Fractalkine Attenuates Excito-neurotoxicity via Microglial Clearance of Damaged Neurons and Antioxidant Enzyme Heme Oxygenase-1 Expression^*

Reagents

l-Glutamate, goat IgG, and LPS were purchased from Sigma (St. Louis, MO). FKN (the chemokine domain) and anti-mouse FKN-neutralizing antibody were obtained from R&D Systems (Minneapolis, MN). Anti-mouse MFG-E8 antibody and JNK peptide inhibitor (L-JNKI) were purchased from MBL (Nagoya, Japan). Hamster IgG was obtained from Jackson ImmunoResearch. The MAPK inhibitors PD98059 (MEK1 inhibitor), U0126 (MEK1/2 inhibitor), SB203580 (p38 inhibitor), and SP600125 (JNK inhibitor) were purchased from Calbiochem (Gibbstown, NJ). Stannus mesoporphyrin (SnMP), a specific inhibitor of HO-1, was obtained from Frontier Scientific (Logan, UT), and dissolved in an arginine-containing solution as previously described (23, 24).

Cell Culture

The protocols for animal experiments were approved by the Animal Experiment Committee of Nagoya University.

Primary neuronal cultures were prepared from the cortices of C57BL/6 mice embryos at embryonic day 17 (E17) as described previously (25). 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 seeded onto 12-mm polyethyleneimine-coated glass coverslips (Asahi Techno Glass Corp., Chiba, Japan) at a density of 5.0 × 10⁴ cells/well in 24-well plates and incubated at 37 °C in a humidified atmosphere containing 5% CO₂. The purity of the cultures was more than 95% as determined by NeuN-specific immunostaining.

Microglia were isolated from primary mixed glial cell cultures prepared from newborn C57BL/6 mice at 14 days in vitro (DIV) using the “shaking off” method, which has been described previously (26). The purity of the cultures was 97–100% as determined by immunostaining for the Fc receptor. Cultures were maintained in DMEM supplemented with 10% fetal calf serum, 5 μg/ml of bovine insulin, and 0.2% glucose. Microglia were seeded at a density of 7.0 × 10⁴ or 1.0 × 10⁵ cells/well in 96- or 48-well plates.

Neuron-microglia co-cultures were prepared by adding 1.0 × 10⁵ microglia in 100 μl of neuronal medium to neuronal cultures (5.0 × 10⁴ neuronal cells) on DIV 14 in 24-well plates. The cultures were maintained in nerve culture medium.

Measurement of Soluble FKN Levels

Secreted soluble FKN from mouse primary microglia and cortical neurons was measured using an ELISA kit (R&D Systems) according to the manufacturer's instructions. Neurons were treated with l-glutamate (1 or 10 μm) for 6–48 h at 37 °C. Supernatants were then collected and assessed for FKN levels.

RT-PCR

Total RNA was extracted from microglia and neurons using an RNeasy Mini Kit (Qiagen, Tokyo, Japan). A first-strand cDNA library was obtained using SuperScript II (Invitrogen, Tokyo) and oligo(dT)_12–18 (Invitrogen) as the first-strand primer. Negative control reactions were performed using the same system after heat denaturation of reverse transcriptase. RT-PCRs to amplify of transcripts encoding mouse FKN, CX3CR1, iNOS, TNF-α, MFG-E8, HO-1, and GAPDH were performed using 0.1 μg of first-strand cDNA, Blend Taq polymerase (Toyobo Co., Osaka, Japan), and oligonucleotide primers (Table 1).

Western Blotting

Cells from the BV-2 mouse microglial cell line were treated with FKN, and cell lysates were boiled after the addition of sample buffer (1 m Tris-HCl, 20% SDS, and 2.5% glycerol). Cytoplasmic and nuclear fractions from the cells were separated using NE-PER^TM Nuclear and Cytoplasmic extraction reagents (Pierce). Fifty micrograms of total protein were separated on a 5–20% Tris glycine SDS-polyacrylamide gel and blotted onto Hybond-P PVDF membranes (GE Healthcare, Buckinghamshire, UK). Membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween 20 for 1 h at room temperature. The antibodies to detect phosphorylated and total MAPK and Hsp90 (Cell Signaling, Danvers, MA), histone H1 antibody (Abcam, Cambridge, UK), or nuclear factor erythroid 2-related factor 2 (Nrf2; Santa Cruz Biotechnology, Santa Cruz, CA) were applied at the concentrations recommended by the manufacturers. Secondary antibodies were horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (GE Healthcare) and were used at a dilution of 1:1000. SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) was used according to the manufacturer's instructions. The intensities of the bands were calculated using CS Analyzer 1.0 (Atto Corporation, Tokyo).

