Evidence That DNA (Cytosine-5) Methyltransferase Regulates Synaptic Plasticity in the Hippocampus
2006; Elsevier BV; Volume: 281; Issue: 23 Linguagem: Inglês
10.1074/jbc.m511767200
ISSN1083-351X
AutoresJonathan M. Levenson, Tania L. Roth, Farah D. Lubin, Courtney A. Miller, I-Chia Huang, Priyanka Desai, Lauren Malone, J. David Sweatt,
Tópico(s)Autism Spectrum Disorder Research
ResumoDNA (cytosine-5) methylation represents one of the most widely used mechanisms of enduring cellular memory. Stable patterns of DNA methylation are established during development, resulting in creation of persisting cellular phenotypes. There is growing evidence that the nervous system has co-opted a number of cellular mechanisms used during development to subserve the formation of long term memory. In this study, we examined the role DNA (cytosine-5) methyltransferase (DNMT) activity might play in regulating the induction of synaptic plasticity. We found that the DNA within promoters for reelin and brain-derived neurotrophic factor, genes implicated in the induction of synaptic plasticity in the adult hippocampus, exhibited rapid and dramatic changes in cytosine methylation when DNMT activity was inhibited. Moreover, zebularine and 5-aza-2-deoxycytidine, inhibitors of DNMT activity, blocked the induction of long term potentiation at Schaffer collateral synapses. Activation of protein kinase C in the hippocampus decreased reelin promoter methylation and increased DNMT3A gene expression. Interestingly, DNMT activity is required for protein kinase C-induced increases in histone H3 acetylation. Considered together, these results suggest that DNMT activity is dynamically regulated in the adult nervous system and that DNMT may play a role in regulating the induction of synaptic plasticity in the mature CNS. DNA (cytosine-5) methylation represents one of the most widely used mechanisms of enduring cellular memory. Stable patterns of DNA methylation are established during development, resulting in creation of persisting cellular phenotypes. There is growing evidence that the nervous system has co-opted a number of cellular mechanisms used during development to subserve the formation of long term memory. In this study, we examined the role DNA (cytosine-5) methyltransferase (DNMT) activity might play in regulating the induction of synaptic plasticity. We found that the DNA within promoters for reelin and brain-derived neurotrophic factor, genes implicated in the induction of synaptic plasticity in the adult hippocampus, exhibited rapid and dramatic changes in cytosine methylation when DNMT activity was inhibited. Moreover, zebularine and 5-aza-2-deoxycytidine, inhibitors of DNMT activity, blocked the induction of long term potentiation at Schaffer collateral synapses. Activation of protein kinase C in the hippocampus decreased reelin promoter methylation and increased DNMT3A gene expression. Interestingly, DNMT activity is required for protein kinase C-induced increases in histone H3 acetylation. Considered together, these results suggest that DNMT activity is dynamically regulated in the adult nervous system and that DNMT may play a role in regulating the induction of synaptic plasticity in the mature CNS. DNA (cytosine-5) methyltransferases (DNMTs) 5The abbreviations used are: DNMT, DNA (cytosine-5) methyltransferase; CNS, central nervous system; LTP, long term potentiation; NMDA, N-methyl-d-aspartate; NMDA-R, NMDA receptor; PDA, phorbol-12,13-diacetate; CS, cutting saline; ACSF, artificial cerebrospinal fluid; Zeb, zebularine; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase; 5-Aza, 5-aza-2-deoxycytidine; PKC, protein kinase C; df, degrees of freedom; fEPSP, field excitatory post-synaptic potential.5The abbreviations used are: DNMT, DNA (cytosine-5) methyltransferase; CNS, central nervous system; LTP, long term potentiation; NMDA, N-methyl-d-aspartate; NMDA-R, NMDA receptor; PDA, phorbol-12,13-diacetate; CS, cutting saline; ACSF, artificial cerebrospinal fluid; Zeb, zebularine; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase; 5-Aza, 5-aza-2-deoxycytidine; PKC, protein kinase C; df, degrees of freedom; fEPSP, field excitatory post-synaptic potential. are a family of enzymes that catalyze the methylation of cytosine residues (1Bestor T. 