2022 Volume 45 Issue 6 Pages 770-779
Neuronal cell death after cerebral ischemia consists various steps including glutamate excitotoxity. Excessive Ca2+ influx through the N-methyl-D-aspartate (NMDA) receptor, which is one of the ionotropic glutamate receptors, plays a central role in neuronal cell death after cerebral ischemia. We previously reported that DNA methylation is transiently increased in neurons during ischemic injury and that this aberrant DNA methylation is accompanied by neuronal cell death. Therefore, we performed the present experiments on glutamate excitotoxicity to gain further insight into DNA methylation involvement in the neuronal cell death. We demonstrated that knockdown of DNA methyltransferase (DNMT)1, DNMT3a, or DNMT3b gene in Neuro2a cells was performed to examine which DNMTs were more important for neuronal cell death after glutamate excitotoxicity. Although we confirmed a decrease in the levels of the target DNMT protein after small interfering RNA (siRNA) transfection, the Neuro2a cells were not protected from injury by transfection with siRNA for each DNMT. We next revealed that the pharmacological inhibitor of DNMTs protected against glutamate excitotoxicity in Neuro2a cells and also in primary cultured cortical neurons. This protective effect was associated with a decrease in the number of 5-methylcytosine (5 mC)-positive cells under glutamate excitotoxicity. In addition, the increased level of cleaved caspase-3 was also reduced by a DNMT inhibitor. Our results suggest the possibility that at least 2 or all DNMTs functionally would cooperate to activate DNA methylation after glutamate excitotoxicity and that inhibition of DNA methylation in neurons after cerebral ischemia might become a strategy to reduce the neuronal injury.
Neuronal cell death after ischemic brain injury occurs by various steps. At first, neurons in the ischemic core undergo a dramatic loss of blood flow, resulting in necrotic cell death. On the other hand, neurons which are located around the ischemic core region, known as the penumbra region, undergo different types of cell death compared with those in the ischemic core.1) They lose ATP, the loss of which causes depolarization of the membrane and excessive release of glutamate into the extracellular space. This abnormal release leads to neurotoxicity.2) The action of the N-methyl-D-aspartate (NMDA) receptor, a glutamate receptor, has been thought to be the major mechanism of neuronal cell death after cerebral ischemia.3) Excessive Ca2+ influx into the intracellular space through the NMDA receptor activates various cell death-related signals, leading to apoptosis of neurons.4) Because apoptosis of neurons in the penumbra is a reason for expansion of the infarct area, preventing this event is necessary for the development of novel therapeutic strategies for stroke.
Epigenetics is an essential mechanism in the process of gene expression in mammals. Epigenetic changes are able to influence on changes in cellular phenotypes without causing changes in DNA sequences. DNA methylation is known to contribute to such a modification and is achieved by DNA methyltransferases (DNMTs) causing the transfer of a methyl group from S-adenosylmethionine to the C5 position of cytosine to form 5-methylcytosine (5 mC). In mammals, 3 types of DNMTs are known. DNMT1 methylates the newly synthesized DNA during DNA replication to maintain the original DNA methylation pattern.5) DNMT3a and DNMT3b are known as de novo DNMTs because these enzymes are able to establish a new methylation pattern to unmodified DNA.5) It has been thought that DNA methylation generally induces gene silencing, which is needed for mammalian development,6) whereas accumulating evidence has also indicated that DNA methylation is involved in the pathogenesis of various diseases, such as not only cancer but also central nervous system diseases.7,8) We previously reported that DNA methylation is transiently increased in neurons after ischemic injury and that this change is related to neuronal cell death.9) The aim of this present study was to gain further insight into the roles played by DNA methylation in cell death after ischemic injury. Therefore, we investigated which types of DNMTs have a more important role in neuronal cell injury by using gene knockdown with each DNMTs small interfering RNA (siRNA) and the effect of a DNMT inhibitor on glutamate excitotoxicity in cells of the mouse neuroblastoma cell line Neuro2a and in cortical neurons in primary culture.
Mouse neuroblastoma Neuro2a cells were obtained from the American Type Culture Collection and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. Differentiation was induced 1 d after seeding by changing the culture medium to DMEM containing 2% FBS, 20 µM retinoic acid (Cat. No. R2625; Sigma-Aldrich, St. Louis, MO, U.S.A.), and 1% penicillin–streptomycin.