Evaluation of Microglial Phagocytosis

Primary mouse cortical neurons in 24-well plates were labeled on DIV 14 with 1 μm CM-DiI (Molecular Probes (Invitrogen)), and treated with 1 or 10 μm glutamate overnight at 37 °C. Less than 30% of the neurons survived, whereas almost 100% of the neurons in the vehicle-treated samples survived. Surviving neurons were identified based on cytoskeletal morphology as previously described (25). After changing the culture medium, microglia were added to these neuronal cultures (1:2 ratio for neurons to microglia) with or without FKN for 24 h. Cells were subsequently fixed in 4% paraformaldehyde. Microglia were stained with Cy5-conjugated rat anti-mouse CD11b monoclonal antibody prior to fixation. In separate experiments, microglia were co-cultured with DiI-stained damaged neurons, FKN, and anti-MFG-E8 antibody for 24 h. Phagocytic uptake of neuronal debris by microglia was estimated based on the detection of DiI-stained neuronal debris (red) in CD11b-positive microglia (green); the phagocytosis index was calculated as the percentage of red staining that overlapped with green staining (shown in yellow) among all of the microglia.

Measurement of HO-1, TNF-α, NO, and Glutamate Levels

Supernatants from microglia were assessed using ELISA kits for TNF-α (BD Pharmingen, Tokyo). Cell extracts from microglia in extraction buffer (1% Nonidet P-40 in PBS) were assayed for HO-1 with an ELISA kit (Takara Bio, Mie, Japan). The level of NO was determined using a Griess reaction, as reported previously (27). To measure glutamate levels, a colorimetric assay kit (Yamasa Corporation, Tokyo) was used as described previously (28).

Immunocytochemistry

Cells were fixed with 4% paraformaldehyde for 15 min at room temperature, blocked, and permeabilized with 0.05% Triton X-100. Neurons were stained with mouse polyclonal anti-MAP-2 antibody (1:1000; Chemicon, Temecula, CA) and secondary antibody conjugated to Alexa 488 (1:1000; Invitrogen). Microglia were stained with Cy5-conjugated rat anti-mouse CD11b monoclonal antibody (1:300, BD Pharmingen) prior to fixation. Images were analyzed using a deconvolution fluorescence microscope system (BZ-8000; Keyence Corporation, Osaka).

Surviving neurons were identified based on their cytoskeletons as described previously (25). Viable neurons were stained strongly with anti-MAP-2 antibody, whereas damaged neurons showed weaker staining. MAP-2-positive neurons were counted in representative areas in each well. During five independent trials, more than 200 neurons were evaluated in each well by a scorer who was blind to the experimental conditions. The number of viable neurons in untreated cultures was set as 100%.

Statistical Analysis

Statistically significant differences between the experimental groups were determined by one-way ANOVA followed by Dunnett or Tukey tests for multiple comparisons. Statistical analysis was performed using the software program Prism 4 for Windows (GraphPad Software, San Diego, CA). p values less than 0.05 were considered to be significant.

Neurons Exposed to Glutamate Release sFKN, Promoting Microglial Clearance of Neuronal Debris

The expression of FKN and its receptor CX3CR1 was examined in neurons and microglia. FKN mRNA was expressed in neurons and CX3CR1 mRNA was mainly expressed in microglia (Fig. 1A). sFKN protein was detected in the supernatant from unstimulated neuronal cultures, but not from microglial cultures, indicating that sFKN was secreted from neurons rather than microglia (Fig. 1B). In a previous report, we confirmed the expression of fractalkine in microglia. However, the expression was found to be less by using a more specific primer in RT-PCR and an ELISA kit in this study.

A previous study in rats showed that cleavage of FKN from neuronal membranes was enhanced by excitotoxic stimuli (29). We therefore examined whether glutamate enhanced sFKN release from mouse primary cortical neurons. As shown in Fig. 1C, both 1 and 10 μm glutamate enhanced sFKN release from these neurons, with the maximum effect observed 6 h after administration of 10 μm glutamate.

We then assessed whether the release of sFKN from neurons damaged by glutamate affected microglial clearance of neuronal debris. We developed a novel microglial phagocytosis assay using primary mouse cortical neurons; microglial phagocytosis has been previously assessed by uptake of fluorescent beads (30, 31). Mouse primary cortical neurons were labeled with fluorescent DiI, and treated with 10 μm glutamate for 24 h to induce damage. Microglia were then added and the cells were cultured for an additional 24 h. Phagocytic uptake of DiI-labeled neuronal debris was examined. Microglia did not markedly phagocytose labeled healthy neurons (Fig. 1D, panel a, and E), an observation that was not affected by treatment with 10 nm sFKN (Fig. 1D, panel b, and E). On the other hand, microglia engulfed neurons that were pretreated with 10 μm glutamate (pre-Glu) (Fig. 1D, panel c, and E). This process was enhanced by treatment with 10 nm sFKN (Fig. 1D, panel d, and E).

