37°C. Following IFN-{gamma} treatment, the change in Ca2+ influx from the baseline was measured as the change in the ratio of fluorescence emission (510 nm) intensities at the excitation wavelengths of 340 and 380 nm. Calculations of Ca2+ concentration were performed according to the equation established by Grynkiewicz (21) : [Ca2+]i = Kd[(R–Rmin)/(Rmax–R)](Sf380/Sb380). To assess the inhibition of Ca2+ influx, cultures were pretreated with each drug for 1 h before IFN-{gamma} challenge. The inhibitors examined were as follows: BAPTA (Calbiochem, San Diego, CA, USA), pan-JAK inhibitor (JAKI, Calbiochem), STAT1-specific inhibitor (F-ara-A, Sigma), PKA-specific inhibitor (H-89, Sigma), Joro spider toxin-3 (JSTx-3, Wako), NBQX (Sigma), and L-NMMA (Calbiochem).

Electrophysiological recording
Membrane currents were recorded using the whole-cell voltage-clamp technique (Axopatch 200A; Molecular Devices, Sunnyvale, CA, USA) at room temperature (23–27°C). The extracellular solution contained (in mM): NaCl, 130; KCl, 3; CaCl2, 1.5; MgSO4, 2; HEPES, 10; glucose, 20; and sodium pyruvate, 2 (pH 7.4, 280–290 mosmol). Patch pipettes fabricated from thin-wall borosilicate glass were filled with the solution (tip resistance, 3–6 M{Omega}) containing (in mm): K-gluconate, 120; NaCl, 10; MgCl2, 4; EGTA, 0.5; HEPES, 10; K2ATP, 4; and Na3GTP, 0.3 (pH 7.35, 283 mosmol). The miniature excitatory postsynaptic currents (mEPSCs) mediated by AMPA receptors were recorded in the presence of 50 µM D-2-amino-5-phosphonovaleric acid (D-APV; Tocris, Bristol, UK), an NMDA receptor antagonist, and 1 µM tetrodotoxin (TTX), a sodium channel blocker, with a resting holding potential of –60 mV during 0–10 min before and 30–40 min after 100 ng/ml IFN-{gamma} application (22) .

Immunofluorescence
To assess the formation of dendritic beads, neurons were stained with a mouse anti-microtubule-associated protein (MAP) -2 antibody (Chemicon, Temecula, CA, USA) as described previously (20 , 23 , 24) . Recombinant IFN-{gamma} (100 ng/ml) was added to cultures for 48 h on day 13 in vitro. To assess the inhibition of bead formation, cultures were pretreated with each drug for 1 h before IFN-{gamma} challenge. The inhibitors examined were as follows: MK801 (Calbiochem), CNQX, NBQX, rat anti-IFNGR antibody, BAPTA, JAKI, F-ara-A, H-89, JSTx-3, and L-NMMA. After fixation with 4% paraformaldehyde (PFA) for 30 min at room temperature (RT), cells were blocked and permeabilized with 0.3% Triton X-100. Cells were stained with an anti-MAP-2 antibody (used in 1:500 dilution), and specific binding was detected using secondary antibodies conjugated to Alexa 488 (Invitrogen, 1:1000). Stained neurons were imaged with an Axioplan 2 microscope (Carl Zeiss MicroImaging GmbH, Göttingen, Germany). The number of bead-bearing neurons, which displayed at least one beading anywhere along their dendritic arbor, was presented as a percentage of total cells in representative areas per well (25) . More than 200 neurons per well were assessed by an investigator blinded to the experimental conditions in five independent experiments.

To assess neuronal death induced by AMPA, NMDA, and IFN-{gamma}, cytoskeletal changes were examined as described (26) . Neuronal cultures were treated with 1 and 10 µM AMPA or NMDA (Calbiochem) with or without 100 ng/ml IFN-{gamma} for 24 h. Viable neurons were immunopositive for MAP-2, whereas damaged neurons stained poorly. The number of MAP-2 positive neurons with intact nuclei in representative areas per well was determined by Hoechst staining. More than 200 neurons per well were examined by an investigator blind to the experimental condition in five independent experiments. The viability of untreated neuronal cells was arbitrarily normalized to 100%.

