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Stroke/brain ischemia is a leading cause of death and long-term disabilities. Increased oxidative stress plays an important role in the pathology of brain ischemia. Hydrogen peroxide (H2O2) is a major oxidant known to cause neuronal injury; however, the detailed mechanism remains unclear. Previous studies have suggested that H2O2-induced injury is associated with increased intracellular Ca2+, mediated by glutamate receptors or voltage-gated Ca2+ channels. Here, we demonstrate that, at concentrations relevant to stroke, H2O2 induces a Ca2+-dependent injury of mouse cortical neurons in the absence of activation of these receptors/channels. With the culture medium containing blockers of glutamate receptors and voltage-gated Ca2+ channels, brief exposure of neurons to H2O2 induced a dose-dependent injury. Reducing [Ca2+]e inhibited whereas increasing [Ca2+]e potentiated the H2O2 injury. Fluorescent Ca2+ imaging confirmed the increase of [Ca2+]i by H2O2 in the presence of the blockers of glutamate receptors and voltage-gated Ca2+ channels. Addition of 2-aminoethoxydiphenyl borate, an inhibitor of transient receptor potential melastatin 7 (TRPM7) channels, or the use of TRPM7-small interference RNA, protected the neurons from H2O2 injury. In contrast, overexpressing TRPM7 channels in human embryonic kidney 293 cells increased H2O2 injury. Our findings indicate that H2O2 can induce Ca2+ toxicity independent of glutamate receptors and voltage-gated Ca2+ channels. Activation of TRPM7 channels is involved in such toxicity. Antioxid. Redox Signal. 14, 1815–1827.
Stroke or brain ischemia is a common neurological disorder. Unfortunately, after decades of active research, there is still no effective treatment for stroke patients other than the use of thrombolisis, which has very limited success. Searching for new cell injury mechanisms and therapeutic targets constitutes major challenge for stroke research. Oxidative stress, a cytotoxic consequence of mismatch between production of reactive oxygen species (ROS) and ability of cells to defend against them, has been implicated in neuronal loss associated with a variety of neurological disorders, including brain ischemia (6, 10, 39). ROS include oxygen-centered radicals possessing unpaired electrons such as superoxide anion (O•−2) and hydroxyl radical (OH•), or covalent molecules such as H2O2. O•−2 is generated in mitochondria by electron-transport process, in cytoplasm catalyzed by xanthine oxidase, or at plasma membrane by activation of phospholipase A2 and NADPH oxidase. H2O2 is formed from O•−2 spontaneously or catalyzed by superoxide dismutase. Highly reactive OH• can be formed from H2O2 by interacting with transitional metals.
A large number of studies have focused on the role of H2O2, one of the primary and the most stable ROS in vivo, in cell injury. In various cell culture models, brief incubation with H2O2 has been shown to induce delayed cell injury (15, 18, 46). Although the detailed mechanism is not fully understood, it has been demonstrated that H2O2-induced injury is associated with an increase in the concentration of intracellular Ca2+ ([Ca2+]i) (15, 18, 46), and the increase of [Ca2+]i by H2O2 involves an entry of Ca2+ from the extracellular space (18, 41).
Activation of voltage-gated Ca2+ channels and glutamate receptors is known to cause [Ca2+]i accumulation, and Ca2+-dependent neuronal injury (7, 44). Recent studies have indicated that the activities of voltage-gated Ca2+ channels can be enhanced by ROS (8, 27). For example, bath application of H2O2 increases the currents of cloned neuronal Ca2+ channels (27). Consistent with an involvement of voltage-gated Ca2+ channels in the effects of H2O2, H2O2-induced increase of [Ca2+]i in smooth muscle cells is reduced by the blockers of these channels (37).
In addition to voltage-gated Ca2+ channels, H2O2 may induce its biological effects through the N-methyl-d-aspartate (NMDA) subtype of glutamate receptors. Although free radicals have been shown to directly inhibit the NMDA currents (2), several studies have indicated that they could also facilitate the NMDA-mediated responses, likely through an increased release of glutamate. Two decades ago, Pellegrini-Giampietro and colleagues first demonstrated that ROS can stimulate the release of glutamate in rat hippocampal slices (36). Later on, Avshalumov and Rice showed that H2O2 exposure can cause activation of normally silent NMDA receptors, possibly via inhibition of redox-sensitive glutamate uptake (3). Mailly et al. also reported that H2O2-induced injury of mouse cortical neurons can be inhibited by (5R,10S)-(+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK801), a specific blocker for NMDA receptor-gated channels (31). Taken together, these findings suggest that secondary activation of voltage-gated Ca2+ channels and the glutamate receptors plays a role in H2O2-induced Ca2+ toxicity. It is, however, unclear whether H2O2 can induce Ca2+ toxicity independent of the activation of glutamate receptors and voltage-gated Ca2+ channels. This question is particularly important following the failure of glutamate antagonists for stroke intervention in clinical trials (4).
Transient receptor potential melastatin 7 (TRPM7) channels are Ca2+-permeable nonselective cation channels that belong to the TRP superfamily (11). They are ubiquitously expressed in almost all tissues and cell types. Activation of TRPM7 channels is important for cellular Mg2+ homeostasis (33). However, its activation is also implicated in Ca2+-mediated neuronal injury under ischemic conditions (1, 42). In addition, our recent study suggested a role for TRPM7 channels in Zn2+-mediated neuronal injury associated with brain ischemia (20). Thus, TRPM7 channels represent a novel therapeutic target for ischemic brain injury.