Measuring Length of Neurite and Cell BodyMicroscope images of Neuro2a cells 2 d after induction of differentiation were taken (BZ-X810, KEYENCE Co., Osaka, Japan), and then the length of neurites and cell body was measured by using an image analyzer (NIH image 1.63, NIH, Bethesda, MD, U.S.A.). Cells cultured for the same duration in the culture medium were used as non-differentiated cells in these experiments.
Glutamate Treatment for Neuro2a CellsGlutamate treatment was performed to induce excitotoxicity of Neuro2a cells. The culture medium was replaced with that containing the desired concentrations of glutamate (Cat. No. G5889; Sigma-Aldrich) at 2 d after inducing differentiation for differentiated cells. MK-801 (1 µM) (Cat. No. M107; Sigma-Aldrich), an NMDA receptor antagonist, and 10 nM zebularine (Cat. No. Z0022; Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), a DNMT inhibitor, were added at 24 h before glutamate treatment. The concentration of zebularine used in the present study was based on the data obtained in our preliminary study.
siRNA Treatment for Neuro2a CellsNeuro2a cells were transfected by electroporation using an electroporator, NEPA21 (Nepa Gene Co., Ltd., Chiba, Japan), according to the recommended protocols. Poring pulses were applied at 130 V (pulse length, 2.5 ms; pulse interval, 50 ms; number of pulses, twice), followed by transfer pulses at 20 V (pulse length, 50 ms; pulse interval, 50 ms; number of pulses, 5 times). After transfection, the cells were cultured in medium and then used for subsequent experiments. Target sequences of siRNAs (Fasmac Co., Ltd., Kanagawa, Japan) used in the present study were as follows: Non-targeting siRNA, UGGUUUACAUGUCGACUAA; Mouse DNMT1 siRNA, CCAUUGGCCUGGAGAUUAA; Mouse DNMT3a siRNA, CGUGUAAGUGUGAAGAUUU; and Mouse DNMT3b siRNA, CCCUGAAACUUUAAAACUU.
WST-1 AssayCell viability of Neuro2a cells after glutamate treatment was determined by performing the WST-1 assay. The cells were incubated in culture medium containing 0.5 mM 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1; 346-06456, Dojindo, Kumamoto, Japan), and 20 µM 1-methoxy-5-methylphenazinium methylsulfate (345-040001, Dojindo) for 1 h at 37 °C. Then, the absorbance at 450 nm was measured with a plate reader (EMax Plus Microplate Reader, Molecular Devices, Sunnyvale, CA, U.S.A.). The relative cell viability was expressed as the ratio of the absorbance at 450 nm of each treatment group against that of the corresponding non-treated group.
Primary Cultures of Rat Cortical NeuronsThe rats were maintained according to the National Institute of Health Guide for the Care and Use of Laboratory Animals and the Guideline for Experimental Animal Care issued by the Prime Minister’s Office of Japan. All experimental procedures were approved by the Committee of Animal Care and Welfare of Tokyo University of Pharmacy and Life Sciences (Permit No. P20-02). All procedures involving animals avoided or minimized discomfort, pain, and stress in the animals. Primary cultures were prepared as previously reported.9) Briefly, Sprague-Dawley pregnant rats (Japan SLC, Inc., Shizuoka, Japan) were sacrificed under the anesthesia with isoflurane and cerebral cortices of fetal rats at embryonic day 16 were dissected and digested with 0.25% trypsin (Invitrogen, Waltham, MA, U.S.A.) in phosphate-buffered saline (PBS) for 20 min at 37 °C. The tissues were then triturated in Neurobasal medium (Invitrogen) containing 10% FBS by use of a fire-polished Pasteur pipet. Isolated cortical cells were suspended in Neurobasal medium containing 0.5 mM glutamine, 2% B27 supplement (Invitrogen), and 1% penicillin-streptomycin (FUJIFILM Wako, Osaka, Japan). These cortical cells were plated at a density of 200000 cells/well in 24-well plates (Falcon, Corning, NY, U.S.A.) coated with poly-D-lysine (FUJIFILM Wako) and then cultured for 10 d before experiments. One half of the culture medium was changed every 2 d.