To examine the surface expression of the IFN-{gamma} receptor and AMPA receptor subunit GluR1, after fixation with 4% PFA and blocking with 10% normal goat serum (NGS) in a 1% BSA solution, neurons were stained with a rabbit polyclonal anti-GluR1 antibody (Upstate Biotechnology, Lake Placid, NY, USA; used in 1:500 dilution) and a hamster anti-IFN-{gamma} receptor 1 antibody conjugated to FITC (2E2, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; 1:50 dilution) overnight at 4°C, followed by secondary antibodies conjugated to Alexa Fluor dyes (Alexa 568 for GluR1) for 1 h at RT. Hochest 33342 was added to secondary antibodies to label nuclei. The samples were mounted and imaged with a confocal laser-scanning microscope (LSM510, Carl Zeiss).

To assess the effects of IFN-{gamma} on neuronal tissue, organotypic slices were treated with 100 ng/ml IFN-{gamma} for 24 h with or without NBQX on day 4 in vitro. Slices were fixed in 4% PFA for 30 min, cryoprotected in 20% sucrose, stored at 4°C, and used for subsequent cryostat sectioning (at 40–50 µm). The sections were rinsed in 0.1 M Tris-buffered saline (TBS) and permeabilized with 1% Triton X-100 and, after blocking with 10% NGS for 30 min, stained for 24 h at 4°C with a rat anti-mouse CD11b antibody for microglia (BD; used in 1:50 dilution), a rabbit anti-glial fibrillary acidic protein (GFAP) antibody for astrocytes (Thermo Scientific; used in 1:500 dilution), and a mouse anti-MAP-2 antibody (used in 1:500 dilution). Thereafter, the sections were rinsed in TBS-Triton X-100 and incubated for 1 h at RT with secondary antibodies conjugated to Alexa Fluor dyes (Alexa 488 for MAP-2, Alexa 568 for GFAP, and Alexa 647 for CD11b) for 1 h at RT with subsequent visualization with a fluorescent microscope system (BZ-8000; Keyence, Osaka, Japan).

Immunoprecipitation and immunoblotting
Immunoprecipitation and immunoblotting were performed as described previously (20 , 27) . Neurons, mixed glial cells, and splenocytes treated with or without IFN-{gamma} were collected in ice-cold PBS with a protease inhibitor cocktail (Complete Mini; Roche Applied Science, Indianapolis, IN, USA), and homogenized on ice. Then, homogenates were centrifuged at 10,000 g for 10 min at 4°C. After removing the pellet, 10 µl of protein G sepharose beads was added to the cell lysate and incubated for 30 min at 4°C. The supernatant was incubated with a rat anti-IFN-{gamma} receptor 1 antibody (PBL; used in 1:50 dilution) or rabbit anti-GluR1 antibody (Upstate; used in 1:500 dilution) overnight at 4°C. Precipitated proteins were separated by SDS-PAGE on 5–20% gradient gels (Bio-Rad, Hercules, CA, USA), and then transferred to PVDF membranes. The membranes were immunoblotted with a rabbit-polyclonal anti-GluR1, anti-GluR2/3, and anti-GluR4 antibody (Upstate; 1:500 dilution) or a rabbit-polyclonal anti-IFN-{gamma} receptor {alpha} antibody (M-20, Santa Cruz; 1:250 dilution). After overnight incubation with primary antibodies at 4°C, each blot was probed with horseradish peroxidase-conjugated anti-rabbit IgG (1:5000, Amersham Biosciences, Piscataway, NJ, USA) for 1 h at room temperature. Blots were visualized with ECL Plus Western blotting detection reagents (Amersham Biosciences).

To assess STAT1 phosphorylation, neurons were collected 30 min after stimulation with IFN-{gamma} or AMPA, and lysed in TNES buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 2 mM EDTA, 0.1% sodium dodecyl sulfate) with protease inhibitor cocktail (Complete Mini EDTA-free, Roche) and phosphatase inhibitor cocktail (Sigma).