Here, we show that, at clinical relevant concentrations, H2O2 can induce substantial neuronal injury mediated by Ca2+ entry through a distinct, glutamate receptor and voltage-gated Ca2+ channels independent pathway. Activation of TRPM7 channels is responsible, at least partially, for such effect of H2O2.
Primary neuronal cultures were prepared from embryonic Swiss mice at 16 days of gestation according to previously described techniques (47). The protocol for neuronal culture using prenatal mouse brains was approved by the Institutional Animal Care and Use Committee of Legacy Research. Briefly, time-pregnant mice were anesthetized with halothane followed by cervical dislocation. Fetuses were rapidly removed and placed in cold Hanks' solution. The cerebral cortices from 10 to 12 embryos were dissected and incubated with 0.05% trypsin–ethylene diamine tetraacetic acid for 10min at 37°C, followed by trituration with fire-polished glass pipettes, and plated on poly-L-ornithine-coated 24-well culture plates or 25mm glass coverslips at a density of 2.5×105 cells per well and 0.5×106 cells per coverslip. Neurons were cultured in the Neurobasal medium supplemented with B27 and maintained at 37°C in a humidified 5% CO2 atmosphere incubator. Toxicity studies were performed at 12–14 days after plating; 5μM 5-fluoro-2-deoxyuridine and 5μM uridine were normally added to the cultures 72h after plating for 2 days to suppress the growth of glial cells. This produces cultures in which ~80% of cells are neurons, as assessed by immunofluorescent staining with the neuron-specific marker neuronal nuclei and the glial-specific marker glial fibrillary acidic protein (not shown).
Neurons were washed three times with fresh, antioxidant-free, Neurobasal medium (Invitrogen), and randomly divided into control and treatment groups. Dilutions of H2O2 were made fresh from 30% stock solution into the Neurobasal medium before each experiment. Exposures to H2O2 were accomplished by incubating cultures with H2O2 for the duration indicated at 37°C in 5% CO2 incubator. Individual wells were then washed three times with the fresh medium and the cultures were incubated at 37°C in an incubator. Unless otherwise specified, the antagonists of glutamate receptors (10μM MK801, 20μM 6-cyano-7-nitroquinoxaline-2,3-dione [CNQX]) and the blocker of L-type Ca2+ channels (5μM nimodipine) were always present in the culture medium during H2O2 exposure. In some cases, they were also present in the medium after H2O2 exposure (see Results).
Quantitative assessment of cell injury was performed by measurement of lactate dehydrogenase (LDH) released into the culture medium as described in our previous studies (47). The LDH value was determined spectrophotometrically using the LDH assay kit (Roche Molecular Biochemicals). Fifty-microliter medium was transferred from each well to a 96-well plate and mixed with 50μl reaction solution. Optical density at 492nm was measured 30min after the mixing utilizing a microplate reader (Spectra Max Plus; Molecular Devices).
Cell viability was determined by simultaneous staining with fluorescein-diacetate (FDA) and propidium iodide (PI) as described previously (47). For staining of live and dead neurons, cultures were incubated in the extracellular solution containing FDA (5μM) and PI (2μM) for 30min, followed by wash three times with dye-free extracellular solution. Live (FDA-positive) and dead (PI-positive) cells were viewed and counted with a fluorescent microscope (Zeiss) at excitation/emission wavelengths of 580nm/630nm for PI, and 500nm/550nm for FDA. Viable cells fluoresce bright green, whereas nonviable cells (nucli) are bright red. Images were collected using an Optronics DEI-730 3-chip camera equipped with a BQ 8000 sVGA frame grabber and analyzed using computer software (Bioquant).
Fluorescent Ca2+-imaging was performed as previously described (47). Cortical neurons grown on 25mm glass coverslips were incubated with 5μM fura-2-acetoxymethyl ester for 40min at room temperature, followed by wash three times and incubated in normal extracellular solution for at least 30min before imaging. Coverslips with fura-2-loaded cells were then transferred to a perfusion chamber on an inverted microscope (TE300; Nikon). Cells were illuminated using a xenon lamp (75W) and observed with a 40× UV fluor oil-immersion objective lens. Video images were obtained using a cooled charged-couple device camera (Sensys KAF 1401; Photometrics). Digitized images were acquired, stored, and analyzed in a computer controlled by Axon Imaging Workbench software (AIW2.1; Axon Instruments). The shutter and filter wheel (Lambda 10-2) were also controlled by AIW to permit timed illumination of cells at either 340 or 380nm excitation wavelengths. Fura-2 fluorescence was detected at an emission wavelength of 510nm. Background-subtracted 340/380 ratio images were analyzed by averaging pixel ratio values in circumscribed regions of cells in the field of view. The values were then exported from AIW to Sigma Plot for further analysis and plotting.