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In the studies described below, we further tested our hypothesis that epigenetic mechanisms regulate memory formation, by determining the effects of DNA methyltransferase inhibitors on synaptic plasticity. Synaptic plasticity is the leading candidate cellular mechanism for storage of memory in vivo. Long term potentiation (LTP) of hippocampal Schaffer collateral synapses has received a great deal of experimental attention, since this form of synaptic plasticity utilizes many of the same mechanisms involved in consolidation of long term memory (35Lynch M.A. Physiol. Rev. 2004; 84: 87-136Crossref PubMed Scopus (1401) Google Scholar). Successful formation of LTP requires engagement of NMDA receptors, activation of the Ras-MEK-ERK signaling cascade, and ultimately post-translational modifications of histones that mediate a transcriptional program resulting in lasting changes in neuronal function (23Levenson J.M. O'Riordan K.J. Brown K.D. Trinh M.A. Molfese D.L. Sweatt J.D. J. Biol. 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We found that inhibition of DNMT alters DNA methylation within the promoter region of both reelin and BDNF1, indicating that acute regulation of DNA methylation occurs in the adult nervous system. Treatment with phorbol-12,13-diacetate (PDA) induced significant regulation of DNMT3A, suggesting that the PKC signaling pathway regulates the expression of DNMT genes. Moreover, inhibition of DNMT blocked induction of LTP, suggesting that DNMT activity is required for induction of synaptic plasticity. Finally, we provide evidence that PKC-mediated changes in histone H3 acetylation are sensitive to DNMT activity. These findings provide the first evidence for DNA (cytosine-5) methylation in dynamic signaling in the adult nervous system and suggest that DNMT activity is required for normal hippocampal synaptic plasticity. Young adult (4–8 weeks) male C57BL/6J mice were used for all experiments. Mice were housed under 12:12 light/dark cycles, with food and water available ad libitum. All procedures were performed in accordance with the Baylor College of Medicine Institutional Animal Care and Use Committee and with national regulations and policies. Animals were sacrificed by cervical dislocation, followed by decapitation. The brain was immersed in oxygenated (95% O2, 5%CO2) ice-cold cutting saline (CS) (110 mm sucrose, 60 mm NaCl, 3 mm KCl, 1.25 mm NaH2PO4, 28 mm NaHCO3, 0.5 mm CaCl2, 7 mm MgCl2, 5 mm glucose, 0.6 mm ascorbate) prior to isolation of the hippocampus. Transverse slices (400 mm) were prepared with a Vibratome (Vibratome 1000, The Vibratome Co., St. Louis, MO). During isolation, hippocampal slices were stored in oxygenated ice-cold CS. After isolation, hippocampal slices were equilibrated in a mixture of 50% CS and 50% artificial cerebrospinal fluid (ACSF) (125 mm NaCl, 2.5 mm KCl, 1.25 mm NaH2PO4, 25 mm NaHCO3, 2 mm CaCl2, 1 mm MgCl2, 25 mm glucose) at room temperature for 45 min prior to use. Transverse hippocampal slices, pooled from 6–8 animals and randomized, were incubated in oxygenated ACSF (32 °C) for 1 h prior to treatment. For DNA methylation assays, slices were treated with either zebularine (100 μm, 40 min), PDA (3 μm, 20 min), or vehicle (0.5% Me2SO). For experiments investigating regulation of DNMT gene expression, slices were treated with either vehicle (0.1% Me2SO) or PDA (3 μm) for 45 min. For DNMT inhibition experiments, slices were treated with either vehicle (0.5% Me2SO) or zebularine (100 μm) or with either vehicle (0.001% CH3COOH) or 5-aza-2-deoxycytidine (30 μm) for 40 min followed by incubation with either vehicle (0.1% Me2SO) or PDA (3 μm) for 20 min. Immediately after all treatments, Area CA1 was isolated in ice-cold CS. For