To assess GluR1 phosphorylation, neurons were collected at each time point (0, 15, 30, 60 min) after stimulation with IFN-{gamma}. In some experiments, neurons were pretreated with JAKI, F-ara-A, H-89 before IFN-{gamma} stimulation. Neurons were applied with 1 µM JAKI or 2 µM H-89 30 min before stimulation, whereas 50 µM F-ara-A were added 24 h before stimulation. Neurons were collected 30 min after IFN-{gamma} stimulation. Cells were lysed in TNES buffer with protease inhibitor cocktail and phosphatase inhibitor cocktail as above. For Western blot analysis, 30 µg of protein from the total lysate was loaded. The primary antibodies used here were as follows: anti-GluR1 rabbit polyclonal antibody, anti-phospho-GluR1 (Ser-831) rabbit polyclonal antibody, anti-phospho-GluR1 (Ser-845) rabbit polyclonal antibody (1:500, respectively, Upstate), anti-STAT1 rabbit monoclonal antibody and anti-phospho-STAT1 (Tyr701) rabbit monoclonal antibody (1:500, respectively; Cell Signaling Technology, Danvers, MA, USA). After overnight incubation with primary antibodies at 4°C, each blot was probed with horseradish peroxidase-conjugated anti-rabbit IgG (1:5000, Amersham Biosciences) for 1 h at room temperature. Blots were then visualized with ECL Plus Western blotting detection reagents.

Statistical analyses
Data were presented as means ± SD. Statistical significance was assessed with a one-way ANOVA followed by post hoc Tukey’s test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IFN-{gamma} reduces intracellular ATP levels and induces the formation of dendritic beads in mouse cortical neurons
First, we investigated the direct effects of IFN-{gamma} on primary cortical neurons. We examined dendritic bead formation and reduction of cytoplasmic ATP as early markers of neuronal dysfunction. The formation of dendritic beads has been observed in vivo in various neurological disorders and is considered to be an early feature of neuronal damage (23 , 28 , 29) . To study the effect of IFN-{gamma} on neuronal cells, recombinant IFN-{gamma} was added to cultures of primary cortical neurons at 13 days in vitro. Stimulation with 100 ng/ml IFN-{gamma} for 72 h did not decrease cell viability as assessed with the MTS assay (Fig. 1 A) nor induce neuronal cell death as determined by MAP-2 staining (Fig. 1B ). However, treatment with IFN-{gamma} for 24, 48, and 72 h significantly reduced intracellular ATP levels in a time-dependent manner (Fig. 1C ). Subsequent to ATP reduction, IFN-{gamma} induced dendritic bead formation (Fig. 1D, E ), and it was predominantly observed in the distal dendrites of pyramidal neurons (Fig. 1E ). Dendritic beads were formed in a time- and dose-dependent manner (Fig. 1F, G ). Treatment with 100 ng/ml IFN-{gamma} for 48 h induced dendritic bead formation in 42% of MAP-2-positive cells (Fig. 1F ).


Figure 1
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  		Figure 1. The direct effects of IFN-{gamma} on cortical neurons. A) Neuronal cell viability with 100 ng/ml IFN-{gamma} treatment as assessed by MTS assay. B) Neuronal cell survival with 100 ng/ml IFN-{gamma} treatment as determined by the number of MAP-2-positive cells. C) IFN-{gamma} decreased intracellular ATP in a time-dependent manner. D) Nontreated cortical neurons stained with MAP-2 had smooth dendrites. E) IFN-{gamma} treatment induced bead-like swelling on dendrites (dendritic bead formation). F, G) IFN-{gamma} increased frequency of beaded neurons in a time- and dose-dependent manner. *P < 0.05 vs. control. Each column indicates mean ± SD; n = 5. Scale bar = 20 µm.

IFN-{gamma}-induced dendritic bead formation is mediated through the IFNGR and AMPA receptor
We characterized the requirements for IFN-{gamma}-induced dendritic bead formation with pharmacological antagonists for various subclasses of glutamate receptors. Although the noncompetitive NMDA receptor antagonist MK-801 (20 µM) did not affect bead formation (Fig. 2 A–C, G), both an AMPA/kainate receptor antagonist CNQX (20 µM) and the specific AMPA/kainate receptor antagonist NBQX (20 µM) lacking inhibitory effects on the NMDA receptor glycine site suppressed the formation of dendritic beads (Fig. 2D, E, G ). Bead formation was also blocked by treatment with an anti-IFNGR antibody (Fig. 2F, G ). These data indicated that IFN-{gamma}-induced dendritic bead formation was mediated through both the AMPA receptor and IFN-{gamma} receptor.


