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. Author manuscript; available in PMC: 2014 Aug 4.
Published in final edited form as: J Neurosci Res. 2013 Mar 29;91(6):828–837. doi: 10.1002/jnr.23208

Reduced Extracellular Zinc Levels Facilitate Glutamate-Mediated Oligodendrocyte Death after Trauma

Joshua T Johnstone 1,6, Paul D Morton 1,6, Arumugam R Jayakumar 2, Valerie Bracchi-Ricard 1, Erik Runko 1, Daniel J Liebl 1,3,6, Michael D Norenberg 2,4,5,6, John R Bethea 1,3,6,7,†
PMCID: PMC4120886  NIHMSID: NIHMS598289  PMID: 23553703

Abstract

Spinal cord injury results in irreversible paralysis, axonal injury, widespread oligodendrocyte death and white matter damage. While the mechanisms underlying this phenomenon are poorly understood, previous studies from our laboratory indicate inhibiting activation of the nuclear factor - κB transcription factor in astrocytes reduces white matter damage and improves functional recovery following spinal cord injury. In the current study, we demonstrate that activation of the nuclear factor - κB transcription factor within astrocytes results in a significant increase in oligodendrocyte death following trauma by reducing extracellular zinc levels and inducing glutamate excitotoxicity. Using an ionotropic glutamate receptor antagonist (CNQX), we show astroglial nuclear factor-κB-mediated oligodendrocyte death is dependent upon glutamate signaling despite no change in extracellular glutamate concentrations. Further analysis demonstrated a reduction in levels of extracellular zinc in astrocyte cultures with functional nuclear factor-κB signaling following trauma. Co-treatment of oligodendrocytes with glutamate and zinc showed a significant increase in oligodendrocyte toxicity in low zinc conditions, suggesting the presence of zinc at specific concentrations can prevent glutamate excitotoxicity. These studies demonstrate a novel role for zinc in regulating oligodendrocyte excitotoxicity and identify new therapeutic targets to prevent oligodendrocyte cell death in central nervous system trauma and disease.

Keywords: astrocytes, zinc, oligodendrocytes, glutamate, excitotoxicity

Introduction

Spinal cord injury (SCI) is a devastating condition that results in the complete or partial loss of sensory, motor, and autonomic function below the level of the injury. Within the first few hours after injury neurons and glia within the lesion site begin to undergo cell death by necrotic and apoptotic mechanisms (Liu et al., 1997; Lu et al., 2000). These injury mechanisms are induced, in part, by glutamate excitotoxicity and subsequent production of reactive oxygen species (ROS) (Liu et al., 1997; Casha et al., 2001; Park et al., 2004; Nashmi and Fehlings, 2001).

Following injury, extracellular glutamate concentrations rise from 10 µM in uninjured animals to as high as 550 µM (Liu et al., 1991; Liu et al., 1999; McAdoo et al., 1999; McAdoo et al., 2005). Slight increases in glutamate concentrations (1–3 µM) can be toxic to neurons and oligodendrocytes by over-stimulation of Ca2+-permeable ionotropic glutamatergic receptors (Lipton and Rosenberg, 1994; Trotti et al., 1998). Oligodendrocytes are particularly susceptible to glutamate level alterations due to the expression of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptors lacking the GluR2 subunit. Typically, RNA editing of the GluR2 subunit renders AMPA receptors permeable only to Na+ ions (Sommer et al., 1991). Since oligodendrocytes express AMPA channels lacking the GluR2 subunit, they are susceptible to Ca2+ influxes and subsequent excitotoxic mechanisms (Agrawal and Fehlings, 1997; Li and Stys, 2000; Park et al., 2003).

During periods of synaptic activity, zinc is packaged and released from glutamatergic synaptic vesicles along with glutamate (Frederickson and Bush, 2001). Importantly, zinc has been shown to inhibit currents through ionotropic glutamate receptors in a dose-dependent manner, presumably by binding to the extracellular N-terminal domain of the receptor and causing a conformational change in the receptor thereby inducing its desensitization (Zhang et al., 2002). In the presence of high extracellular glutamate concentrations (200 µM), low levels of zinc fail to inhibit Ca2+ currents through AMPA receptors. Supplementation of extracellular zinc is sufficient to attenuate the currents in a dose-dependent manner (Zhang et al., 2002). The ability of zinc to inhibit glutamate signaling was determined to be due to the GluR3 subunit specific composition of the receptors (Dreixler and Leonard et al., 1994). The presence of the GluR2 subunit within the AMPA receptor composition abolished the elevated receptor currents in the presence of glutamate and low levels of extracellular zinc. This result demonstrates that the inhibitory effect of zinc on glutamate signaling is isolated to the Ca2+ permeable isoform of the AMPA receptor. As a result, at low extracellular zinc concentrations, cells expressing the GluR3 subunit of the AMPA receptor (such as oligodendrocytes) are susceptible to glutamate-mediated AMPA receptor activation and cell death. Despite the documented role of zinc in modulating glutamate signaling, little is known about factors that mediate the inhibitory action of zinc on excitotoxicty.