DNA methylation assays and experiments measuring DNMT gene expression, tissue was immediately frozen on dry ice. For experiments measuring histone acetylation, tissue was immediately processed for histone extraction. DNA was isolated from hippocampal tissue, purified (Wizard genomic DNA purification kit; Promega, Madison, WI), and processed for bisulfite modification (CpGenome DNA modification kit; Chemicon, Temecula, CA). Quantitative real time PCR was used to determine the DNA methylation status of the Reelin and BDNF1 promoter. Methylation-specific PCR primers were designed using Methprimer software (available on the World Wide Web at www.urogene.org/methprimer/). One set of primers targeted a large CpG island present in the reelin promoter (see also Ref. 37Mitchell C.P. Chen Y. Kundakovic M. Costa E. Grayson D.R. J. Neurochem. 2005; 93: 483-492Crossref PubMed Scopus (62) Google Scholar), and another set of primers targeted two CpG islands detected in silico in the promoter upstream of exon I in the mouse BDNF gene. Detection of Unmethylated DNA—Detection of unmethylated DNA in the reelin promoter was performed using the following primers: Region 1, forward (5′-GGTTGTAGTTGGTGTAGATAAAGAATAGT-3′) and reverse (5′-CCCAAAAAAACAAAACTACTCAAC-3′); Region 2, forward (5′-TTTTTGTTTGAGGAATTTTTTAGTAATG-3′) and reverse (5′-AAACCAAATCATCAAACTTCTCAAC-3′). Detection of unmethylated DNA in the BDNF promoter was performed using the following primers: CpG island 1, forward (5′-GGGTAGTGATTTTGGGGAGGAAGTAT-3′) and reverse (5′-CAACCTCTATACACAACTAAATCCACC-3′); CpG island 2, forward (5′-TTGGGGAAATTTGAAAAGTAGTGT-3′) and reverse (5′-CACAACAAAACAAAAAAACCAAA-3′). PCRs were performed in a total volume of 20 μl, consisting of 2 μl of bisulfite-modified DNA, 10 μl of iQ SYBR Green Supermix (Bio-Rad), 1 μl of primer (18 μm), and 7 μl of diethylpyrocarbonate-treated double-distilled H2O. Reactions were performed in a Chromo4 real time PCR system (Bio-Rad) using the following cycling conditions: 95 °C for 3 min, 40 cycles of 95 °C for 15 s, 60 °C for 1 min, and either 77.5 °C (reelin, Region 1), 72.5 °C (reelin, Region 2), 74 °C (BDNF, CpG island 1), or 60 °C (BDNF, CpG island 2) for 15 s. Detection of the fluorescent products occurred at the end of the 15-s temperature step. For a melting curve analysis, PCR products were melted by increasing the temperature in 1 °C increments beginning at 60 °C. To further verify specificity of the final product, 10 μl of the amplified products were analyzed by electrophoresis on a 2% agarose gel stained with ethidium bromide and visualized under UV light. Detection of Methylated DNA—Detection of unmethylated DNA was performed using primers complementary to Region 1 of reelin and CpG Islands 1 and 2 of BDNF listed above. Sequences for primers to the reelin promoter were as follows: forward, 5′-GTAGTCGGCGTAGATAAAGAATAGC-3′; reverse, 5′-CCGAAAAAACAAAACTACTCGAC-3′. Primer sequences to detect methylated DNA in the BDNF1 promoter were as follows: CpG island 1, forward (5′-GTAGCGATTTTGGGGAGGAAGTAC-3′) and reverse (5′-CAACCTCTATACGCGACTAAATCCG-3′); CpG island 2, forward (5′-ATCGGGGAAATTTGAAAAGTAGC-3′) and reverse (5′-GCAACGAAACAAAAAAACCGAA-3′). Reactions were performed as described above, with the final step at 75.5 °C (BDNF, CpG island 1) or 60 °C (BDNF, CpG island 2) for 15 s. Due to low levels of methylation at the region of the reelin promoter, a “kick start” method was used to ensure detection of a product. First, a conventional PCR reaction was performed in a total volume of 20 μl, consisting of 6 μlof bisulfite-modified DNA, 10 μl of iQ SYBR Green Supermix, 1 μl of primer (18 μm), 3 μl of diethylpyrocarbonate-treated double-distilled H2O. Reactions were carried out in a PTC-200 Peltier Thermal Cycle (Bio-Rad) with the following cycling conditions: 95 °C for 3 min, 20 cycles of 95 °C for 15 s, 60 °C for 1 min, 72 °C for 1 min, and a final 10 min cycle at 72 °C. The product from the conventional PCR was then further amplified with the same primer sequence and similar cycling