DNA fragmentation in apoptotic cells was observed by terminal deoxyribonucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate (dUTP)-biotin nick end labeling (TUNEL). Cultures were air-dried, fixed with 10% formalin for 15min, washed three times in PBS, and permeabilized with 1% Triton X-100 for 20min. Neurons were subsequently incubated with the reaction mixture containing fluorescein isothiocyanate–dUTP and 300U/ml terminal deoxy-transferase for 90min at 37°C. Cultures were then mounted with 4′,6′-diamidino-2-phenylindole containing media (Vector Labs) and viewed with fluorescent microscope at an excitation/emission wavelength of 500/550nm (green) for fluorescein isothiocyanate–TUNEL-labeled cells.
To construct the plasmid for silencing mouse TRPM7, two oligonucleotides were annealed and inserted into pSilencer 1.0-U6 (Ambion) according to manufacturer's instruction. RNA directed to nucleotides 5152–5172 of cording region of TRPM7 (GenBank accession number NM021450) (20). A fragment cut with BamHI was excised and inserted into BamHI site of pCAGGS-enhanced green fluorescent protein (eGFP) (kindly provided by Dr. J. Miyazaki; Division of Stem Cell Regulation Research, Osaka University Medical School, Osaka, Japan) to express both eGFP and shRNA. For the negative control, a fragment cut with BamHI from pSilencer 1.0-U6 was inserted into pCAGGS-eGFP. For transfection, NeuroFect (Genlantis) was used for cortical neurons at DIV 8 in accordance with the manufacturer's instruction.
H2O2, MK801, CNQX, nimodipine, benzamil, dimethyl sulfoxide (DMSO), dithiothreitol (DTT), and 2,2′-dithiobis-5-nitropyridine (DTNP) were purchased from Sigma Chemical Co. 2-(2-(4-(4-Nitrobenzyloxy)phenyl)ethyl)isothiourea (KB-R7943) was purchased from Tocris. H2O2 was freshly prepared from 30% stock just before each experiment. DTNP was dissolved in acetone before it was added into the culture medium. The final concentration of acetone (3%) was tested to be ineffective (see Results).
All data are expressed as mean±SEM. Student's t-test or ANOVA was employed to examine the statistical significance. The criterion of significance was set at p<0.05.
To determine whether H2O2 can induce neuronal injury independent of the activation of glutamate receptors and voltage-gated Ca2+ channels, neurons were treated with H2O2 in the presence of the blockers of glutamate receptors and the voltage-gated Ca2+ channels. Twelve to 14 days after plating, mouse cortical neurons grown in 24-well culture plates were treated with H2O2 in the presence of 10μM MK801, 20μM CNQX, and 5μM nimodipine. In some cases, 3μM ω-conotoxin MVIIA, a nonselective blocker for N-, P/Q-, and R-type Ca2+ channels, was also added (see below). Cell injury was assayed by the measurement of LDH released into the culture medium at various time points and normalized to the maximal releasable LDH in each well. To obtain the maximal amount of releasable LDH in each well, 1% triton X-100 was added, at the end of each experiment, to permeabilize the cell membrane. Percentage of total LDH release was presented. As shown in Figure 1a, 1h incubation of neurons with H2O2 in the presence of MK801, CNQX, and nimodipine induced a dose-dependent cell injury, with a threshold concentration of ~10μM H2O2. The relative LDH release, measured at 24h after 1h H2O2 incubation, was 0.11±0.01, 0.11±0.02, 0.45±0.05, 0.50±0.02, 0.55±0.02, and 0.60±0.05 for 0μM (control), 10, 30, 50, 100, and 300μM H2O2, respectively (n=7–8 wells). These data indicate that H2O2 can induce a significant neuronal injury independent of the activation of glutamate receptors and L-type Ca2+ channels. Addition of high concentration of ω-conotoxin MVIIA (3μM), a nonselective blocker for N-, P/Q-, and R-type Ca2+ channels, did not affect H2O2-induced LDH release, indicating that activation of these channels is not responsible for H2O2-induced glutamate-independent neuronal injury (n=4, not shown). Previous studies have shown that, after brain ischemia, the concentration of H2O2 in the brain can reach above 100μM (19). Therefore, the concentrations used here are pathophysiologically relevant.
In rat hippocampal slice, Avshalumov and Rice have shown that the activity of NMDA receptors is enhanced after washout of H2O2 (3). To exclude the possibility that activation of the NMDA receptors after H2O2 exposure is involved in H2O2-induced injury, in a separate experiment, MK801, CNQX, and nimodipine were present both during and after H2O2 exposure. This measure, however, did not reduce H2O2-induced LDH release (n=4, not shown), suggesting that a subsequent activation of the glutamate receptors after washout of H2O2 is not responsible for the injury of cultured mouse cortical neurons.
We then determined the duration of H2O2 incubation required to induce neuronal injury. Neurons were incubated with the medium containing 100μM H2O2 for 10min, 30min, 1h, 6h, or 24h. LDH release was measured at 6 and 24h after the start of H2O2 treatment. As shown in Figure 1b, 10min incubation of neurons with 100μM H2O2 induced a similar proportion of neuronal injury as 1 or 6h treatment. Six hours after the start of H2O2 incubation, for example, relative LDH release of 0.4±0.03, 0.38±0.02, 0.49±0.06, and 0.46±0.06 were detected for 10min, 30min, 1h, and 6h H2O2 exposures (n=4 wells for each group). At 24h after the start of H2O2 incubation, relative LDH release of 0.62±0.06, 0.57±0.04, 0.65±0.06, and 0.55±0.07 were detected for 10min, 30min, 1h, and 24h H2O2 exposure (n=4 wells for each group). No statistically significant differences were detected in LDH release induced by 10min, 30min, 1h, or continuous H2O2 treatment. These data indicate that a brief incubation (e.g., <10min) with H2O2 is sufficient to induce substantial delayed, glutamate-independent neuronal injury. This finding is consistent with previous reports that brief (>5min) incubations with H2O2 caused a significant loss of viability of cultured rat forebrain and cortical neurons (15, 45). Since a substantial neuronal injury is detected at 6h after H2O2 exposure and the background LDH release is very low at this time point, for the rest of experiments we focused on the effects of H2O2 at 6h time point.