The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) family of transcription factors regulates the expression of genes associated with apoptosis, inflammation, cell proliferation (Viatour et al., 2005) and is a key regulator of secondary injury mechanisms following SCI (Brambilla and Bracchi-Ricard et al., 2005). Previously, our laboratory demonstrated that inactivation of the NF-κB transcription factor within astrocytes resulted in a significant recovery of locomotor behavior, reduced lesion volume, and significant preservation of white matter at 8 weeks following a contusive SCI (Brambilla and Bracchi-Ricard et al., 2005). Astrocytes are well known for their role in maintaining extracellular ion and amino acid concentrations under naïve conditions. However, the mechanism by which astrocytes modulate ionic concentrations, excitotoxicity, and oligodendrocyte fate after SCI is still relatively unknown.

Methods

Mice and genotyping

Transgenic mice expressing the inhibitor of κBα (IκBα)-dn transcript were produced and characterized as previously described (Brambilla and Bracchi-Ricard et al., 2005). Wild-type mice from the same breeding groups were used as controls. Animals were housed in a 12 hr light/dark cycle in a virus/antigen free environment. All animals had access to food and water throughout the study

SCI

Surgical procedures were performed in the Animal and Surgical Core Facility of The Miami Project to Cure Paralysis, according to the protocols approved by the Institutional Animal Care and Use Committee of the University of Miami (IACUC). The studies shown here were approved by the University of Miami IACUC. Adult female mice 3–5 months of age (20–24g in weight) underwent a moderate contusion injury to the T9 segment of spinal cord using the Electromagnetic Spinal Cord Injury Device (developed at Ohio State University). Mice were injured with a predetermined spinal cord displacement of 0.5 mm. Following injury, the mice were treated with topical antibiotics and subcutaneous Ringer’s solution to reduce fluid loss, and given nutritional supplements. Bladder expression was performed twice daily and gentamicin was administered to prevent urinary tract infections.

Astrocyte cultures

Astrocyte cultures were prepared as previously described (Ducis et al., 1990). Primary astrocyte cultures were isolated from one to two day old mice. Briefly, cortices were removed, cleared of meninges, minced, dissociated by trituration, passed through sterile nylon sieves, and plated with Dulbecco’s modified Eagle medium (DMEM; Life Technologies, Gaithersburg, MD) containing penicillin, streptomycin, and fetal bovine serum. Cultures were incubated at 37°C in a humidified incubator provided with 5% CO2 and 95% air. Following 10 days in culture, the bovine serum was replaced with horse serum. After 14 days in culture, astrocytes were treated and maintained with dibutyryl cyclic adenosine monophosphate (Sigma, St. Louis, MO) to enhance cell maturation. Experiments were carried out when cultures were at least 3 weeks old. Cultures consisted of 98% astrocytes as previously described (Jayakumar et al., 2008).

In vitro injury model

The in vitro trauma was induced using a fluid percussion device developed by Sullivan et al. (1976) and later modified for cell culture (Shepard et al., 1991). Five atmospheres of pressure was applied twice to the cultures for a 25 millisecond duration each. Pressure was monitored with a PowerLab system (ADInstruments, Inc., Colorado Springs, CO) with a high-speed pressure transducer. Uninjured controls were treated the same as injured cultures except they did not receive the trauma.