conditions as described above with the following changes: 50 cycles instead of 20 and the final step occurring at 79 °C. Likewise, a melting curve analysis and gel electrophoresis was carried out to verify specificity of the final product. For every quantitative PCR, samples were assayed in triplicate, and the Ct value for each sample was chosen within the linear range. Samples were normalized to β-tubulin-4 (forward, 5′-GGAGAGTAATATGAATGATTTGGTG-3′; reverse, 5′-CATCTCCAACTTTCCCTAACCTACTTAA-3′), and the comparative Ct method was used to calculate differences in gene expression between samples (38Livak K.J. Schmittgen T.D. Methods. 2001; 25: 402-408Crossref PubMed Scopus (116613) Google Scholar, 39Pfaffl M.W. Nucleic Acids Res. 2001; 29: e45Crossref PubMed Scopus (24586) Google Scholar). RNA was isolated from tissue using Trizol (Invitrogen). RNA samples were further purified with CHCl3, and concentrations were determined spectrophotometrically. Real time, reverse transcription quantitative PCR was performed in one step using commercially available reagents (iScript one-step supermix; Bio-Rad) and Taqman probes (Applied Biosystems, Foster City, CA). All probes were designed to span exon boundaries, ensuring amplification of only mRNA. Equal amounts of RNA were analyzed in triplicate for each probe used; equal loading was confirmed by amplification of β-tubulin 4. Ct values were chosen in the linear range of amplification, and the comparative Ct method was used to calculate differences in gene expression between samples (38Livak K.J. Schmittgen T.D. Methods. 2001; 25: 402-408Crossref PubMed Scopus (116613) Google Scholar, 39Pfaffl M.W. Nucleic Acids Res. 2001; 29: e45Crossref PubMed Scopus (24586) Google Scholar). Hippocampal tissue was homogenized with a Dounce homogenizer using six strokes in 1 ml of ice-cold homogenizing buffer (250 mm sucrose, 50 mm Tris, pH 7.5, 25 mm KCl, 0.5 mm phenylmethylsulfonyl fluoride, 1% protease inhibitor mixture (Sigma), 0.9 mm Na+-butyrate). Tissue homogenates were centrifuged at 7,700 × g for 1 min. The supernatant was taken as the cytoplasmic fraction. Pelleted nuclei were resuspended in 250 μl of 0.4 n H2SO4, incubated on ice for 30 min, and then centrifuged at 4 °C again for 10 min at 14,000 × g. The supernatant was transferred to a fresh tube, and proteins were precipitated with 125 μlof 100% trichloroacetic acid containing 4 mg/ml deoxycholic acid (Na+ salt; Sigma) by incubation on ice for 30 min. The supernatant was discarded, and the protein pellet was washed with 1 ml of ice-cold acidified acetone (0.1% HCl) followed by 1 ml of ice-cold acetone for 5 min each. Protein precipitates were collected between washes by centrifugation at 14,000 × g for 5 min. The resulting purified proteins were resuspended in 10 mm Tris (pH 8.0) and stored at –80 °C. Protein concentrations were determined using the Bio-Rad protein assay reagent. Protein extracts (1 μg) were separated by SDS-PAGE on a 15% resolving gel with a 4% stacking gel and transferred onto polyvinylidene difluoride membrane. After transfer, the polyvinylidene difluoride membranes were briefly rinsed with 100% methanol, air-dried for 15 min, and washed with 25 ml of TTBS (0.1% Tween 20, 50 mm Tris-HCl, pH 7.5, 150 mm NaCl) for 5 min at room temperature. The membranes were then incubated overnight at 4 °C with an antiserum against proteins of interest in primary buffer (0.1% Tween 20, 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, and 5% bovine serum albumin). This was followed by three washes in TTBS and incubation for 2 h with an horseradish peroxidase-conjugated secondary anti-rabbit antibody in TTBS (1:10,000) at room temperature. The membranes were washed in TTBS, and blots were developed using ECL and exposed to film (BioMax; Eastman Kodak Co.). The anti-rabbit primary antibodies used and their dilutions were as follows: anti-histone H3 (1:1,000), anti-acetyl-histone H3 (Lys-14; 1:1,000), anti-histone H4 (1:1,000), and anti-acetyl-histone H4 (Lys-5/Lys-8/Lys-12/Lys-16; 