Intracellular Ca2+ accumulation plays an important role in neuronal injury associated with brain ischemia. To determine whether the H2O2-induced glutamate receptor and voltage-gated Ca2+ channel-independent neuronal injury depends on the entry of Ca2+ into neurons, membrane impermeable Ca2+ chelator ethylene glycol tetraacetic acid (EGTA) (2mM) was added to the culture medium to reduce the concentration of the extracellular Ca2+ in the medium. After the addition of EGTA, which lowers pH, pH was readjusted back to 7.3 by titration with NaOH. With 2mM EGTA and a total Ca2+ of 1.8mM in the medium, a free Ca2+ concentration of ~0.8μM was calculated (38). To determine the effect of extracellular Ca2+ on H2O2-induced neuronal injury, neurons were exposed to 100μM H2O2 for 1h in the absence and presence of EGTA. As shown in Figure 2a, addition of 2mM EGTA in the culture medium dramatically reduced H2O2-induced relative LDH release from 0.43±0.05 to 0.20±0.03 (n=4 wells in each group, p<0.01). Addition of the same concentration of EGTA in the control medium did not affect the background LDH readings (0.075±0.01 vs. 0.080±0.01, n=4 wells in each group, p>0.05), suggesting that the decrease of LDH release by EGTA was due to its inhibition of H2O2-induced neuronal injury. In contrast to EGTA, adding Ca2+ to the culture medium potentiated the H2O2-induced neuronal injury (Fig. 2b). In the control medium containing 1.8mM Ca2+, 1h incubation with 100μM H2O2 induced a relative LDH release of 0.39±0.04 (n=8). With the culture medium containing 2.8, 3.8, or 6.8mM Ca2+, 1h incubation with 100μM H2O2 induced 0.45±0.03, 0.54±0.05, and 0.71±0.09 of relative LDH release (n=8 for each group, p<0.05 between control and 3.8mM Ca2+ group; p<0.01 between control and 6.8mM Ca2+ group). The medium with 2.8 or 3.8mM Ca2+ in the absence of H2O2 did not show different background LDH readings compared with the medium containing 1.8mM Ca2+, though the medium containing 6.8mM Ca2+ showed a slightly increased background LDH of 0.11±0.02 (n=8). Together, these data suggest that H2O2-induced glutamate and voltage-gated Ca2+ channel-independent neuronal injury likely involves an entry of extracellular Ca2+ into neurons. Addition of EGTA did not completely prevent the H2O2 injury (Fig. 2a), indicating that Ca2+ entry-independent mechanism may be partially responsible for H2O2-induced glutamate receptor and voltage-gated Ca2+ channel-independent neuronal injury.
In addition to LDH release, H2O2-induced injury was also studied by fluorescent staining of live (FDA-positive) and dead cells (PI-positive) (21). Neurons were cultured on 25mm coverslips and used for the studies 12 days after plating. Six hours after H2O2 treatment, PI (2μM) and FDA (5μM) were added into the culture medium for 30min before observation with fluorescent microscope for PI- or FDA-positive cells. As shown in Figure 3, treatment with H2O2 (100μM, 1h), in the presence of MK801, CNQX, and nimodipine induced a significant increase in PI-positive cells. Addition of 2mM EGTA substantially decreased the total number of PI-positive cells (Fig. 3). Six hours after H2O2 incubation, the percentages of PI-positive cells were 4.3%±1.0%, 68.2%±3.8%, and 28.5%±7.6% for control, H2O2 alone, and H2O2 in the presence of 2mM EGTA (six to eight fields from 2 to 3 coverslips in each group, p<0.01 between H2O2 alone and H2O2 with 2mM EGTA).
In L929 cells, H2O2-induced Ca2+ toxicity involves Ca2+ entry through Na+-Ca2+ exchange, operated in the reversed mode (22). To determine whether a similar mechanism is involved in H2O2-induced glutamate receptor-independent injury of cultured mouse cortical neurons, we tested the effect of KB-R7943, a selective blocker for the reversed Na+-Ca2+ exchanger, on H2O2-induced neuronal injury; 10μM KB-R7943 was added into culture wells 10min before and during the treatment with 100μM H2O2. As shown in Supplementary Figure 1 (Supplementary Data are available online at www.liebertonline.com/ars), addition of KB-R7943 had little effect on H2O2-induced neuronal injury. The relative LDH release at 6h after 1h H2O2 exposure was 0.41±0.02 in the absence of KB-R7943 and 0.44±0.04 in the presence of 10μM KB-R7943 (n=4 and 8, p>0.05). Similarly, addition of benzamil (100μM), a common blocker for Na+-Ca2+ and Na+-H+ exchange, did not affect H2O2-induced neuronal injury. In the presence of benzamil, the relative LDH release induced by H2O2 was 0.42±0.03 (n=4, p>0.05 compared to H2O2 treatment alone). Together, these data indicate that activation of Na+-Ca2+ exchange system is not responsible for H2O2-induced glutamate receptor-independent injury of cultured mouse cortical neurons.