NMDA Treatment for Cortical NeuronsPrimary cultures of cortical neurons at 10 d in vitro (DIV) were washed twice for 15 min each time at 37 °C with Hank’s balanced salt solution (HBSS; Invitrogen) containing 2.4 mM CaCl2 and 20 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) without magnesium, which can block the NMDA receptor (HBSS buffer). Next, the neurons were incubated with 30 µM NMDA and 10 µM glycine, a co-activator of the NMDA receptor, in HBSS buffer for 15 min at 37 °C. Cortical neurons incubated with HBSS buffer lacking both NMDA and glycine were used for control experiments. After treatment, cortical neurons were cultured for the desired times in the culture medium. Cells were pretreated with 50 µM zebularine at 24 h before inducing cell injury. The concentration of zebularine in primary cultures of cortical neurons was based on the data obtained in our preliminary study. Treatment with 25 µM zebularine decreased the number of dead neurons to a lesser degree than that with 50 µM. In all experiments, age-matched cultured cortical cells without any treatment were used as the “non-treated group.”
Propidium Iodide StainingPropidium iodide (PI) staining of nuclei was performed for estimating the number of dead neurons at 24 h after NMDA treatment. Cells were washed twice with PBS and then incubated in 4 µM PI (FUJIFILM Wako) in PBS containing Hoechst33342 (1 : 2000, Cat. No. 346–07951; Dojindo) for 15 min at 37 °C. The PI-positive cells were counted in random 4 areas (1.3 mm2 each) of each well and indicated as percentages of the total number of Hoechst-positive cells.
Immunocytochemistry for 5 mCCells were fixed with 4% paraformaldehyde for 10 min and then washed twice with PBS. Subsequently, the cells were treated with deoxyribonuclease (DNase) I for 5 mC immunostaining as previously described with minor modification.10,11) Briefly, the cells were incubated for 30 min at 37 °C with 100 U/mL DNase I (Cat. No. LS002139, Worthington Biochemical Co., Lakewood, NJ, U.S.A.) in DNase I buffer comprising 10 mM MgCl2 in 50 mM Tris–HCl (pH 7.5). After that, the buffer was replaced with 50 mM Tris–HCl (pH 7.5) pre-heated for 10 min at 70 °C to inhibit DNase I activity and then cooled on ice for 3 min. Permeabilization was performed by incubation with 0.2% Triton X-100 containing PBS (PBS-T) for 10 min at room temperature. The cells were blocked with 10% goat serum and 1% bovine serum albumin (BSA) in PBS-T for 30 min at room temperature and then incubated with the primary antibody, rabbit monoclonal anti-5-mC (Cat. No. 28692, Cell Signaling Technology Inc., Danvers, MA, U.S.A.) at 4 °C. The next day, the cells were incubated with the secondary antibody, Alexa Fluor 594-labeled goat anti-rabbit immunoglobulin G (IgG) antibodies (Cat. No. A11037; Invitrogen) for 1 h at room temperature. Fluorescence was detected by using an Olympus fluorescence microscope (IX-71; Olympus, Tokyo, Japan). Images of 4 random areas (1.3 mm2 each) of each well were taken, and the fluorescent images were then loaded into the MetaMorph software program (Molecular Devices). The number of 5 mC-positive cells was counted based on the background fluorescence and the size of nuclei detected by the MetaMorph software program.
Western ImmunoblottingCells were washed thrice with PBS. After that the cells were harvested in lysis buffer containing 1% TritonX-100, 0.1% deoxycholic acid, and 1 mM ethylenediaminetetraacetic acid (EDTA) in 50 mM Tris-buffered saline with protease inhibitors (Roche Diagnostics Co., Mannheim, Germany) or sample buffer comprising 62.5 mM Tris–HCl (pH 6.8), 10% glycerol, 2% sodium dodecyl sulfate (SDS), and 5% β-mercaptoethanol. Measuring the protein concentration was performed for cells collected in lysis buffer. Then, the samples were heated at 95 °C for 5 min in sample buffer.
