Ca2+/Calmodulin-dependent Protein Kinase IV Links Group I Metabotropic Glutamate Receptors to Fragile X Mental Retardation Protein in Cingulate Cortex*

Fragile X syndrome is caused by a lack of fragile X mental retardation protein (FMRP) due to silencing of the FMR1 gene. The metabotropic glutamate receptors (mGluRs) in the central nervous system contribute to higher brain functions including learning/memory, persistent pain, and mental disorders. Our recent study has shown that activation of Group I mGluR up-regulated FMRP in anterior cingulate cortex (ACC), a key region for brain cognitive and executive functions; Ca2+ signaling pathways could be involved in the regulation of FMRP by Group I mGluRs. In this study we demonstrate that stimulating Group I mGluRs activates Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) in ACC neurons. In ACC neurons of adult mice overexpressing CaMKIV, the up-regulation of FMRP by stimulating Group I mGluR is enhanced. The enhancement occurs at the transcriptional level as the Fmr1 mRNA level was further elevated compared with wild-type mice. Using pharmacological approaches, we found that inhibition of CaMKIV could attenuate the up-regulation of FMRP by Group I mGluRs. CaMKIV contribute to the regulation of FMRP by Group I mGluRs probably through cyclic AMP-responsive element binding protein (CREB) activation, as manipulation of CaMKIV could simultaneously cause the change of CREB phosphorylation induced by Group I mGluR activation. Our study has provided strong evidence for CaMKIV as a molecular link between Group I mGluRs and FMRP in ACC neurons and may help us to elucidate the pathogenesis of fragile X syndrome.

FMRP, an mRNA-binding protein, is involved in activity-dependent synaptic plasticity through regulation of local protein synthesis at synapses (6 -12). The function of Group I metabotropic glutamate receptor (mGluR) activation require the translation of pre-existing mRNA near active synapses. The abnormal functions of Group I mGluR-dependent synaptic plasticity have been observed in hippocampus of Fmr1 knockout (KO) mice (8,(13)(14)(15). Because FMRP normally functions as a repressor of translation of specific mRNAs (6,7,14,16), it is supposed that the protein synthesis-dependent functions of Group I mGluRs are exaggerated because of the lack of FMRP in fragile X syndrome (13,14,17).
The anterior cingulate cortex (ACC) plays an important role in cognitive learning, fear memory, and persistent pain (18 -24). Previous studies have shown that trace fear memory is impaired in Fmr1 KO mice accompanied by alterations in synaptic plasticity in ACC (25,26). These findings suggest that the dysfunction of ACC due to lack of FMRP may be responsible for certain types of mental disorders in fragile X syndrome. Electrophysiological and behavioral studies in animals found that the mGluRs in ACC may contribute to activity-dependent synaptic plasticity and behavioral fear memory (27,28). The regulation of FMRP by mGluRs has been mostly studied in hippocampal neurons (9,13,14,29,30) (see Table 1). Our recent study has found that activation of Group I mGluRs regulates the expression of FMRP in ACC neurons; Ca 2ϩ -dependent signaling is involved in the regulation of FMRP by Group I mGluRs (31). These findings indicate a possible signaling pathway linking mGluRs to FMRP in ACC. Loss of this signaling pathway may contribute to the pathogenesis of fragile X syndrome.
In the present study we have demonstrated that Ca 2ϩ / calmodulin-dependent protein kinase IV (CaMKIV), a key neuronal Ca 2ϩ signaling molecule in synaptic plasticity (32)(33)(34)(35)(36), is critical for regulation of FMRP by Group I mGluRs in ACC neurons. Stimulating Group I mGluRs with DHPG, an agonist of Group I mGluRs, activates CaMKIV in cingulate cortex. Using transgenic mice overexpressing CaMKIV (35), we found that CaMKIV contributes to the up-regulation of FMRP by Group I mGluRs. By measuring Fmr1 mRNA, we found that overexpression of CaMKIV enhanced the transcription of Fmr1 gene. In addition, we provided evidence that CaMKIV is involved in regulation of FMRP by Group I mGluRs through activation of cyclic AMP-responsive element binding protein (CREB). These findings were further confirmed by pharmacological inhibition of CaMKIV. We propose that CaMKIV is a key molecule in regulation of FMRP Group I mGluRs through the Ca 2ϩ -stimulated CREB signaling pathway. CaMKIV may play an isoform-specific role in regulation of FMRP by Group I mGluRs in ACC neurons, as the CaMK inhibitor did not show effect in CaMKIV KO mice.