Oxidization of sulfhydryl groups on cysteine residues has been implicated in many physiological or pathological effects of ROS. To know whether H2O2-induced glutamate receptor-independent injury of mouse cortical neurons involves an oxidization of the sulfhydryl groups, we tested the effect of DTT, a thiol-reducing agent (5), on H2O2-induced neuronal injury. As shown in Figure 4a, addition of DTT during H2O2 exposure produced a concentration-dependent reduction of H2O2-induced neuronal injury. In the absence of DTT, 1h incubation with 100μM H2O2 induced a relative LDH release of 0.39±0.07 at 6h. However, in the presence of 1.0 or 2.0mM DTT, the same concentration of H2O2 induced a relative LDH release of only 0.30±0.03 or 0.16±0.01, respectively (n=4 wells each, p<0.05 between H2O2 alone and H2O2 with 2mM DTT). One hour treatment with either 1.0 or 2.0mM DTT alone without H2O2 did not affect the background LDH release (n=4). This finding suggests that H2O2-induced glutamate receptor-independent neuronal injury likely involves the oxidization of sulfhydryl groups on cysteine residues. An alternative explanation could be that DTT reacts with H2O2 directly, thus attenuating the effect of H2O2. To provide further evidence that H2O2-induced glutamate receptor-independent neuronal injury involves the oxidization of sulfhydryl groups, we determined whether incubation of neurons with DTNP, a lipophilic cysteine-specific oxidizing agent, can induce similar injury as H2O2. As shown in Figure 4b, 1h treatment of neurons with 100μM DTNP in the presence of MK801, CNQX, and nimodipine induced a 0.39±0.03 of relative LDH release at 6h (n=8 wells, p<0.01 compared with the control group), providing further evidence that oxidization of sulfhydryl groups on cysteine residues might be involved in H2O2-induced glutamate-independent neuronal injury.
H2O2 may induce its biological effect through a direct oxidation of its substrate, or indirectly through its reactive bi-product OH• (13). To know whether H2O2-induced glutamate receptor-independent neuronal injury involves the formation of OH•, the effect of DMSO, a scavenger for OH• (48), on H2O2-induced neuronal injury was examined. As shown in Supplementary Figure 2a, addition of 0.1%–2.0% DMSO (~1.5 to 35mM) to the medium did not affect H2O2-induced glutamate receptor-independent LDH release. In the absence of DMSO, 1h incubation with 100μM H2O2 induced a relative LDH release of 0.49±0.09 at 6h (n=4). In the presence of 0.1%, 0.5%, or 2% of DMSO, the relative LDH release was 0.46±0.04, 0.47±0.09, or 0.44±0.03, respectively (n=4 wells in each group, p>0.05 between control and different DMSO groups).
Since the formation of OH• from H2O2 requires the presence of transition metal iron, we have also examined the effect of iron chelating agent deferoxamine (DFO) on H2O2-induced neuronal injury; 100μM DFO was added into culture wells 10min before and during H2O2 incubation. As shown in Supplementary Figure 2b, addition of DFO did not provide any protection against the H2O2 toxicity. In the absence of DFO, 1h incubation with H2O2 induced a relative LDH release of 0.39±0.03 at 6h. In the presence of 100μM DFO, the relative LDH release was 0.45±0.02 (n=8 in each group, p>0.05). Together, these data suggest that the formation of OH• is not required for H2O2-induced glutamate receptor-independent neurotoxicity in cultured mouse cortical neurons.
To provide a direct evidence that H2O2 can induce an increase of [Ca2+]i independent of the activation of glutamate receptors and voltage-gated Ca2+ channels, fura-2 fluorescent Ca2+-imaging was performed to determine whether application of H2O2 can produce an increase of [Ca2+]i in the presence of the blockers for glutamate receptors and L-type Ca2+ channels. As shown in Figure 5, in the presence of MK801, CNQX, and nimodipine, perfusion of 100μM H2O2 increased the 340/380 ratio from 0.73±0.11 to 1.94±0.26 within 10min (n=9, p<0.01). This increase of [Ca2+]i can be inhibited by removing the extracellular Ca2+ (Fig. 5). With no added Ca2+ in the extracellular solution, perfusion of 100μM H2O2 for 10min only increased the 340/380 ratio from 0.48±0.02 to 0.63±0.04 (n=12). The slight increase of [Ca2+]i by H2O2 in the absence of added Ca2+ could be due to residual contaminating Ca2+ in the extracellular solution. Together, these data suggest that a glutamate receptor and voltage-gated Ca2+ channel-independent Ca2+ entry pathway is activated by H2O2.