Western immunoblotting was performed according to standard protocols. Proteins applied onto the gel were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred to polyvinylidene difluoride membranes at 80 V for 2 h. The following primary antibodies were used: rabbit monoclonal anti-DNMT1 (dilution, 1 : 1000; Cat. No. 5032, Cell Signaling Technology Inc.), rabbit monoclonal anti-DNMT3a (dilution, 1 : 1000; Cat. No. 3598, Cell Signaling Technology Inc.), rabbit polyclonal anti-DNMT3b (dilution, 1 : 1000; Cat. No. ab2851, Abcam, Cambridge, U.K.), rabbit polyclonal anti-cleaved caspase-3 (dilution, 1 : 1000; Cat. No. 9661, Cell Signaling Technology Inc.), and mouse monoclonal anti-β-actin (dilution, 1 : 10000; Cat. No. A5441, Sigma-Aldrich) antibody. Subsequently, the protein blots were washed and incubated with the appropriate secondary antibodies (dilution, 1 : 5000; Pierce Biotechnology, Rockford, IL, U.S.A.). Immunoreactive proteins were detected by using ImmunoStar basic (FUJIFILM Wako), ImmunoStar zeta (FUJIFILM Wako) or West Femto (Pierce Biotechnology). Quantification was performed by using computerized densitometry (LuminoGraph II, ATTO Co., Tokyo, Japan) and an image analyzer (CS Analyzer, ATTO Co.).
Statistical AnalysisAll obtained data were analyzed by using GraphPad Prism software (version 5, GraphPad Software, San Diego, CA, U.S.A.) and presented as the means ± standard error of the mean. The unpaired Student’s t-test was performed for the comparison between 2 groups. Differences among multiple groups were evaluated by using ANOVA followed by Tukey’s test as a post hoc test. p-Values of less than 0.05 were considered to indicate statistical significance.
Neuro2a cells differentiate into neuron-like cells when they are cultured in retinoic acid-containing, low-serum medium12,13) (Figs. 1A, B). We first confirmed the morphological changes in the cells at 2 d after differentiation. The distribution of the number of neurites in non-differentiated Neuro2a cells was maximum at 11–20 µm. On the other hand, those of differentiated Neuro2a cells were maximum at 31–40 µm (Fig. 1C). The average length of all measured neurites of non-differentiated and differentiated Neuro2a cells were 15.0 ± 0.5 and 50.0 ± 1.5 µm, respectively (Fig. 1D). Moreover, we measured the major and minor axes of the cell body. The distribution of the number of cells for the major axis in non-differentiated Neuro2a cells was maximal at 16–20 µm. On the other hand, that of the differentiated Neuro2a cells was greatest at 21–25 µm (Fig. 1E). The average length of the major axis of differentiated Neuro2a cells (25.1 ± 0.3 µm) was slightly longer than that of the non-differentiated ones (20.2 ± 0.4 µm; Fig. 1F). The distribution of the number of cells for the minor axis did not differ regardless of the induction of differentiation (Figs. 1G, H).
(A, B) Representative images of non-differentiated (A) and differentiated (B) Neuro2a cells. Scale bar shows 50 µm. (C, D) Changes in neurite length induced by differentiation. Distribution of neurite length is expressed as the ratio of the number to total neurites. White and black bars indicate non-differentiated and differentiated Neuro2a cells, respectively. A total of 1562–1633 neurites were counted in this experiment (C). Average length of neurites is shown (D). The number of neurites counted in each well was 306–428. Changes in lengths of major axis (E, F) and minor axis (G, H) induced by differentiation. Distribution of number of cells with length of the major axis (E) and minor axis (G) are expressed as the ratio of the number to total cells. White and black bars indicate non-differentiated and differentiated Neuro2a cells, respectively. Cells counted were 2102–2393 (E) and 2088–2301 (G) in this experiment. Average length of major axis (F) and minor axis (H) are shown. The number of cells counted was 456–715 (F) and 448–691 (H) in each well. Results are the means ± standard error of the mean (S.E.M.) (n = 4 independent experiments). ND, non-differentiated; D, differentiated. p-Values of less than 0.05 were considered to indicate statistical significance.
We next showed a dose-dependent decrease in cell viability by glutamate treatment of both non-differentiated and differentiated Neuro2a cells (Figs. 2A, B). Based on this result, 150 mM glutamate for non-differentiated cells and 125 mM glutamate for differentiated cells were used in the following study. We next confirmed that treatment with the NMDA receptor antagonist MK-801 (1 µM), which was added 24 h before the glutamate treatment, protected cells against glutamate excitotoxicity only in the case of the differentiated Neuro2a cells (Figs. 2C, D). This result indicates that glutamate excitotoxity in the differentiated Neuro2a cells was mainly mediated by NMDA receptors and was similar with respect to our previously reported results using primary cultured cortical neurons.14,15) On the basis of these results, differentiated cells were used in the following study.