1:1000). All antibodies were obtained from Upstate Biotechnology, Inc. Electrophysiology was performed in an interface chamber (Fine Science Tools, Foster City, CA). Oxygenated ACSF (95% O2, 5%CO2, 30 °C) was perfused into the recording chamber at a rate of 1 ml/min. Electrical signals were amplified (model 1800; A-M Systems, Sequim, WA), digitized (Digidata 1320A; Axon Instruments, Union City, CA), and stored on a PC (Clampex; Axon Instruments). Extracellular stimuli were delivered (model 2200 stimulus isolator; A-M Systems) by placing isonel enamel-coated, bipolar platinum-tungsten (92%:8%, 0.0011-inch diameter) electrodes on the border of Areas CA3 and CA1 along the Schaffer collaterals. fEPSPs were recorded in stratum radiatum with an ACSF-filled glass recording electrode (1–3 megohms). All analyses of electrophysiologic traces were performed using Clampfit (Axon Instruments). The relationship between the left slope of the fiber volley (presynaptic depolarization) and the left slope of the fEPSP (postsynaptic depolarization) over various stimulus intensities was used to assess base-line synaptic transmission. All subsequent experimental stimuli were set to an intensity that evoked an fEPSP that had a left slope of 50% of the maximum fEPSP left slope. NMDA receptor-mediated synaptic transmission was assessed as per base-line synaptic transmission above in the presence of the AMPA-R antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (20 μm) and a modified ACSF (same as above, except 0 mm MgCl2, 4 mm CaCl2). These conditions allow unblocking of the NMDA-R and allow assessment of NMDA-R function by measuring base-line fEPSPs mediated by this receptor. Paired pulse facilitation was investigated at various interstimulus intervals (20, 50, 100, 200, and 300 ms). LTP was induced by administering three trains of θ-burst stimulation. Each train consisted of 10 sets of bursts (4 stimuli, 100 Hz) with an interburst interval of 200 ms. There was 20 s between each stimulus train. Synaptic efficacy was monitored 30 min prior to and 180 min following induction by recording fEPSPs every 20 s (traces were averaged for every 2-min interval). Drug or vehicle was administered to slices for the duration of the experiment beginning 20 min prior to LTP induction. Slices that did not exhibit stable fEPSP slopes during the first 10 min of recording were excluded from the study. Zebularine and 5-aza-2-deoxycytidine were purchased from Calbiochem. 6-Cyano-7-nitroquinoxaline-2,3-dione was purchased from Tocris Cookson, Inc. (Ellisville, MO). PDA was purchased from Sigma. One-sample t tests were used to assess changes in the methylation state of regions 1 and 2 of the reelin and BDNF1 promoters and changes in expression of DNMT genes in the hippocampus. One-way analysis of variance combined with Bonferroni's multiple comparison test was used to analyze changes in unmethylated DNA between Regions 1 and 2 of the reelin promoter. Data relating fEPSP slope to fiber volley slope was fit with a single exponential function (Y = TOP × (1 – e–K × X)); TOP and K were compared between treatment groups using an F test. Paired-pulse facilitation and LTP experiments were analyzed using a two-way analysis of variance with repeated measures. Analysis of histone acetylation was performed using analysis of variance; post hoc comparisons were made using the Tukey-Kramer test. Significance was set at p ≤ 0.05 for all tests. We hypothesized that (cytosine-5) DNA methylation in the CNS is, at least in part, a dynamic process that plays a role in information storage. Therefore, we investigated whether genes in the hippocampus could undergo acute changes in methylation status in response to inhibition of DNMT. Reelin is a large (∼340-kDa) extracellular protein that was first discovered in the context of neural development but has since been implicated in formation of long term memory, synaptic plasticity, and cognition (40Impagnatiello F. Guidotti A.R. Pesold C. Dwivedi
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