Oligodendrocyte isolation and culture

This procedure for producing pure oligodendrocyte cultures was modified from an existing protocol (Cohen et al., 2003). The spinal cords were dissected out dorsally from adult female mice (2–3 months old), the meninges removed, and rinsed twice in ice-cold DMEM (Gibco). The spinal cords were chopped and digested in dissociation buffer [1 × Hanks’ Balanced Salt Solution (HBSS, Gibco) containing 10 U/ml papain (Sigma), 5.5 mM cysteine (Sigma), 2.5 mM ethylenediaminetetraacetic acid (EDTA; Sigma), 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Sigma), 0.01 N sodium nydroxide (NaOH)], at 37°C for 45 min. At 15 min intervals, the dissociated spinal cord was triturated with a 10 ml pipet followed by a 5 ml pipet. The dissociation was stopped by adding equal volume of stop buffer [1× HBSS containing 250 U/ml deoxyribonuclease I (Sigma), 0.2% bovine serum albumin (Sigma), 10 µg/ml gentamicin (Gibco), and 20 mM HEPES (Sigma), pH 7.4] for a 15 minute incubation at 37°C. The dissociated spinal cord was filtered through a 100 micron filter mesh (BD Biosciences) followed by a 40 micron filter mesh. The cells were spun at 1500 rpm for 5 min, resuspended in 10% Percoll (GE Healthcare) solution made in DMEM, applied to a discontinuous 15%/60% Percoll gradient in an Oak Ridge centrifugation tube (Nalgene), and centrifuged at 30,000 g for 30 minutes at 4°C in a fixed angle JA-20 rotor in a Beckman Coulter J2-MC centrifuge. The 15%/60% cloudy interface was removed, washed twice with 10 ml of DMEM and spun at 1500 rpm for 5 min at 4°C. The cells were then resuspended in DMEM with 10% fetal bovine serum (ThermoScientific) and 10 µg/ml gentamicin and cultured on 100 µg/ml poly-L-lysine (Sigma) coated 6 well plates (Corning). After two days, the cells were rinsed in 1X HBSS and cultured for 5 days with 30% B104 conditioned media (CM) and 70% serum-free media (SFM). B104 neuroblastoma cells were used as a source of soluble gliogenic factors (Bottenstein et al., 1988) and were obtained from Dr. David Schubert (Salk Institute). B104 cells were grown in DMEM with 10% fetal bovine serum and 10 µg/ml gentamicin. SFM is serum free media (1× DMEM) containing 25 µg/ml transferrin (Sigma), 30 nM triiodothyronine (T3; Calbiochem), 20 nM hyrdrocortisone (Sigma), 20 nM progesterone (Sigma), 10 nM biotin (Sigma), 1× Trace Element Mix B (Mediatech), 30 nM selenium (Sigma), 5 µg/ml insulin (Sigma), 1 µg/ml putrescine (Sigma), 0.1% BSA (Sigma), 10 µg/ml gentamicin. These progenitor cultures were trypsinized (0.05 % trypsin-EDTA, Gibco), split 1:3 and replated in 30% B104 CM/70% SFM for up to 5 passages. These small and bipolar progenitor cells label with A2B5. To produce mature oligodendrocytes, starting from a confluent plate of progenitor cells, the media was changed to SFM for a period of 7–10 days resulting in cells with large, flat processes (label with O4, O1, myelin basic protein, and galactocerebrosidase). Cultures were negative for glial fibrillary acidic protein (GFAP; astrocytes) and CD11b (microglia) immunoreactivity (Supplementary Figure 1).

Cell Survival

Cell survival was assessed by using the trypan blue exclusion assay in which dying cells, whose membrane integrity has been compromised, would be permeable to the trypan blue dye (Antony et al., 2004). Following treatment with injured astrocyte media, oligodendrocytes were trypsinized with 0.05% trypsin-EDTA (Gibco), centrifuged at 1,500 rpm, and resuspended in phosphate-buffered saline (PBS). Cells were stained with .4% Trypan blue, and counted with a hemacytometer.

Glutamate assay

Extracellular glutamate concentrations were determined using a colorimetric glutamate assay kit (BioVision) following the manufacturer’s instructions.

Zinc assay

Extracellular free zinc concentrations were determined using the Quantichrom Zinc Assay Kit (BioAssay Systems) following the manufacturer’s instructions.

Terminal deoxynucleotidyltransferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) staining

DNA fragmentation was determined by using fluorescent TUNEL staining with the Apoptag Apoptosis Detection Kit (Millipore) following manufacturer’s instructions.

Immunohistochemistry

Injured animals were perfused with 4% paraformaldehyde in 0.1 M PBS. A 1 cm segment (5mm rostral and 5mm caudal of the lesion epicenter; lesion site was identified by the contusion of the spinal cord induced by the impact injury) of the spinal cord was removed and post-fixed overnight. Tissues were placed in 0.1 M PBS containing 25% sucrose for cryoprotection and cut into 20 or 25 µm thick sections. Sections were stored in blocking buffer (5% normal goat serum in .4% Triton-X100 in PBS) for 1 hr at room temperature and were incubated at 4°C overnight in primary antibody [rabbit anti-glutathione-S-transferase π (GSTπ ; Millipore; 1:2000)]. Samples were incubated in secondary species-specific fluorescent antibodies (Alexa Fluor 594, 1:750; Molecular Probes, Eugene, OR) for 1 hr at room temperature. Slides were mounted using 4’, 6-diamidino-2-phenylindole (DAPI) mounting medium (Vectashield, Vector Laboratories, Burlingame, CA) and stored at -20°C. Sections lacking treatment of primary antibodies were used as negative controls.