Figure 2
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  		Figure 2. Inhibition of IFN-{gamma}-induced dendritic bead formation by AMPA/kainate receptor antagonists but not by an NMDA receptor antagonist. IFN-{gamma}-induced bead formation was evaluated with MAP-2 staining (A–F). A) nontreated neurons. B, C) IFN-{gamma}-induced bead formation was not inhibited by the NMDA receptor antagonist MK-801. D, E) The AMPA/kainate receptor antagonist, CNQX (D), and a more specific AMPA/kainate receptor antagonist lacking inhibitory effects on the NMDA receptor glycine site, NBQX (E), inhibited bead formation. F) Bead formation was also blocked by treatment with an anti-IFN-{gamma} receptor (IFNGR) antibody. G) The frequency of beaded neurons was significantly reduced in the presence of CNQX (20 µM), NBQX (20 µM), and IFNGR (0.2 µg/ml). *P < 0.05 vs. IFN-{gamma}-treated neurons. Each column indicates the mean ± SD; n = 5. Scale bar = 20 µm.

IFNGR and AMPA receptor GluR1 colocalize and form a unique complex and IFN-{gamma} phosphorylates GluR1 at serine 845
We examined the cellular distributions of IFNGR receptor and the AMPA receptor subunit GluR1 by double-labeling cortical neurons with specific antibodies against IFNGR and GluR1. GluR1 (Fig. 3 A) and IFNGR (Fig. 3B ) colocalized in most cortical neurons treated with IFN-{gamma} (Fig. 3C ). To confirm colocalization of IFNGR and GluR1, we performed coimmunoprecipitation experiments in neurons, mixed glial cells, and splenocytes treated with IFN-{gamma}. IFNGR-associated proteins were collected by immunoprecipitation with an anti-IFNGR antibody. These samples were analyzed by immunoblotting with an anti-GluR1 antibody. In a reciprocal experiment, proteins immunoprecipitated with an anti-GluR1 antibody were immunoblotted with anti-IFNGR. We detected GluR1 in IFNGR immunoprecipitates in neuron, whereas GluR2, GluR3, and GluR4 were not detected in the immunoprecipitates. Similarly, IFNGR were detected in the immunoprecipitates with an anti-GluR1 antibody (Fig. 3D ). The colocalization of IFNGR and GluR1 was not detected in mixed glial cells and splenocytes (Fig. 3D ). Thus, IFNGR and GluR1 colocalize and form a unique complex exclusively in neuron. This complex was hardly detected in neuron without IFN-{gamma} treatment (data not shown). Western blot analysis revealed that IFN-{gamma} induced STAT1 phosphorylation, but AMPA did not affect STAT1 phosphorylation (Fig. 3E ). Moreover, IFN-{gamma} induced neuronal GluR1 phosphorylation at serine 845 position (S845) (Fig. 3F ), whereas IFN-{gamma} did not affect GluR1-S831 phosphorylation (data not shown).


Figure 3
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  		Figure 3. Neuron-specific coupling of IFNGR and AMPA receptor GluR1. A–C) Colocalization of IFNGR and GluR1 in IFN-{gamma}-treated cortical neurons. The distributions of IFNGR and GluR1 were examined by double-immunostaining with specific antibodies and Alexa Fluor-conjugated secondary antibodies. GluR1 (A) and IFNGR (B) have similar distributions (C). Scale bar = 20 µm. D) Immunoprecipitation and immunoblotting experiments reveal that IFNGR and GluR1 form a unique complex. Neuronal proteins immunoprecipited (IP) with an anti-IFNGR antibody were analyzed by immunoblotting (IB) with an anti-IFNGR antibody, anti-GluR1, anti-GluR2/3, and anti-GluR4 antibody. S = with antibody; C = without antibody. Neuronal proteins were collected by IP with an anti-GluR1 antibody and then analyzed by IB. Mixed glial cells and splenocytes proteins were collected by IP with an anti-IFNGR antibody and analyzed by IB with an anti-IFNGR antibody. E) Western blot analysis revealed that AMPA does not affect neuronal STAT1 phosphorylation. F) Western blot analysis revealed that IFN-{gamma} induces neuronal GluR1 phosphorylation at serine 845 position (S845).

IFN-{gamma} phosphorylates GluR1 S845 through JAK1·2/STAT1 and PKA pathway, and IFN-{gamma}-induced neurotoxicity is mainly mediated by calcium influx and subsequent NO production
The IFN-{gamma}-induced phosphorylation of GluR1-S845 was blocked by JAKI), F-ara-A, and H-89 (Fig. 4 A). These observations indicate that IFN-{gamma}-induced GluR1-S845 phosphorylation is mediated by the activation of JAK1·2/STAT1 and PKA. IFN-{gamma} did not affect AMPA-induced spontaneously occurring mEPSCs in frequency and amplitude (Fig. 4B ).


