Roles of CREB in the regulation of FMRP by group I metabotropic glutamate receptors in cingulate cortex

Phosphorylated CREB (pCREB) binds to cAMP response element (CRE) site in gene promoters and activates gene transcription[41, 42, 45, 51, 52]. It has been reported that the FMR1 gene promoter contains the CRE site[53, 54]. Our recent study had found that (RS)-3, 5-Dihydroxyphenylglycine ((RS)-3, 5-DHPG) treatment could upregulate FMRP and increase the pCREB levels in ACC slices, suggesting that the regulation of FMRP by Group I mGluRs in ACC neurons likely occurs through CREB activation[37, 38].

Overexpression of dominant active CREB mutant in the forebrain could positively regulate memory consolidation and enhance memory performance by upregulating the expression of Brain derived neurotrophic factor (BDNF)[47], which is well known as a CREB target gene[40, 42, 55]. To further investigate whether CREB is involved in the upregulation of FMRP caused by stimulating Group I mGluRs, we then tested the expression of FMRP induced by the Group I mGluR agonist DHPG (100 μM, 30 min) treatment in ACC slices from mice overexpressing CREB. By Western blot, we found that there was no difference in the basal levels of FMRP in ACC slices between WT and CREB overexpression mice (P > 0.05, compared with WT mice, n = 5, Figure1A). DHPG treatment could increase expression of FMRP in ACC slices; the increase of FMRP was further enhanced in ACC slices from mice overexpressing CREB compared to WT mice (198 ± 11% and 248 ± 14% of the WT control levels for WT and CREB overexpression mice, respectively. In two-way ANOVA analysis, for genotype, F = 13.39, P < 0.01; for treatment, F = 254.87, P < 0.01; genotype X treatment, F = 8.26, P < 0.05; n = 5 for each group, Figure1B). The data indicates that overexpression of CREB can enhance the upregulation of FMRP induced by Group I mGluR activation. It provides further evidence that CREB is involved in the regulation of FMRP by Group I mGluRs in ACC neurons.

Since transcriptional repressor DREAM interacts with CREB in a Ca²⁺ dependent manner and prevents the recruitment of CREB-binding protein (CBP) blocking CRE-dependent gene transcription[48, 56], we next checked whether DREAM might be involved in the regulation of FMRP by Group I mGluRs through CREB signaling pathway. To explore the role of DREAM in the upregulation of FMRP by stimulating Group I mGluRs, we have taken the advantage of transgenic mice overexpressing a Ca²⁺-insensitive DREAM mutant (TgDREAM)[49, 50]. The TgDREAM mice could develop normally and did not exhibit any abnormalities in brain structures. However, overexpression of mutant DREAM impaired NMDA receptor-mediated synaptic plasticity and contextual fear memory[50].

We next tested the effect of DHPG (100 μM, 30 min) treatment in ACC slices from TgDREAM mice. Importantly, no difference in the basal levels of FMRP in ACC slices was observed between WT and TgDREAM mice (P > 0.05, compared with WT mice, n = 5, Figure1C). Furthermore, the increase of FMRP after DHPG treatment was not affected in ACC slices from TgDREAM mice compared to WT mice (199 ± 10% and 201 ± 9% of the WT control levels for WT and TgDREAM mice, respectively. In two-way ANOVA analysis, for genotype, F = 0.10, P = 0.75; for treatment, F = 249.81, P < 0.01; for genotype X treatment, F = 0.001, P = 1.00; n = 5 for each group, Figure1D). The data indicate that overexpression of Ca²⁺-insensitive mutant form of DREAM does not affect the upregulation of FMRP induced by Group I mGluR activation, suggesting that DREAM might not be involved in the CREB-dependent regulation of FMRP by Group I mGluRs in ACC neurons.

To identify conserved sequences, 20 kb of mouse genomic sequence including the FMR1 transcription start site (TSS) was aligned among multiple mammalian species using the UCSC Genome browser (Figure2). Sequences of multiple mammalian species were then scanned for matches to the consensus sequence of CRE (TGACGTCA). Two putative CREs (upstream CRE, -48 ~ −45; downstream CRE, +106 ~ +113) were found in the highly conserved regions across multiple mammals (Figure2v). The upstream putative CRE has been reported as a potential CRE in human FMR1 promoter[53, 54]. Comparisons of putative CRE sequences among mammalian species are shown in Figure2B. These data support our finding that the FMR1 is a target gene of CREB.

Adult male C57Bl/6 mice were used in most of experiments. The transgenic mice overexpressing dominant active mutant CREB (Y134F) or Ca²⁺ insentive DREAM were generated and maintained as reported previously[47, 50]. All mice were housed under a 12:12 light cycle with food and water provided ad libitum. All mouse protocols are in accordance with NIH guidelines and approved by the Animal Care and Use Committee of University of Toronto.

(RS)-3, 5-DHPG was purchased from Tocris Bioscience (Ellisville, MO). phosphatase inhibitor cocktail 1 and 2 were purchased from Sigma-Aldrich (St. Louis, MO). The anti-FMRP antibody, horseradish peroxidase-linked goat anti-mouse IgG and goat anti-rabbit IgG for Western blot were purchased from Chemicon International (Temecula, CA). The anti-phospho-threonine antibody, anti-CREB antibody and anti-phosph CREB antibody were purchased from Cell Signaling Technology (Danvers, MA). The anti-actin antibody was from Sigma-Aldrich (St. Louis, MO).

Mice were anesthetized with 2% halothane and brain slices (300 μm) containing ACC were cut at 4°C using a Vibratome, in oxygenated artificial cerebrospinal fluid [ACSF; containing the following (in mM): 124 NaCl, 4 KCl, 26 NaHCO₃, 2.0 CaCl2, 1.0 MgSO₄, 1.0 NaH₂PO₄, 10 D-glucose, pH 7.4]. The slices were slowly brought to final temperature of 30°C in ACSF gassed with 95% O₂/5% CO₂ and incubated for at least 1 hour before experiments. Slices then were exposed to different compounds of interest for the indicated times and snap frozen over dry ice. For biochemical experiments, the ACC regions were microdissected and sonicated in ice-cold homogenization buffer containing phosphatase and protease inhibitors.

Western blot was conducted as previously described[25, 38]. The brain tissues were dissected and homogenized in lysis buffer containing 10 mM Tris–HCl (pH 7.4), 2 mM EDTA, 1% SDS, 1X protease inhibitor cocktail, and 1X phosphatase inhibitor cocktail 1 and 2. Protein concentration was measured by Bradford protein assay (Bio-Rad, Hercules, CA). Electrophoresis of equal amounts of total protein was performed on NuPAGE 4-12% Bis-Tris Gels (Invitrogen, Carlsbad, CA). Separated proteins were transferred to polyvinylidene fluoride membranes (Pall Corporation, East Hills, NY) at 4°C for analysis. Membranes were probed with 1:3000 dilution of anti-FMRP, or 1:1000 dilution of anti-phospho-CREB (Ser133) and anti-CREB antibodies. The membranes were incubated in the appropriate horseradish peroxidase-coupled secondary antibody diluted 1:3000 for 2 h followed by enhanced chemiluminescence (ECL) detection of the proteins with Western Lightning Plus-ECL (PerkinElmer Life and Analytical Science Inc., Waltham, MA) according to the manufacturer’s instructions. To verify equal loading, membranes were also probed with 1:3000 dilution of anti-actin antibody. The density of immunoblots was measured using NIH ImageJ program.

All data were presented as the mean ± S.E.M. Statistical comparisons were performed by paired t-test or two-way ANOVA. In all cases, P < 0.05 is considered statistically significant.