Stereology

Serial 20–25 µM horizontal sections spaced 500 µm throughout the width of the spinal cord were counted using Stereo Investigator (MBF Biosciences) software with a 63× objective. For each section, a 40 × 40 µM counting frame was used to count cells within 200 µM2 area intervals. Investigator was blind to experimental groups during quantitation.

In vitro treatments

6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), L-glutamic acid, zinc chloride, and 1,10-phenanthroline (all Sigma) were dissolved in dH2O and used at the indicated concentrations.

Statistics

Statistical analysis was performed with one or two-way ANOVA, followed by appropriate post-test. Student’s T test was used for single comparisons. Error bars depicted indicate the standard error of the mean.

Results

Astroglial-NF-κB inactivation reduces oligodendrocyte death following SCI

SCI results in the phosphorylation and activation of the NF-κB transcription factor within the first hour after injury and phosphorylated levels continue to increase in a time-dependent manner (Bethea et al., 1998; Brambilla and Bracchi-Ricard et al., 2005). Inhibition of NF-κB activation within astrocytes using a transgenic mouse model (GFAP-IκBα-dn) significantly improves functional recovery and reduces white matter damage following SCI (Brambilla and Bracchi-Ricard et al., 2005). To determine if the reduction in white matter damage in GFAPI-κBα-dn mice could be due to reduced levels of oligodendrocyte death following SCI, spinal cord sections were double-labeled with TUNEL and the mature oligodendrocyte nuclear marker GSTπ (Figure 1) during the peak of oligodendrocyte death at 1 week after injury (Liu et al., 1997). Cells with nuclear staining positive for both labels were quantified using stereological principles to determine the number of oligodendrocytes undergoing cell death. To be certain that any potential changes in oligodendrocyte numbers would not be the result of developmental abnormalities due to expression of the GFAP-IκBα-dn transgene, total oligodendrocytes were quantified by GSTπ labeling (Figure 1C) in naïve WT and GFAP-IκBα-dn spinal cord sections. No significant differences in the total number of oligodendrocytes were detected (WT = 121,329 ± 2,413.4; GFAP-IκBα-dn = 122,061 ± 336.1). At one week following SCI, NF-κB inactivation (GFAP-IκBα-dn) resulted in a 50% reduction in the number of GSTπ/TUNEL double-labeled cells when compared to mice with functional NF-κB (WT) (Figure 1D).

Figure 1. Astroglial-NF-κB activation induces oligodendrocyte death following trauma.

Figure 1

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Spinal cord sections from (A) WT and (B) GFAP-IκBα-dn mice were labeled with TUNEL (marker for cell death) and the oligodendrocyte marker GSTπ to quantify the number of oligodendrocytes undergoing cell death at 1 week after injury. Scale bars = 100 µM. (C) Total GSTπ counts revealed no significant change in total number of oligodendrocytes between naïve WT (n=5) and GFAP-IκBα-dn (n=4) animals. (D) Oligodendrocyte death was assessed by quantifying the number of oligodendrocytes staining positive for TUNEL and GSTπ. Results are expressed as the number of cells labeled with both markers (n = 3–4). [*, p ≤ 0.05 compared to WT].

Astroglial NF-κB activation induces oligodendrocyte death in vitro

In order to test the direct contribution of injured astrocytes to oligodendrocyte death, we utilized an in vitro fluid percussion device to injure astrocytes cultured from WT or GFAP-IκBα-dn mice (Jayakumar et al., 2008). At various time points after injury, WT or GFAP-IκBα-dn astrocyte conditioned media was removed and added to WT oligodendrocytes cultured from the adult spinal cord in a 1:1 ratio with oligodendrocyte media for the corresponding time. Conditioned media from uninjured astrocytes was used as a control. Treatment of oligodendrocytes with media from uninjured astrocytes of either genotype resulted in no significant changes in cytotoxicity at any time point measured when compared to oligodendrocyte media alone (Figure 2). Following treatment with 1 hr post-trauma astrocyte media, there were no significant changes in oligodendrocyte cytotoxicity in either the WT (19.77 ± 4.07%) or GFAP-IκBα-dn (14.52 ± 2.82%) condition. Treatment with injured WT media resulted in a significant increase in oligodendrocyte toxicity over control levels at 3 hrs (52.7 ± 3.43%), 1 d (59.7 ± 2.95%), and 3 d (61.2 ± 3.38%) post-trauma. Importantly, we observed no change in oligodendrocyte death from control levels following treatment with media from injured GFAP-IκBα-dn astrocytes at any time point measured.