(A, B) Cell viability was determined by using the WST-1 assay. Absorbance at 450 nm was measured and the values expressed as the ratio of the absorbance at 450 nm of the non-treated group. Results are the means ± S.E.M. (n = 4–5 independent experiments). #, ## Significant difference against the 0 mM glutamate-treatment group (#p < 0.01, ##p < 0.001). NT, non-treated group. (C, D) Effect of NMDA receptor antagonist MK-801 (1 µM) on glutamate (125 mM for non-differentiated or 150 mM differentiated Neuro2a cells) excitotoxicity. Cell viability was examined by performing WST-1 assay. Absorbance at 450 nm was measured and the values expressed as the ratio of the absorbance at 450 nm of the non-treated group. Results are the means ± S.E.M. (n = 4 independent experiments). NT, non-treated group; Glu, glutamate. p-Values of less than 0.05 were considered to indicate statistical significance.
Next, we performed knockdown of DNMT genes through siRNA transfection by electroporation in differentiated Neuro2A cells. At first, we confirmed that transfection with DNMT1 (Figs. 3A–D), DNMT3a (Figs. 3E–H) or DNMT3b (Figs. 3I–L) siRNA effectively decreased the specific protein level compared with non-targeting siRNA-transfected cells. Because siRNA transfection was specific for the target gene, we next determined the effect of the transfection with each siRNA on DNA methylation and the cell viability after glutamate treatment (125 mM). The number of 5 mC-positive cells was increased after glutamate treatment. Unexpectedly, there was no effect of the transfection with DNMT siRNAs on the number of 5 mC-positive cells (Figs. 4A, B, D, E, G, H). In addition, the decreased cell viability of differentiated Neuro2A cells after glutamate treatment (125 mM) was not affected by the transfection with DNMT siRNAs (Figs. 4C, F, I).
Effect of transfection with DNMT1 (A–D), DNMT3a (E–H), and DNMT3b (I–L) siRNAs on the protein level of each DNMTs 3 d after transfection. Bands corresponding to DNMT1, DNMT3a, DNMT3b, and β-actin were scanned, and the scanned bands were normalized by the non-treated group on the same blot. β-actin was used as a loading control. Results are the means ± S.E.M. (n = 4–5 independent experiments). NT, non-treated group. p-Values of less than 0.05 were considered to indicate statistical significance.
Effects of DNMT1 (A, B), DNMT3a (D, E), and DNMT3b (G, H) siRNA transfection on glutamate (125 mM)-induced increase in the DNA methylation 2 h after treatment in differentiated Neuro2a cells. (A, D, G) Representative images of 5 mC staining are shown. The scale bar indicates 50 µm. (B, E, H) 5 mC-positive cells were counted and expressed as the ratio to total cells (Hoechst-positive cells). The number of cells counted was 572–1687 (B), 641–1046 (E) and 567–1020 (H) in each well. Images of 4 random areas (each area of 1.3 mm2) of each well were taken. Results are the means ± S.E.M. (n = 4 independent experiments). Effect of transfection with DNMT1 (C), DNMT3a (F), and DNMT3b (I) siRNAs on glutamate (125 mM) excitotoxicity in differentiated Neuro2a cells. Cell viability was determined by using the WST-1 assay. Absorbance at 450 nm was measured and the values expressed as the ratio of the absorbance at 450 nm of the non-treated group. Results are the means ± S.E.M. (n = 4 independent experiments). 5 mC, 5-methylcytosine; NT, non-treated group; CTL, control; Glu, glutamate. p values of less than 0.05 were considered to indicate statistical significance.
We next examined the effect of pharmacological inhibition of DNMTs against glutamate (125 mM)-induced DNA methylation and excitotoxicity in differentiated Neuro2A cells. The increase in the number of 5 mC-positive cells after glutamate treatment was reduced by treatment with 10 nM zebularine (Figs. 5A, B). The decrease in cell viability of differentiated Neuro2A by glutamate treatment was also attenuated by zebularine treatment (Fig. 5C). Furthermore, the increase in the amount of cleaved caspase-3 protein, which regulates apoptosis,16,17) under glutamate excitotoxicity was suppressed by zebularine treatment (Fig. 5D).