EXPERIMENTAL PROCEDURES
Animals-Adult male C57Bl/6 mice were used in most of experiments. The transgenic mice overexpressing CaMKIV and CaMKIV KO mice were generated and maintained as reported previously (32,33,35). All mice were housed under a 12:12 light cycle with food and water provided ad libitum. All mouse protocols are in accordance with National Institutes of Health guidelines and approved by the Animal Care and Use Committee of University of Toronto.
Brain Slice Preparations-Mice were anesthetized with 2% halothane, and brain slices (300 m) containing ACC were cut at 4°C using a Vibratome, in oxygenated artificial cerebrospinal fluid containing 124 mM NaCl, 4 mM KCl, 26 mM NaHCO 3 , 2.0 mM CaCl 2 , 1.0 mM MgSO 4 , 1.0 mM NaH 2 PO 4 , 10 mM D-glucose, pH 7.4]. The slices were slowly brought to final temperature of 30°C in artificial cerebrospinal fluid gassed with 95% O 2 , 5% CO 2 and incubated for at least 1 h 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.
Immunoprecipitation-For detection of CaMKIV phosphorylation, the solubilized protein samples were prepared with lysis buffer and precipitated with 50 l of protein G-agarose (Sigma-Aldrich) precoupled with anti-CaMKIV antibody for 4 h at 4°C. The reaction mixtures were then washed three times, eluted by boiling in loading buffer, and subjected to Western blot using anti-phosphothreonine antibody.
Western Blot Analysis-Western blot was conducted as previously described (21,37). The brain tissues were dissected and homogenized in lysis buffer containing 10 mM Tris-HCl, pH 7.4, 2 mM EDTA, 1% SDS, 1ϫ protease inhibitor mixture, and 1ϫ phosphatase inhibitor mixture 1 and 2. Protein concentration was measured by Bradford protein assay (Bio-Rad). Electrophoresis of equal amounts of total protein was performed on NuPAGE 4 -12% Bis-Tris gels (Invitrogen). Separated proteins were transferred to polyvinylidene fluoride membranes (Pall Corp., East Hills, NY) at 4°C for analysis. Membranes were probed with a 1:3000 dilution of anti-FMRP, or a 1:1000 dilution of anti-phospho-CREB (Ser-133) and anti-CREB, or a 1:2000 dilution of anti-CaMKIV antibodies and anti-phosphothreonine antibody. 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 Sciences) according to the manufacturer's instructions. To verify equal loading, membranes were also probed with a 1:3000 dilution of anti-actin antibody. The density of immunoblots was measured using the NIH ImageJ program.
RT-PCR-The total RNA from the ACC was isolated using RNAspin Mini kit (GE Healthcare). RT-PCR was carried out using Qiagen One-Step RT-PCR kit (Qiagen, Hilden, Germany). A 25-l PCR reaction contained 0.5 g of total RNA, 5 l of 5ϫ RT-PCR buffer, 400 M dNTP, and 0.5 M concentrations of each primer and 1 l of RT-PCR enzyme. PCR conditions were adjusted to be in a linear range of amplification. The PCR cycles consisted of initial incubation at 94°C for 1 min, denaturation at 94°C for 45 s, annealing at 56°C for 45 s, and extension at 72°C for 1 min, for 30 cycles, and a final extension at 72°C for 10 min. The primers for Fmr1 used in this experiment were as follows: sense, 5Ј-CCGAACAGATAATCGTC-CACG-3Ј; antisense, 5Ј-ACGCTGTCTGGCTTTTCCTTC-3Ј. Glyceraldehyde-3-phosphate dehydrogenase was amplified as an internal control by using the primer sets: sense, 5-AAC-GACCCCTTCATTGAC-3Ј; antisense, 5Ј-TCCACGACAT-ACTCAGCAC-3Ј. RT-PCR products were electrophoresed on 1.5% agarose gels and visualized under UV light by ethidium bromide staining. The relative density of bands was analyzed by the NIH ImageJ program.