Figure 2. Injured astrocyte modulate oligodendrocyte death at different time points.

Figure 2

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Oligodendrocytes treated with (A) 1 hr post-injury astrocyte media for 1 hr (n = 6–9), (B) 3 hr post-injury astrocyte media for 3 hr (n = 6), (C) 1 day post-injury astrocyte media for 1 day (n = 9–12), (D) 3 day post-injury astrocyte media for 3 days (n = 6–9), (E) 1 hr post-trauma media for 3 hrs (n = 3–6), and (F) 3 hr post-trauma media for 1 hr (n = 4). Results are expressed as the percent cytotoxicity. OM = oligodendrocyte media [**, p ≤ 0.01, ***, p ≤ 0.001 compared to OM; #, p ≤ 0.05, ##, p ≤ 0.01, ###, p ≤ 0.001 compared to injured WT].

To determine if the failure of the 1 hr post-trauma media to induce oligodendrocyte death was due to the exposure time of the cultures to the injured media, 1 hr post trauma media was added to oligodendrocyte cultures for 3 hrs (Figure 2E). Treatment of oligodendrocytes for 3 hrs elicited similar results to the 3 hr, 1d, and 3d treatment conditions. Exposure of oligodendrocytes to injured astrocyte media produced no significant change in cytotoxicity in the GFAP-IκBα-dn (25.35 ± 4.44%) condition. Treatment of oligodendrocytes with injured WT astrocyte media induced a significant increase in oligodendrocyte death (52.07 ± 4.20%). In addition, treatment of oligodendrocytes with 3 hr post-trauma media for 1 hr failed to induce oligodendrocyte toxicity (Figure 2F). These results suggest that astrocytes release or deplete factors from the media that regulate oligodendrocyte survival within the first hour after injury, and it takes hours for oligodendrocyte death to occur following treatment with injured astrocyte media.

Astrocyte-induced oligodendrocyte death is mediated by ionotropic glutamate receptors

Stimulation of Ca2+-permeable AMPA receptors on oligodendrocytes induces a robust Ca2+ influx, which can result in glutamate-mediated excitotoxic cell death (Park et al., 2003; Grossman et al., 1999). Since one of the key functions of astrocytes is to regulate extracellular glutamate levels, we wanted to determine if trauma-induced oligodendrocyte cell death could be due to astrocyte-derived glutamate. In order to test this theory, oligodendrocytes were pre-treated with the AMPA receptor antagonist CNQX for 10 minutes prior to treatment with either injured WT or GFAP-IκBα-dn astrocyte media (Figure 3A). As previously demonstrated, treatment with 3 hr or 1 d post-injured WT astrocyte media induced a significant increase in oligodendrocyte cytotoxicity (65.9 ± 3.77% and 63.7 ± 4.70% respectively). Importantly, pre-treatment of oligodendrocyte cultures with CNQX was sufficient to return cell death to control levels in both the 3 hr and 1 d conditions (41.3 ± 4.99% and 34.6 ± 3.56%). Cultures treated with media from GFAP-IκBα-dn astrocytes again failed to induce any significant changes in oligodendrocyte death compared to controls. These results show that astrocytes with functional NF-κB signaling can directly induce oligodendrocyte death at multiple time points following trauma by activation of ionotropic glutamate receptors on oligodendrocytes.

Figure 3. Injured astrocytes induce oligodendrocyte death through a glutamate-mediated mechanism.

Figure 3

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(A) Pre-treatment of oligodendrocytes with 30 µM CNQX was able to reduce the astrocyte-induced cell death to control levels (n = 4–8). Results are expressed as percent cytotoxicity. OM = oligodendrocyte media [***, p ≤ 0.05 compared to OM; ##, p ≤ 0.01, ###, p ≤ 0.001 compared to injured WT media]. (B) Glutamate concentrations in media from uninjured or injured WT or IκBα-dn astrocyte media at 3hrs and 1 d (n = 5–8) (UAM = unconditioned astrocyte media). [*, p ≤ 0.05, ***, p ≤ 0.001 compared to UAM; #, p ≤ 0.05 compared to 3 hr groups; †††, p ≤ 0.001 compared to 1 d injured WT].