Figure 4
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  		Figure 4. IFN{gamma}-induced GluR1-S845 phosphorylation is mediated by the activation of JAK1·2/STAT1 and PKA. A) Western blot analysis revealed that the phosphorylation GluR1-S845 is blocked by the pan-JAK inhibitor (JAKI), the STAT1-specific inhibitor (F-ara-A), and the PKA-specific inhibitor (H-89). B) The frequency and amplitude of mEPSCs remained unchanged 0–10 min before and 30–40 min after IFN{gamma} application. Cells were voltage-clamped at –60 mV in the presence of 1 µM TTX. C) Ca2+ influx by IFN-{gamma}. Intracellular Ca2+ concentration in neurons was measured by means of Fura-2-based fluorometry with a Wallac 1420 ARVOMX. The change in intracellular Ca2+ from baseline was measured as the change in the ratio of fluorescence emission (510 nm) intensities at the excitation wavelengths of 340 and 380 nm. IFN-{gamma} treatment significantly increased intracellular Ca2+ in a dose-dependent manner. *P < 0.05 vs. nontreated neurons. D) Inhibition of IFN-{gamma}-induced Ca2+ influx and dendritic bead formation by the calcium chelator (10 µM BAPTA), JAK1·2/STAT1, and PKA signaling blockers (1 µM JAKI, 0.5 µM F-ara-A, 2 µM H-89), AMPA receptor blockers (5 µM JSTx-3, 20 µM NBQX), and NOS inhibitor (1 mM L-NMMA). Bead formation was evaluated with MAP-2 staining. *P < 0.05 vs. IFN-{gamma}-treated neurons. Each column indicates the mean ± SD; n = 5–8.

Next, we examined the involvement of Ca2+ in the IFN-{gamma}-induced dendritic bead formation because intracellular Ca2+ levels have been implicated in the bead formation (30) . IFN-{gamma} increased Ca2+ influx in a dose-dependent manner (Fig. 4C ). Calcium chelator BAPTA, JAKI, F-ara-A, H-89, and NBQX inhibited IFN-{gamma}-induced Ca2+ influx and dendritic bead formation. Importantly, treatment with JSTx-3 (31 32 33) , a selective blocker of Ca2+-permeable AMPA receptors that lack the edited GluR2 subunit also inhibited IFN-{gamma}-induced Ca2+ influx and dendritic bead formation. Neuronal NO synthase (nNOS) activity is regulated by increases in intracellular Ca2+ via calmodulin binding (34) . The NOS inhibitor L-NMMA did not inhibit IFN-{gamma}-induced Ca2+ influx but reduced bead formation (Fig. 4D ). Therefore, Ca2+ influx and subsequent NO production through IFNGR and GluR1 complex are involved in the direct effect of IFN-{gamma} on neurons.

IFN-{gamma} enhances AMPA-induced neurotoxicity
We investigated the effect of IFN-{gamma} on AMPA-induced neurotoxicity. Treating cultures with AMPA for 24 h decreased neuronal survival in a dose-dependent manner (83% at 1 µM AMPA treatment and 72% at 10 µM, Fig. 5 A). Addition of IFN-{gamma} enhanced AMPA-induced neurotoxicity. Survival rates were significantly decreased with addition of IFN-{gamma}: 83% at 1 µM AMPA, 63% at 1 µM AMPA, and 100 ng/ml IFN-{gamma}, 72% at 10 µM AMPA, 48% at 10 µM AMPA and 100 ng/ml IFN-{gamma} treatment (Fig. 5A ). However, IFN-{gamma} treatment did not enhance NMDA toxicity (Fig. 5B ). The enhancement of AMPA-induced neurotoxicity by IFN-{gamma} was suppressed by concomitant treatment with NBQX or an anti-IFNGR antibody (Fig. 5C ). Thus, IFN-{gamma} enhances neurotoxicity mediated by AMPA receptors but not NMDA receptors.


Figure 5
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  		Figure 5. Enhancement of AMPA-mediated neurotoxicity by IFN-{gamma}. Neuronal survival rates were assessed with MAP-2 staining. A) AMPA treatment induced neuronal death. Addition of 100 ng/ml IFN-{gamma} enhanced AMPA-induced neuronal death. *P < 0.05 vs. AMPA-treated neurons. B) IFN-{gamma} did not enhance NMDA-mediated neurotoxicity. C) NBQX and IFNGR inhibited the enhancement of AMPA-induced neurotoxicity by IFN-{gamma}. *P < 0.05 vs. both AMPA and IFN-{gamma}-treated neurons. Each column indicates the mean ± SD; n = 8.