H2O2 treatment, in the absence of glutamate receptor antagonists, has been shown to induce apoptotic cell death (15, 45). Since neuronal injury by activation of glutamate receptors also involves an apoptotic process (26), it is not clear whether H2O2 can also induce apoptotic cell injury when glutamate receptors are blocked. For this reason, we have examined whether H2O2 treatment can induce apoptosis in the presence of the blockers of glutamate receptors and voltage-gated Ca2+ channels. Neurons grown on 25mm coverslips were treated with H2O2 (100μM, 1h) in the presence of 10μM MK801, 20μM CNQX, and 5μM nimodipine. TUNEL staining was performed 6h after H2O2 exposure. As shown in Figure 6, H2O2 treatment induced a substantial increase in TUNEL-positive cells. Chelating the extracellular Ca2+ by addition of 2mM EGTA in the medium significantly reduced the total number of TUNEL-positive cells induced by H2O2. The percentage of TUNEL-positive cells was 3.2%±1.4%, 30.3%±1.9%, and 15.1%±2.2% for control, H2O2 alone, and H2O2 in the presence of 2mM EGTA (n=6–7 fields from three coverslips in each group, p<0.01 between control and H2O2 groups, H2O2 alone and H2O2/2mM EGTA groups). In contrast, adding 2mM Ca2+ into the medium (final concentration: 3.8mM) increased the total number of TUNEL-positive staining (data not shown). Together, these data indicate that H2O2 can induce apoptotic cell injury independent of the activation of glutamate receptors, and that the Ca2+ entry from extracellular space is involved in such injury. The finding that the percentage of TUNEL-positive staining (Fig. 6), which is an indication of apoptotic cell injury, is lower than the PI-positive staining (Fig. 3), which detects both necrotic and the late phase of apoptotic cell injury, suggests that both necrotic and apoptotic cell injury processes are involved in H2O2-induced glutamate receptor-independent injury of mouse cortical neurons.
Next, we investigated the potential Ca2+-entry pathways responsible for H2O2-induced glutamate receptor-independent cell injury. We have recently demonstrated that activation of TRPM7, a Ca2+-permeable nonselective cation conductance, is involved in prolonged hypoxia-induced glutamate receptor-independent neuronal injury (1). It was also suggested that increased production of reactive nitrogen species by hypoxia, for example, nitric oxide (NO), mediates the activation of TRPM7 channels and the resultant neuronal injury. Unlike NO, however, H2O2 was less involved (1).
Recent studies also recognized the importance of TRPM2 channels, a nonselective cation channel activated by ADP-ribose, in H2O2-induced responses in some cell types (14). For example, transfection of human embryonic kidney (HEK) cells with rat TRPM2 increased H2O2-induced injury (23). It has also been shown that injury of rat cortical neurons by high concentrations of H2O2 (e.g., 1mM) is reduced by small interference RNA (siRNA)-TRPM2 (23). To determine whether H2O2-induced glutamate-independent injury of mouse cortical neurons at physiologically relevant concentrations involves the activation of TRPM2 or TRPM7 channels, we first determine whether addition of 2-aminoethoxydiphenyl borate (2-APB), a commonly used nonspecific blocker for TRPM7 (28) and TRPM2 channels (43), can inhibit H2O2-induced injury of mouse cortical neurons. As shown in Figure 7, addition of 30μM 2-APB, a concentration known to completely inhibit the TRPM2 current (43) but has minor effect on the TRPM7 current (28), did not provide significant protection against H2O2-induced injury. Increasing 2-APB to 100μM, however, significantly reduced H2O2-induced neuronal injury (Fig. 7), indicating that activation of TRPM7 channels might be involved in H2O2-induced glutamate receptor and voltage-gated Ca2+ channel-independent neuronal injury. Neuronal injury induced by higher concentration of H2O2 (500μM) was not protected by 100μM 2-APB, suggesting that nonphysiologically high concentrations of H2O2 may activate additional cell injury mechanisms, for example through nonspecific lipid peroxidation.
Consistent with a lack of TRPM2 involvement in H2O2-induced injury of mouse cortical neurons, incubation of cells with N-(p-amylcinnamoyl)anthranilic acid, another inhibitor of TRPM2 channels (25), did not have significant effect on H2O2-induced cell injury (Fig. 8a, n=8 wells).
To further determine whether activation of TRPM7 channels might be involved in H2O2-mediated cell injury, we questioned whether increasing expression of TRPM7 channels increased the sensitivity of cells to H2O2. Due to limited success in transfecting native neurons with plasmid encoding TRPM7 channels, we used a HEK293 cell line with inducible expression of TRPM7 channels (20, 34). We compared the degree of H2O2-induced injury of HEK293 cells with and without overexpression of TRPM7 channels. HEK293 cells stably transfected with Flag-murine TRPM7/pCDNA4-TO construct (34) were grown in 24-well culture plates with DMEM supplemented with 10% fetal bovine serum, blasticidin (5μg/ml), and zeocin (0.4mg/ml). TRPM7 expression was induced by adding 1μg/ml tetracycline to the culture medium, as described in our previous studies (20). Induced expression of TRPM7 was confirmed by Western blot (20). Forty-eight hours after the induction of TRPM7 expression, H2O2-induced cell injury was analyzed. We expected that, if activation of TRPM7 was involved in H2O2-mediated cell injury, increased expression of these channels would make them more sensitive to H2O2-induced increase of LDH release. As shown in Figure 8b, without overexpression of TRPM7 channels, HEK293 cells were relatively resistant to H2O2 injury as shown by small nonsignificant increase of LDH release. However, after induction of TRPM7 expression, incubation of cells with 100μM induced large increase of LDH release (n=15–16). These data further suggest that TRPM7 channels play an important in mediating H2O2-induced glutamate-independent cell injury.