Effects of DNMT inhibitor zebularine (10 nM) on increase in DNA methylation 2 h after glutamate (125 mM) treatment in differentiated Neuro2a cells. (A) Representative images of 5 mC staining are shown. The scale bar indicates 50 µm. (B) 5 mC-positive cells were counted and expressed as the ratio to total cells (Hoechst-positive cells). The number of cells counted was 419–2029. Images of 4 random areas (each area of 1.3 mm2) of each well were taken. Results are the means ± S.E.M. (n = 6 independent experiments). Glu, glutamate; Zeb, zebularine. (C) Effect of DNMT inhibitor zebularine (10 nM) on glutamate (125 mM) excitotoxicity in differentiated Neuro2a cells. Cell viability was examined by using the WST-1 assay. Absorbance at 450 nm was measured and the values expressed as the ratio of the absorbance at 450 nm of the non-treated group. Results are the means ± S.E.M. (n = 6 independent experiments). NT, non-treated group; Glu, glutamate; Zeb, zebularine. (D) Effect of DNMT inhibitor zebularine (10 nM) on the protein level of cleaved caspase-3 at 24 h after start of glutamate (125 mM) treatment. Bands corresponding to cleaved caspase-3 and β-actin were scanned, and the scanned bands were normalized by the non-treated group on the same blot. β-Actin was used as a loading control. Results are the means ± S.E.M. (n = 5 independent experiments). 5 mC, 5-methylcytosine; CTL, control group; NT, non-treated group; Glu, glutamate; Zeb, zebularine. p-Values of less than 0.05 were considered to indicate statistical significance.
We previously demonstrated using cortical neurons in primary culture that the number of 5 mC-positive cells increases transiently after NMDA treatment.9) Therefore, we next examined whether the nucleoside inhibitor zebularine can affect the DNA methylation and cell injury under excitotoxicity using cortical neurons in primary culture. We revealed that the increase in the number of 5 mC-positive neurons induced by 30 µM NMDA was attenuated by treatment with 50 µM zebularine (Figs. 6A, B). Zebularine also protected primary cultured cortical neurons against NMDA-induced neuronal injury (Figs. 6C, D). Next, we determined the protein level of cleaved caspase-3. The up-regulation of cleaved caspase-3 protein induced by 30 µM NMDA was suppressed by the zebularine (50 µM) treatment in primary cultured cortical neurons (Fig. 6E).
Effects of DNMT inhibitor zebularine (50 µM) on NMDA (30 µM)-induced DNA methylation 30 min after start of treatment in primary cultured cortical neurons. (A) Representative images of 5 mC staining are shown. The scale bar indicates 50 µm. (B) 5 mC-positive cells were counted and are expressed as the ratio to total cells (Hoechst-positive cells). The number of cells counted in each well was 647–1895. Images of 4 random areas (each area of 1.3 mm2) of each well were taken. Results are the means ± S.E.M. (n = 4 independent experiments). Effects of DNMT inhibitor zebularine (50 µM) on NMDA (30 µM)-induced cell injury. (C) Representative images of PI staining are shown. The scale bar indicates 50 µm. (D) PI-positive cells were counted and are expressed as the ratio to total cells (Hoechst-positive cells). The number of cells counted in each well was 959–1585. Images of 4 random areas (each area of 1.3 mm2) of each well were taken. Results are the means ± S.E.M. (n = 4 independent experiments). (E) Effect of DNMT inhibitor zebularine (50 µM) on the protein level of cleaved caspase-3 8 h after 30 µM NMDA treatment in primary cultured cortical neurons. Bands corresponding to cleaved caspase-3 and β-actin were scanned, and the scanned bands were normalized by the non-treated group on the same blot. β-Actin was used as a loading control. Results are the means ± S.E.M. (n = 5 independent experiments). 5 mC, 5-methylcytosine; CTL, control group; NT, non-treated group; Zeb, zebularine. p-Values of less than 0.05 were considered to indicate statistical significance.