In order to assess whether differences in extracellular glutamate concentrations between WT and GFAP-IκBα-dn astrocyte media could account for the observed AMPA receptor mediated cell death, a colorimetric glutamate assay was performed on media from uninjured and injured astrocytes (Figure 3B). Surprisingly, there was no change in the levels of extracellular glutamate concentrations between uninjured and injured astrocytes of either genotype at the 3 hr time point. However, by 1 d post-injury GFAP-IκBα-dn astrocytes significantly reduced extracellular glutamate concentrations (96.9 ± 6.91 µM) from WT levels (161.0 ± 3.0 µM). Since there was no difference in glutamate levels between WT and GFAP-IκBα-dn cultures at 3 hrs after injury, extracellular glutamate concentrations alone could not account for the observed astrocyte-mediated oligodendrocyte death.

Injured astrocytes induce oligodendrocyte death by reducing extracellular zinc concentrations

In the presence of high levels of glutamate (200 µM), extracellular zinc has been shown to inhibit Ca2+ currents through AMPA receptors in a dose-dependent manner (Zhang et al., 2002). Therefore, we hypothesized that injured WT astrocyte media may have reduced extracellular zinc concentrations when compared to uninjured controls and injured GFAP-IκBα-dn astrocyte media. To test this hypothesis, a colorimetric zinc assay was performed on uninjured and injured astrocyte media (Figure 4A). There was no significant difference in extracellular zinc concentrations between uninjured WT and GFAP-IκBα-dn astrocyte media at either 3 hrs or 1 d; however, trauma resulted in a significant reduction in extracellular zinc levels in WT astrocyte media when compared to injured GFAP-IκBα-dn media and controls in both the 3 hr (WT: 25.3 ± 8.07 µM; GFAP-IκBα-dn media: 67.2 ± 9.8 µM) and 1 d (WT: 26.8 ± 5.01 µM; GFAP-IκBα-dn media: 56.5 ± 7.97 µM) conditions.

Figure 4. Reduced extracellular zinc concentrations in injured astrocyte media induce oligodendrocyte death.

Figure 4

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(A) Zinc assay detected a significant reduction in the levels of zinc present in injured WT astrocyte media at 3 hrs and 1 d (n = 4–6). [*, p ≤ 0.05 compared to uninjured controls and injured IκBα-dn media]. (B) Oligodendrocytes were treated with 1 µM 1,10-phenanthroline alone or in combination with injured GFAP-IκBα-dn astrocyte media. (n = 4–9). Zinc chelation of injured GFAP-IκBα-dn astrocyte media containing high levels of glutamate increased the levels of oligodendrocyte death. Results are expressed as the percent cytotoxicity. OM = oligodendrocyte media [**, p ≤ 0.01 compared to OM + Phenanthroline].

Here we show that injured astrocytes can induce oligodendrocyte death through activation of ionotropic glutamate receptors on oligodendrocytes. We also observed a reduction in extracellular zinc levels in injured WT astrocyte media, which in the presence of high extracellular glutamate could facilitate AMPA receptor activation on oligodendrocytes. To confirm that zinc can modulate oligodendrocyte death by injured WT astrocyte media, we reduced zinc levels in injured GFAP-IκBα-dn media by adding the zinc chelator 1,10-phaneanthroline prior to oligodendrocyte treatment (Figure 4B). If zinc chelation of injured GFAP-IκBα-dn media results in cell death equivalent to injured WT astrocyte media, we could conclude that injured astrocyte-mediated oligodendrocyte toxicity was due to reduced extracellular zinc levels. Since oligodendrocyte media is devoid of zinc, treatment with 1,10-phenanthroline alone caused no significant changes in cell death (28.8 ± 2.19%) when compared to oligodendrocyte media alone (29.4 ± 6.21%). Co-treatment of oligodendrocytes with injured GFAP-IκBα-dn astrocyte media and1,10-phenanthroline significantly increased oligodendrocyte toxicity (54.2 ± 2.80%). Importantly, oligodendrocyte toxicity was increased to levels equivalent to the injured WT astrocyte media condition (Figure 2). This data indicates that zinc can modulate oligodendrocyte survival following astrocyte trauma.