To provide more convincing evidence that TRPM7 channels are required for H2O2-mediated glutamate-independent injury of neurons, we determined whether knockdown the expression of TRPM7 channels with TRPM7-siRNA affects the H2O2-induced neuronal injury. At 8 days after culture, mouse cortical neurons were transfected with TRPM7-siRNA-GFP or control-siRNA-GFP, as described in our previous studies (20). H2O2-induced injury was analyzed 3 days after transfection. Live/dead cells were counted based on the morphology of cells and confirmed by PI staining. To minimize variations, individual cells were followed through before and after H2O2 treatment. In 11 neurons (from three separated cultures) transfected with TRPM7-siRNA-GFP, all of them stayed alive 6h after 1h incubation with 100μM H2O2 (Fig. 9a). In contrast, in 13 neurons transfected with control-siRNA-GFP, only 7 stayed alive (Fig. 9b, p<0.05, Chi square test).
Intracellular Ca2+ accumulation plays a critical role in the pathology of brain ischemia. Ca2+ entry through glutamate receptors, particularly the NMDA subtype of the glutamate receptors, has been considered as the main source of [Ca2+]i accumulation associated with ischemic brain injury (7). Unfortunately, clinical trials failed to show a satisfactory neuroprotection by the antagonists of glutamate receptors (16). Although multiple factors may have contributed to the failure of the trials, emerging new studies also support an involvement of glutamate receptor-independent Ca2+ entry pathways, for example, TRPM7 channels and acid-sensing ion channels, in ischemic brain injury (1, 42, 47).
The objective of the present study was to determine whether H2O2, the most stable and one of the primary ROS in vivo, induces a glutamate receptor-independent Ca2+ toxicity. Using cultured mouse cortical neurons, we studied the H2O2-induced cell injury in the presence of the blockers of glutamate receptors and voltage-gated Ca2+ channels. We demonstrated that brief exposures to H2O2, at clinical relevant concentrations, induced substantial neuronal injury independent of the activation of glutamate receptors and voltage-gated Ca2+ channels. We further demonstrated that such effect of H2O2 involves an entry of Ca2+ from the extracellular space. This was supported by the finding that chelating the extracellular Ca2+ reduced, whereas increasing the Ca2+ potentiated, neuronal injury. Consistent with activation of a Ca2+ entry pathway, fluorescent Ca2+-imaging demonstrated a H2O2-induced increase of [Ca2+]i in the presence of the blockers of glutamate receptors and voltage-gated Ca2+ channels, and that the increase of [Ca2+]i was diminished with extracellular solutions containing no added Ca2+. More importantly, we demonstrated that H2O2-induced neuronal injury was inhibited by TRPM7 blockade or TRPM7-siRNA, which supports the involvement of TRPM7 channels.
TRPM7 is a member of the large TRP channel superfamily expressed in almost every tissue and cell type (9). The TRP superfamily of ion channels are divided into six subfamilies according to their sequence homology: TRPC, TRPM, TRPV, TRPP, TRPML, and TRPA (9). TRPM subfamily of TRP channels has eight members, TRPM1–8. Different members of the TRPM subfamily appear to have different gating and regulatory mechanisms, along with different ion selectivity and expression patterns. Increasing evidence suggests that activation of TRPM7 channels contributes to various physiological and pathophysiological processes. Notably, we demonstrated that activation of TRPM7 channels by oxygen-free radicals plays a critical role in hypoxia-induced glutamate-independent neuronal injury (1).
Previous studies have indicated activation of nonselective conductance by H2O2 (32, 41). However, the molecular identity of the conductance was unclear. Mendez and Penner demonstrated that conditions that mimic oxidative stress, for example, exposure to ultraviolet light or direct perfusion of H2O2 activated a nonselective cation current in several mammalian cell lines, including RBL, mast, HEK, PC12, and 3T3 cells (32). The H2O2-activated current demonstrated no voltage dependency and little selectivity among monovalent cations with substantial Ca2+ permeability. In rat striatal neurons, Smith and colleagues reported that, at nonphysiological high concentrations (>10mM), H2O2 activated a Ca2+-permeable nonselective cation channel with a single-channel conductance of 70–90pS (41). Activation of this conductance was suggested to be responsible for the injury of a sub-population of striatal neurons by H2O2. Since the concentrations of H2O2 used in our studies are ~100 times lower than that required to activate the cation conductance in striatal neurons (41), it is not clear whether the same channel was activated in both studies.
Recent studies by Kaneko et al. have suggested an involvement of TRPM2 channels in H2O2-induced neuronal injury (23). They demonstrated that, in rat cortical neurons, high concentrations of H2O2 (e.g., 1mM) induced cell injury that was attenuated by siRNA-TRPM2 (23). Our studies suggest that, at physiologically relevant concentrations, H2O2-induced glutamate-independent injury of mouse cortical neurons does not involve the activation of TRPM2 channels. This was supported by the finding that addition of 30μM 2-APB, a concentration known to completely inhibit the TRPM2 current (43), did not provide significant protection against H2O2-induced neuronal injury, and that addition of N-(p-amylcinnamoyl)anthranilic acid, another inhibitor of TRPM2 channels (25), had little effect on H2O2-induced neuronal injury.