We previously reported that DNA methylation in neurons was increased after ischemic injury and that a DNMT inhibitor protected neurons against NMDA-induced injury by inhibiting this increase.9) In that study, the non-nucleoside inhibitor RG108, showed a protective effect. Therefore, by using gene knockdown with each DNMTs siRNA, we investigated which types of DNMTs have a more important role in neuronal cell injury. Although we expected that knockdown of each of the DNMTs would also show a protective effect against glutamate excitotoxicity, none of them inhibited DNA methylation or cell death. Considering that DNA methylation induced by glutamate excitotoxity was probably induced by de novo DNMTs, it is possible that DNMT3a and DNMT3b were associated with this reaction. Accumulated evidence suggests that DNMT3a is involved in the increase in DNA methylation in a sciatic nerve avulsion model as well as in an in vivo neuropathic pain model.18,19) It was reported that hippocampal CA1 region-specific deletion of DNMT3b, which is thought to just be only slightly expressed in mature neurons, impairs recognition memory.20–22) Therefore, DNMT3b has the ability to have an influence on changes in DNA methylation in mature neurons. In addition, DNMT1, which is generally associated with maintenance DNA methylation, is related to de novo activity in some cases.23) That study also indicates that DNMT3a initiates de novo methylation of DNA and then DNMT1 becomes activated and further induces DNA methylation.23) Therefore, our results suggest the possible involvement of at least 2 or 3 DNMTs isoforms that functionally cooperate during DNA methylation after glutamate excitotoxicity. Although we confirmed decreases in the target DNMT protein levels after transfection with siRNA, the decrease in each DNMT protein might not have been sufficient to rescue neurons from glutamate excitotoxicity.
Accumulating evidence showed a nucleoside inhibitor, zebularine, can influence DNA methylation levels in neurons.24,25) In the present study, we revealed that zebularine had a protective effect against excitotoxic injury along with inhibition of DNA methylation in Neuro2a cells and also in primary cultured cortical neurons. It has generally thought that nucleoside inhibitors must be incorporated into DNA in order to inhibit DNMTs. Although the reason why the nucleoside DNMT inhibitor zebularine was protective against excitotoxicity in non-dividing cells, such as neurons, is not yet fully understood, we may propose a possible mechanism. DNA demethylation occurs in enzymatic DNA base modification and nucleotide replacement via base excision repair (BER).26) It is possible that nucleoside DNMT inhibitors are phosphorylated by cytidine kinases and could then be incorporated into DNA instead of cytosine during BER.27) This abnormal DNA reduces DNA methylation activity.28) It was also reported that the central nervous system contains the highest levels of 5-hydroxylmethylcytosine (5hmC), an intermediate of DNA demethylation, of all of the tissues in the adult mouse.29) Moreover, 5hmC-positive cells co-localize with NeuN-positive mature neurons in the cerebellum and hippocampus and the number of these double-positive cells increases with neurodevelopment.30) Actually, DNA demethylation is associated with synaptic plasticity in primary cultures of cortical neurons.31) These reports suggest that DNA demethylation plays a pivotal role in the activity of neurons constitutively. Therefore, nucleoside DNMT inhibitors might be able to be incorporated into DNA through the processes of DNA demethylation. Zebularine is a newly characterized compound as a nucleoside inhibitor and shows high stability and low cytotoxic effects compared with other nucleoside DNMT inhibitors such as 5AdC.32,33) Therefore, zebularine might have a protective effect against excitotoxicity in cultured cortical neurons.
Based on our previous and present results, we demonstrated a pivotal role of DNA methylation in neuronal cell death after ischemic injury. In addition, we revealed that the increase in DNA methylation in neurons under excitotoxic injury was involved in caspase-3 dependent cell death in the present study. Caspase-3 is one of the most important caspases in the apoptotic pathway, because activation of caspase-3 leads to DNA fragmentation by cleaving the inhibitor of caspase-activated DNase (ICAD) from caspase-activated DNase (CAD).16) It was previously reported that activated caspase-3 plays an important role in neuronal apoptosis following cerebral ischemia.34) In addition, DNA methylation increases rapidly in motor neurons during apoptosis in a sciatic nerve avulsion model.18) Moreover, the number of apoptotic cells is increased by gene knockdown of ten-eleven translocation 1 (Tet1), which is known as a methylcytosine dioxygenase that induces DNA demethylation, in cerebellar granule cells under oxidative stress.35) These reports indicate that DNA methylation is related to apoptotic cell death, which is consistent with the results in our studies. Therefore, DNA methylation in neurons after cerebral ischemia might be related to neuronal injury and become therapeutic targets.
In conclusion, our findings suggest that at least 2 or all DNMTs might functionally cooperate to activate DNA methylation under glutamate excitotoxicity and that inhibition of DNA methylation by treatment with zebularine might become a strategy to reduce ischemic neuronal injury.
We thank Min Wang for her technical assistance.
The authors declare no conflict of interest.