We also confirmed that sFKN-treated microglia engulfed pre-Glu neurons by immunostaining for the phagocytic marker Rab7, which signals phagosome maturation. Neuronal debris that was engulfed after pretreatment with 10 μm glutamate colocalized with Rab-7 in the cytoplasm of sFKN-treated microglia (supplemental Fig. S1).

We also assessed the effects of sFKN on microglial activation. sFKN did not induce the production of such neurotoxic molecules as glutamate, NO, and TNFα in microglia (Fig. 1, F–H).

sFKN Up-regulates Microglial Phagocytosis of Excitotoxically Damaged Neurons via MFG-E8 Production

We examined how sFKN enhanced microglial phagocytosis of excitotoxically damaged neurons. sFKN up-regulated the mRNA and protein expression of the PS receptor MFG-E8 in microglia in a dose-dependent manner (Fig. 2A).

We next assessed whether anti-MFG-E8 neutralizing antibody prevented sFKN-induced microglial phagocytosis. Unstimulated microglia did not appear to phagocytose pre-Glu neurons (Fig. 2B, panel a), whereas 100 nm sFKN markedly enhanced microglial phagocytosis (Fig. 2B, panel b). Although anti-MFG-E8 neutralizing antibody at a concentration of 10 μg/ml had no effect on phagocytosis (Fig. 2B, panel c), 20 μg/ml of anti-MFG-E8 neutralizing antibody completely blocked sFKN-induced microglial uptake of DiI-labeled neuronal debris (Fig. 2B, panel d). However, 20 μg/ml of hamster IgG, isotype-matched control of anti-MFG-E8 antibody, had no effect on phagocytosis of microglia (Fig. 2B, panel e). Quantitative analysis confirmed these findings (Fig. 2C). These results indicate that sFKN enhances microglial phagocytic uptake of excitotoxically damaged neuronal cells via MFG-E8 expression.

sFKN-treated Microglia Exhibit Neuro-protective Activity against Glutamate-induced Excitotoxicity

Phagocytic clearance of neuronal debris in response to sFKN may contribute to the stability of neuronal networks. We next examined whether sFKN directly promotes neuronal survival in neuronal cultures (Fig. 3A, panels a–e) or neuron-microglia co-cultures (Fig. 3A, panels f–j). Unstimulated neurons stained with anti-MAP-2 antibody had no detectable morphologic abnormalities and possessed intact cell bodies and dendrites; microglia stained with anti-CD11b antibody also appeared normal (Fig. 3A, panels a and f, and B). Glutamate (10 μm) induced neuronal cell damage in both types of cultures, with the survival rates decreasing to 32.1 and 47.1%, respectively (Fig. 3, A, panels b and g, and B). Treatment with 100 nm sFKN rescued ∼70% of neurons from excitotoxic death in the co-cultures, but not in the neuronal cultures (Fig. 3, A, panels c and h, and B). Addition of anti-FKN antibody significantly attenuated neuronal survival in the co-cultures (22.6%) (Fig. 3, A, panel i, and B). However, addition of goat IgG, an isotype-matched Ig control for anti-FKN antibody, had no significant effect on the survival rate (Fig. 3A, panels e and j, and B). We confirmed that sFKN by itself did not affect neuronal survival even after long exposures, whereas 10 nm sFKN was also neuroprotective in neuron-microglia co-cultures (Fig. 3B and supplemental Fig. S2, A and B). Additionally, the neuroprotective activity of sFKN was evaluated by Annexin V and PI staining. Glutamate treatment remarkably increased the number of Annexin V and PI positive-staining neurons, whereas sFKN treatment significantly decreased the number of Annexin V and PI positive-staining neurons in the co-cultures (supplemental Fig. S2, C–E).

The Neuroprotection of sFKN-treated Microglia Is Mediated by Enhanced Phagocytosis and the Clearance of Damaged Neurons via MFG-E8

Furthermore, we examined whether the enhanced phagocytosis and the clearance of damaged neurons by FKN-treated microglia would promote survival of neurons. Glutamate-induced neuronal cell damage (Fig. 4, A, panels a and b, and B) was inhibited by 100 nm sFKN in neuron-microglia co-cultures (Fig. 4, A, panel c, and B). Addition of 20 μg/ml of anti-MFG-E8 neutralizing antibody significantly inhibited neuroprotection by sFKN (Fig. 4, A, panels d, and B). On the other hand, hamster IgG, isotype-matched control of this antibody, had no effect on sFKN-induced neuroprotection (Fig. 4, A, panel e, and B). Both anti-MFG-E8 antibody alone and IgG control alone did not affect the neuronal survival rate (Fig. 4, A, panel f, and B).