IFN-{gamma} induces dendritic bead formation in organotypic slice cultures
We confirmed the ability of IFN-{gamma} to promote dendritic bead formation in mouse organotypic slice cultures. Dendrites and cell bodies of cortical neurons in slice cultures were stained with an anti-MAP-2 antibody (Fig. 6 A–C, M–O). As we observed in primary neuronal cultures, treating slice cultures with 100 ng/ml IFN-{gamma} for 24 h induced dendritic bead formation (Fig. 6N , arrows). IFN-{gamma}-induced bead formation was inhibited by coapplication of 20 µM NBQX (Fig. 6O ). To examine neuronal damage and glial responses, cortical slices were stained with an anti-GFAP antibody (Fig. 6D –F) and an anti-CD11b antibody (Fig. 6G-I ) to identify astrocytes and microglia, respectively. IFN-{gamma} treatment markedly increased the intensity anti-GFAP and anti-CD11b staining, suggesting activation of astrocytes and microglia (Fig. 6J, K ). Similarly, the responses of these glial cells were inhibited by 20 µM NBQX (Fig. 6J, L ).


Figure 6
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  		Figure 6. Representative images of IFN-{gamma}-induced dendritic bead formation in organotypic slice cultures. A–L) Cortical slices were stained with anti-MAP-2 antibody (A–C), anti-GFAP antibody (D–F), and anti-CD11b antibody (G–I) to identify neurons, astrocytes, and microglia, respectively. IFN-{gamma} treatment markedly increased the intensity of anti-GFAP and anti-CD11b staining, suggesting activation of these glial cells (J, K). These glial responses were inhibited by 20 µM NBQX (J, L). M) Nontreated control culture. N) 100 ng/ml IFN-{gamma} for 24 h induced bead formation (arrows). O) NBQX inhibited IFN-{gamma}-induced bead formation. Scale bars = 50 µm.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The aim of this study is to examine the direct effect of IFN-{gamma} on neuronal cells. The present study is the first to demonstrate that IFN-{gamma} directly induces neuronal dysfunction through a unique neuron-specific IFNGR and AMPA receptor GluR1 complex and that IFN-{gamma} phosphorylates GluR1 by JAK1·2/STAT1 pathway. Figure 7 shows the proposal mechanism of IFNGR and AMPA receptor signaling. Exclusively in neurons, IFNGR couples with AMPA receptor GluR1. First, IFN-{gamma} activates JAK1·2/STAT1. Then, activated STAT1 induces cAMP elevation and the following PKA activation. The phosphorylation of GluR1 at S845 may potentiate GluR1-containing AMPA receptors (35) . Our results demonstrate that AMPA receptors coupled with IFNGR lack GluR2 and that IFN-{gamma} facilitates Ca2+-permeable AMPA receptors. The enhanced Ca2+ entry promotes NO production, leading to the reduction in intracellular ATP level by inhibition of mitochondrial respiratory chain. Because IFN-{gamma} produced no effect on AMPA receptor-mediated mEPSCs, IFNGR-GluR1 receptor complex may be located extrasynaptically rather than synaptically. This subcellular localization may explain why IFN-{gamma} alone produced only weak excitotoxicity.


Figure 7
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  		Figure 7. Proposal mechanism of IFNGR and AMPA receptor GluR1 complex signaling. IFNGR couples with AMPA receptor GluR1 exclusively in neurons. IFN-{gamma} activates JAK1·2/STAT1. Then, activated STAT1 induces cAMP elevation and the following PKA activation. The phosphorylation of GluR1 at Ser-845 position (S845) elicits Ca2+ influx and the subsequent NO production. NO reduces the intracellular ATP level by inhibition of mitochondrial respiratory chain, leading to neuronal dysfunction.

Ca2+ influx through AMPA receptor is reported to be neurotoxic (36) . AMPA increases Ca2+ influx, and endogenous NO modulates glutamate signaling in organotypic cultures of the rat hypothalamic paraventricular nucleus (37) . AMPA-mediated up-regulation of nNOS and release of NO by inhibitory interneurons play a prominent role in transsynaptic neuronal degeneration in limbic cortex (38) . These reports support our findings.