In order to confirm that zinc could functionally participate in glutamate-mediated oligodendrocyte death, oligodendrocytes were treated with the high concentration of glutamate measured in uninjured and injured astrocyte media (130 µM), in combination with increasing zinc concentrations (Figure 5). Oligodendrocytes in normal media were treated with glutamate (130 µM) in addition to various levels of zinc (0 – 100 µM). Since oligodendrocyte media lacks zinc, the addition of zinc to the media at different concentrations will demonstrate a dose dependent effect of zinc in regulating glutamate-mediated oligodendrocyte death. Cytotoxicity increased significantly in cultures treated with no or low zinc (0, 5, 10, and 25 µM) in combination with 130 µM glutamate. Importantly, cultures treated with glutamate plus the concentration of zinc measured in injured WT astrocyte media (25 µM) possessed significantly higher levels of oligodendrocyte death when compared to controls, indicating that the extracellular levels of glutamate and zinc found following astrocyte trauma are sufficient to induce oligodendrocyte death. Alternatively, oligodendrocytes co-treated with glutamate and with the higher concentration of zinc measured in injured GFAP-IκBα-dn and control media (50 µM) resulted in no significant increases in cell death over oligodendrocyte media control levels. In addition, zinc treatment alone was able to significantly decrease oligodendrocyte viability only at 100 µM. These results demonstrate that low extracellular zinc levels are not toxic to oligodendrocytes. However, the combination of low zinc levels and glutamate is sufficient to induce oligodendrocyte cytotoxicity through ionotropic glutamate receptor activation.

Figure 5. Astrocyte-induced oligodendrocyte death is mediated by glutamate in the presence of reduced extracellular zinc levels.

Figure 5

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Oligodendrocytes were treated with 130 µM glutamate along with varying levels of zinc (white bars). Gradual increases in extracellular zinc concentrations protected oligodendrocytes from high glutamate levels (n = 3). Zinc treatment alone induced oligodendrocyte death only at 100 µM (black bars). X-axis refers to the zinc concentration added to the oligodendrocyte media. Results are expressed as percent cytotoxicity. OM = oligodendrocyte media [*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001 compared to OM; #, p ≤ 0.05 compared to 0 µM Zinc].

Discussion

Using an in vitro trauma model to injure cultured astrocytes from WT and GFAP-IκBα-dn mice, we show that trauma-induced glutamate excitotoxicity in oligodendrocytes is regulated by changes in extracellular zinc concentration dependent upon NF-κB activation within astrocytes. Oligodendrocytes treated with injured WT astrocyte media were significantly susceptible to cytotoxicity when compared to controls and GFAP-IκBα-dn media conditions. Treatment of oligodendrocytes with the AMPA receptor antagonist CNQX prior to injured astrocyte media application was sufficient to attenuate oligodendrocyte death equivalent to control levels; thereby implicating glutamate in promoting oligodendrocyte toxicity in this model. Interestingly, there was no difference in the concentration of extracellular glutamate levels at 3 hrs after injury.

Within the first hour after SCI, the initial trauma induces a robust release of glutamate from damaged cells and axons within the lesion site (Liu et al., 1991; Liu et al., 1999; McAdoo et al., 1999; McAdoo et al., 2005). This finding suggests that cells within the lesion site would be subjected to glutamate excitotoxicity immediately after injury. Interestingly, peak oligodendrocyte death during the initial phase of injury does not occur until 4 hrs to 1 day after injury (Liu et al., 1997), suggesting that other factors may be involved in the excitotoxic cell death process.

Previous findings have demonstrated that alterations in extracellular zinc concentrations can modulate currents through the AMPA receptor in the presence of high extracellular glutamate levels (Zhang et al., 2002). In addition, low levels of zinc (< 35 µM) have been shown to induce cell death in cortical neuron cultures (Kim et al., 1999a; Lobner et al., 2000). Given the reports documenting a role for zinc in regulating glutamatergic signaling, we hypothesized that WT astrocyte media contained lower levels of zinc when compared to controls. Quantification of zinc concentrations from WT and GFAP-IκBα-dn astrocyte media showed significantly lower levels of zinc in media from injured WT astrocytes at 3 hrs and 1 d when compared to uninjured controls and injured GFAP-IκBα-dn astrocyte media.

In order to confirm of low zinc levels can enhance oligodendrocyte death in the presence of high glutamate levels, we treated oligodendrocytes with injured astrocyte media in combination with the zinc chelator 1,10-phenanthrolline. Alone phenanthroline had no effect on cytotoxicity. When oligodendrocytes were treated with injured GFAP-IκBα-dn astrocyte media along with phenanthroline, cell death was significantly elevated. We previously showed that injured GFAP-IκBα-dn astrocyte media did not significantly reduce cell survival. By chelating extracellular zinc in the injured GFAP-IκBα-dn astrocyte media, we were able to increase oligodendrocyte death to levels measured following treatment with injured WT astrocyte media. This data lends strong support for our hypothesis that reducing extracellular zinc concentrations in the presence of glutamate potentiates cytotoxicity.