We have demonstrated previously that activation of TRPM7, a nonselective cation conductance with high Ca2+ permeability, is involved in prolonged hypoxia-induced neuronal injury (1). We further showed that, an increased production of NO was likely responsible for the activation of TRPM7 channels (1). Unlike NO, however, H2O2 was less involved (1). The lack of clear involvement of H2O2 in hypoxia-induced glutamate-independent neuronal injury maybe due to a lack of sufficient production of H2O2 in the cell culture condition. In the present studies, we show that incubation of neurons with exogenous H2O2, at concentrations relevant to brain ischemia (19), produced substantial neuronal injury independent of glutamate receptors and voltage-gated Ca2+ channels, and that such H2O2-induced neuronal injury was inhibited by TRPM7 blockade or TRPM7 knockdown.
TRPM7 channels are highly expressed in brain cells (1, 42). Although a specific agonist for this channel has not been identified, various biochemical changes associated with brain ischemia, for example, reduced extracellular Ca2+, decreased cellular ATP, and increased production of NO, can facilitate the opening of these channels (1, 11). Our present study discloses a new activator for TRPM7 channels. In addition to being activated by these changes, one recent study has shown that expression of TRPM7 channels is also increased after brain ischemia, and that reduced expression is associated with neuroprotection by electric acupuncture (50). Therefore, TRPM7 channels may represent a novel, glutamate receptor-independent, Ca2+ entry pathway responsible for ischemic brain injury.
In addition to Ca2+, TRPM7 channels have substantial Zn2+ permeability (33). Our recent studies suggested that entry of Zn2+ through these channels plays an important role in Zn2+-mediated neuronal cell death (20). Therefore, activation of these channels likely contributes to both Ca2+ and Zn2+ toxicity associated with brain ischemia. A recent study by Hwang and colleagues has shown an increase of intracellular Zn2+ associated with H2O2-mediated neuronal injury (17). Although Zn2+ entry from glutamate receptors and/or release from Zn2+-binding proteins could contribute to the increase of [Zn2+]i, our present studies suggest that activation of TRPM7 channel by H2O2 could be an alternative pathway mediating this H2O2-Zn2+ toxicity.
After brain ischemia, the production of ROS is dramatically enhanced in the central nervous system. Increased production of O•−2 and H2O2, for example, is induced through the action of xanthine oxidase, leakage from mitochondrial electron transport chain, and activation of phospholipase A2 (29). Using microdialysis or electron spin resonance, a number of studies have measured increase of ROS production after ischemia and after reperfusion. Hyslop and colleagues, for example, reported that in the rat brain, a baseline concentration of H2O2 is at low micromolar range, whereas the peak H2O2 concentration after global ischemia is ~100μM (19). The concentrations of H2O2 used in our studies are, therefore, close to the in vivo ischemic condition. We show that a threshold concentration for H2O2 to produce glutamate-independent neuronal injury is at ~10μM. Thus, this glutamate receptor-independent Ca2+ toxicity by H2O2 should contribute to the overall ischemic brain injury in vivo.
The mechanism of how H2O2 activates TRPM7 channels is not clear and is the subject of future studies. H2O2 may induce its biological effect by a direct oxidation of its substrate, or indirectly through its more reactive by-product OH•. Our studies suggest that H2O2-induced glutamate-independent neuronal injury does not involve OH•. This was based on the following evidence: (a) DMSO, a scavenger for OH•, had no effect on H2O2-induced injury; (b) addition of iron chelator DFO did not affect the H2O2-induced neuronal injury. It is known that generation of OH• from H2O2 requires transition metals such as iron or copper. If the formation of OH• is involved in the effect of H2O2, then the presence of DFO would have attenuated the effect of H2O2. Oxidization of sulfhydryl groups on cysteine residues has been implicated in many physiological or pathological effects of ROS. Our data suggest that such mechanism may be involved in H2O2-induced glutamate receptor-independent neuronal injury. This is supported by the findings that thiol-reducing agent DTT effectively prevented the effect of H2O2, whereas cysteine oxidizing agents DTNP mimicked its effect.
The exact mechanism underlying ROS-mediated cell damage is not fully understood. ROS can markedly alter protein structure and induce protein cross-linking, thereby increase rates of proteolysis (40). Recent studies also suggested important roles of NADPH oxidase and poly(ADP-ribose) polymerase-1 in oxidative stress-mediated cell injury (24). One key link between oxidative stress and cell death is excessive activation of poly(ADP-ribose) polymerase-1, which causes NAD+ depletion and brain damage (35, 49). It has also been postulated that ROS may disrupt the integrity of cell membranes in a nonspecific manner through lipid peroxidation (12). In addition, recent new findings suggest that the majority effects of ROS may be mediated by specific signaling pathways rather than nonspecific damage of cell membrane or intracellular molecules (30). Our present studies also support the involvement of a specific signaling pathway in H2O2-induced cell injury; that is, activation of TRPM7 channels is required for H2O2-induced glutamate receptor-independent Ca2+ toxicity.
This work was supported by grants from National Institutes of Health (R01NS47506 and R01NS49470) and American Heart Association (0840132N). We thank Dr. A. Scharenberg (University of Washington) for providing HEK293 cells with inducible expression of TRPM7.
No competing financial interests exist.