Neuronal dysfunction induced by IFN-{gamma} is represented as the formation of dendritic beads. Dendritic bead formation is observed following brain ischemia, epilepsy, CNS trauma, and EAE and is one of the earliest signs of glutamate receptor-mediated excitotoxic injury. Sodium, chloride, and water entry contribute to bead formation, while calcium entry through NMDA receptors results in lasting structural changes in damaged dendrites (25) . Previously, we have shown that glutamate released from activated microglia induces dendritic bead formation via NMDA receptor signaling, and this morphological change is a characteristic of neuronal cell dysfunction that precedes neuronal death. Dendritic beads contain motor proteins and collapsed cytoskeletal structures arising from impaired neuronal transport secondary to cellular energy loss (20) . The mechanism of bead formation induced by IFN-{gamma} may resemble that induced by activated microglia as to Ca2+ influx, NO elevation, and subsequent ATP reduction. Although IFN-{gamma} induced neuronal cell dysfunction through this receptor complex, it did not induce cell death. However, IFN-{gamma} works synergistically with glutamate to promote neuronal excitotoxicity through this receptor complex. Although previous studies have shown that IFN-{gamma} alters AMPA-mediated synaptic activity in rat hippocampal neurons (39) and induces changes in synaptic activity and AMPA receptor clustering (40) , this is the first report to show that IFNGR and AMPA receptor GluR1 form a neuron-specific complex and interact to mediate excitotoxicity.

Several proinflammatory cytokines have been reported to modulate glutamate excitotoxicity. TNF-{alpha} up-regulates expression of GluR1 in mouse hippocampus and cerebral cortex neurons (41) and exacerbates AMPA-induced neuronal death at high doses (26) . IL-1β also enhances NMDA-mediated neurotoxicity and NMDA-induced Ca2+ influx (42) . Although IL-6 treatment of developing cerebellar granule neurons increases the excitotoxicity of NMDA (43) , it has a neuroprotective effect in hippocampal or cortical cultures exposed to NMDA (44) . It is uncertain that these cytokines function through cytokine/glutamate receptor complex like IFN-{gamma}.

In organotypic slice cultures, IFN-{gamma} induced bead formation, similar to that observed in primary neuronal cultures, followed by activation of microglia and astrocytes. Both bead formation and glial reactions were inhibited by NBQX treatment. In our previous study (20) , we observed that the NMDA receptor antagonist MK-801 but not the AMPA receptor antagonist NBQX suppressed neurotoxicity induced by activated microglia. In addition, in this study we demonstrate that the IFNGR did not colocalize with GluR1 in mixed glial cells. Thus, glial activation in slice cultures may be considered as a secondary reaction to IFN-{gamma}-induced neuronal damage.

In EAE, an animal model of MS, dendritic bead formation is present in the motoneuron dendrites, which extend into ventrolateral white matter of the lumbosacral spinal cord during acute EAE episodes and EAE relapses but exhibit marked recovery during remission. Thus, the formation of dendritic beads is associated with inflammatory cell infiltration at different EAE stages (29) . Glutamate excitotoxicity mediated by the AMPA/kainate receptors damages both neurons and oligodendrocytes. Treatment with the AMPA antagonist NBQX ameliorates EAE by increasing survival of oligodendrocytes and reducing axonal damage (12) . Our results are consistent with these findings. It is possible that IFN-{gamma}, together with AMPA receptors, may contribute to the neuronal dysfunction observed in EAE and MS.

Only immunocytes such as T cells or natural killer cells have been considered as a source of IFN-{gamma}. However, microglia produce IFN-{gamma} on stimulation with IL-12 and/or IL-18 (45) . Therefore, the synergistic neurotoxicity of IFN-{gamma} and glutamate may occur without inflammatory cell infiltration, for example in neurodegenerative diseases such as Alzheimer’s disease or Parkinson’s disease. Antagonizing IFN-{gamma} function may represent a useful strategy to inhibit excitotoxicity in neuroinflammatory and neurodegenerative diseases.


   ACKNOWLEDGMENTS
 
This work was supported in part by a Grant-in-Aid for Scientific Research (grant C), and the 21st Century COE program Integrated Molecular Medicine for Neuronal and Neoplastic Disorders from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and for Research on Specific Diseases from the Ministry of Health, Labor, and Welfare of Japan.


   FOOTNOTES
 
1 These authors contributed equally to this work.

Received for publication October 15, 2007. Accepted for publication December 23, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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