To further confirm if high extracellular glutamate in combination with low zinc concentrations (as in injured WT astrocyte media) can induce oligodendrocyte death, oligodendrocytes were treated with 130 µM glutamate in addition to varying levels of zinc. In the presence of glutamate, oligodendrocyte death was potentiated in the presence of 0 – 25 µM zinc. In addition, co-treatment with 100 µM zinc induced cell death. It is important to note that treatment with 100 µM zinc alone was sufficient to increase oligodendrocyte toxicity. This result is consistent with the literature documenting that excessive levels of zinc (100 – 300 µM) can be cytotoxic, typically through the induction of necrotic mechanisms (Kim et al., 1999b). In the presence of elevated extracellular glutamate levels, zinc administration was able to reduce oligodendrocyte death, suggesting a role for zinc in regulating AMPA receptor signaling in oligodendrocytes.

This data was supported by previous findings demonstrating a dose-response of extracellular zinc in inhibiting currents through Ca2+-permeable AMPA receptors (Zhang et al., 2002). Zhang et al. (2002) showed that exposure to constant levels of glutamate potentiated currents through the AMPA receptor in the presence of low levels of zinc. As zinc levels were increased, AMPA receptor currents were attenuated. These results support our findings demonstrating an increase in glutamate-mediated oligodendrocyte death following treatment with injured astrocyte media containing low levels of zinc, as well as a protective effect of increasing zinc concentrations. These studies indicate a role for zinc in regulating glutamatemediated oligodendrocyte excitotoxicity after trauma.

In the current study, we provide evidence demonstrating that activation of the NF-κB transcription factor within astrocytes leads to a reduction in extracellular zinc levels. The method in which astrocytes regulate extracellular zinc levels after injury remains unclear, but previous studies have shown that zinc uptake can occur through voltage-gated Ca2+ channels, AMPA and N-methyl-D-aspartic acid receptors, and the solute light carrier 39 (SLC39) family of zinc transporters (Frederickson et al., 1988; Tonder et al., 1990; Weiss et al., 1993; Sensi et al., 1997; Weiss and Sensi, 2000; Eide, 2006). Studies investigating zinc cellular permeability have shown L-type voltage-gated Ca2+ channels as being the main factor influencing zinc uptake (Atar et al., 1995; Kim et al., 2000; Nolte et al., 2004). Pharmacological opening of L-type voltage-gated Ca2+ channels resulted in the rapid intracellular accumulation of zinc in astrocytes (Nolte, 2004), and expression of these channels is increase following traumatic brain injury (MacVicar, 1984; Westenbroek et al., 1998). Future studies will be able to better elucidate the potential link between astroglial NF-κB activation and zinc influx through L-type voltage-gated Ca2+ channels.

Although we provide evidence demonstrating the role of zinc in modulating oligodendrocyte death, we cannot disregard the possibility that other factors released by astrocytes may be playing a role in our model. We show that in the presence of high glutamate levels, reducing extracellular zinc concentrations is sufficient to induce oligodendrocyte death. Alternatively, oligodendrocytes are sensitive to changes in a variety of factors, including: cytokines (tumor necrosis factor-α and interleukin 1β), growth factors (nerve growth factor), and glutathione, many of which are secreted by astrocytes and regulated by the NF-κB transcription factor (Casaccia-Bonnefil et al., 1996; Juurlink, 1997; Takahashi et al., 2003; McTigue and Tripathi, 2008; Steelman and Li, 2011). Regardless of these potential contributing factors, we clearly present a causal role of glutamate/zinc dependent oligodendrocyte cell death following trauma. Future studies will be able to elucidate if the release of these factors from astrocytes plays a role in oligodendrocyte glutamate excitotoxicity.

This study demonstrates that oligodendrocyte excitotoxicity is mediated by astroglial NF-κB activation in astrocytes following trauma. The expression of functional NF-κB signaling resulted in a reduction in extracellular zinc levels, thereby inducing glutamate-induced cell death through activation of AMPA receptors. These findings describe novel targets responsible for oligodendrocyte death following trauma and identify potential therapeutic targets for treatment in central nervous system trauma and disease.

Supplementary Material

Supp Figure 1

Acknowledgments

Funding: This work was supported by National Institutes of Health grant R01-NS051709 (J.R.B.) and the Lois Pope LIFE Fellowship (J.T.J.).

Footnotes

The authors have no conflicting interests.

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