sFKN Neuroprotection Is Mediated by Microglial Production of HO-1

Interestingly, we found that sFKN dose dependently increased HO-1 mRNA and protein levels (Fig. 5, A and B). Glutamate-induced neuronal cell damage (Fig. 5C, panel b), which was compared with intact cultures (Fig. 5C, panel a), was almost completely inhibited by 100 nm sFKN in neuron-microglia co-cultures (Fig. 5C, panel c). This neuroprotective effect of sFKN was abolished by treatment with 10 μm SnMP, a HO-1 inhibitor (Fig. 5C, panel e); a lower concentration of SnMP did not exert this effect (Fig. 5C, panel d). 10 μm SnMP alone was not toxic for neurons in the presence of microglia (Fig. 5C, panel f). Quantitative analysis confirmed the effects of sFKN and the HO-1 inhibitor (Fig. 5D).

sFKN Exerts Neuroprotective Effects via Activation of ERK and JNK MAPK Signaling

FKN is reportedly involved in the activation of various intracellular signaling pathways, including ERK1/2, JNK, and p38 MAPKs, and PI3K (19, 32, 33). We explored which MAPK signaling pathways contributed to the neuroprotective effects of FKN. BV-2 cells were treated with 100 nm sFKN for various time periods, revealing time-dependent phosphorylation of ERK1/2 (p42/44 MAPK) and JNK, but not p38 MAPK (Fig. 6A). Moreover, the activation level of these MAPKs by 10 nm sFKN treatment was lower than that by 100 nm sFKN (supplemental Fig. S3A). The ERK activation level was similar to results obtained following LPS treatment. On the other hand, glutamate treatment had no effect on the activation of these MAPKs (supplemental Fig. S3B).

The neuroprotective effects of 100 nm sFKN (Fig. 6B, panel a) was almost completely canceled by treatment with the MEK1/2 inhibitor U0126 (Fig. 6B, panel b) or the JNK peptide inhibitor L-JNKI (Fig. 6B, panel c), whereas the p38 inhibitor SB203580 had little effect (Fig. 6B, panel d). Thus, U0126 or L-JNKI treatment alone had no effect on the survival of neurons (Fig. 6B, panels e and f), and other MAPK inhibitors were not toxic for neuron-microglia co-cultures (Fig. 6C). U0126 inhibits both MEK1 and MEK2, and thereby completely suppresses the activation of ERK, a kinase downstream of MEK. On the other hand, the ERK inhibitor PD98059 inhibits only MEK1. JNK inhibitors primarily act at two sites: targeting the JNK peptide (e.g. L-JNKI) or JNK kinase (e.g. SP600125). We found that these inhibitors of MEK (PD98059) or JNK (SP600125) also blocked sFKN-induced neuroprotection (Fig. 6C).

sFKN Induces Microglial HO-1 Production via JNK-Nrf2 Signaling

We examined whether MAPK signaling pathways are involved in HO-1 production by microglia. Inhibitors of JNK MAPK (JNK peptide, L-JNKI; JNK kinase, SP600125) significantly suppressed sFKN-induced HO-1 expression (Fig. 7A). Inhibitors of ERK MAPK (MEK1/2, U0126; MEK1, PD98059) or p38 MAPK (SB203580) did not suppress HO-1 expression (Fig. 7A). Similarly, the PI3K inhibitor wortmannin had no effect on sFKN-induced HO-1 expression (supplemental Fig. S4).

Nrf2 has been implicated in the induction of HO-1 expression and reportedly plays a pivotal role in cellular survival (34). Indeed, HO-1 expression is regulated by nuclear translocation of Nrf2 (35). We therefore examined whether sFKN induces Nrf2 translocation from the cytoplasm to the nucleus via activation of MAPK pathways. Using Western blot analysis, nuclear expression of Nrf2 was increased by 100 nm sFKN (Fig. 7B). Among the various MAPK inhibitors, only the JNK inhibitor L-JNKI inhibited Nrf2 translocation (Fig. 7, B and C). Cytoplasmic Nrf2 expression was not affected significantly by these inhibitors (Fig. 7, B and D). Immunofluorescence staining of Nrf2 in microglia confirmed these results. 100 nm sFKN promoted nuclear translocation of Nrf2 (Fig. 7E, panels a, b, f, g, k, and l). Among the various MAPK inhibitors, only the JNK inhibitor L-JNKI disrupted Nrf2 translocation (Fig. 7E, panels c–e, h–j, and m–o). We confirmed that Nrf2 was translocated to the nucleus by sFKN in the neuron-microglia co-cultures treated with glutamate (supplemental Fig. S5).