Data Analysis-All data were presented as the mean Ϯ S.E. Statistical comparisons were made using the paired t test. In all cases p Ͻ 0.05 is considered statistically significant.

Activation of CaMKIV by Group I mGluRs in the ACC
Neurons-Activation of Group I mGluRs cause the increase of intracellular Ca 2ϩ and initiates the Ca 2ϩ signaling pathways (31, 38 -43). CaMKIV, a key Ca 2ϩ signaling molecule in synaptic plasticity, can be activated by the increase of intracellular Ca 2ϩ (31,33,34,44,45). The phosphorylation of CaMKIV at threonine residues is known to be critical for its kinase activity (44, 46 -48). Can CaMKIV be activated by stimulating Group I mGluRs in ACC neurons? To address this we investigated the effects of mGluR activation on the phosphorylation of CaMKIV at threonine residues in ACC slices from adult mice. We found that application of Group I mGluR agonist DHPG (100 M) to ACC slices for 15 min caused an increase in the phosphorylation of CaMKIV at threonine site in ACC slices (218 Ϯ 14% of the control levels, p Ͻ 0.01, compared with control, n ϭ 6, Fig.  1B). By contrast, neither application of Group II mGluR agonist (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (10 M) nor Group III mGluR agonist (1S,3R,4S)-1-aminocyclopentane-1,3,4-tricarboxylic acid (100 M) for 15 min affected the phosphorylation of CaMKIV in ACC slices (p Ͼ 0.05, compared with control, n ϭ 4, Fig. 1C). DHPG (100 M) increased the phosphorylation of CaMKIV in a time-dependent manner, the increase could be observed at 5 min, and the highest level was reached at 15 min (p Ͻ 0.01 or p Ͻ 0.05, compared with control, n ϭ 4, Fig. 1D). However, the presence of DHPG (100 M) did not affect the expression of CaMKIV in ACC slices (p Ͼ 0.05, compared with control, n ϭ 4, Fig. 1E). These data indicate that stimulating Group I mGluRs activates CaMKIV in the ACC neurons. To our knowledge this is the first time that it has been shown that CaMKIV can be activated by stimulating Group I mGluRs. The activation of CaMKIV suggests that CaMKIV might be a downstream effector for Group I mGluRs in cingulate cortex.
There are two subtypes, including mGluR1 and mGluR5, in Group I mGluRs (13, 49 -51). To identify which Group I mGluR subtype(s) was responsible for the activation of CaMKIV caused by DHPG, we applied DHPG (100 M) to ACC slices in the presence of either a selective mGluR1 antagonist MPMQ (10 M) or a specific mGluR5 antagonist of ACDPP (10 M). We found that the phosphorylation of CaMKIV caused by DHPG was only partially blocked by MPMQ or ACDPP (p Ͻ 0.05, compared with DHPG treatment, n ϭ 6, Fig. 2, A and B). The presence of Group I mGluR antagonist DL-AP3 (100 M) completely blocked the phosphorylation of CaMKIV caused by DHPG in ACC slices (221 Ϯ 13 and 102 Ϯ 12% of the control levels for DHPG or DL-AP3 treatment, respectively, p Ͻ 0.01, compared with DHPG treatment, n ϭ 6, Fig. 2C). However, the application of MPMQ, ACDPP, or DL-AP3 alone did not affect the basal phosphorylation levels of CaMKIV in ACC slices (p Ͼ 0.05, compared with control, n ϭ 4, Fig. 2D). These data further confirm that stimulating Group I mGluRs can activate CaMKIV, and both mGluR1 and mGluR5 are involved in the phosphorylation of CaMKIV caused by DHPG in ACC neurons.
Calcium Is Critical for the Activation of CaMKIV by Group I mGluRs-The activation of Group I mGluRs can induce Ca 2ϩ release from trisphosphate-sensitive intracellular stores (41,49,52,53). The sarco/endoplasmic reticulum Ca 2ϩ /ATPase (SERCA) pump inhibitor cyclopiazonic acid depletes intracellular stores of Ca 2ϩ by blocking Ca 2ϩ re-uptake into the stores (41,53). To examine the role of Ca 2ϩ release from intracellular stores in the phosphorylation of CaMKIV induced by DHPG, cyclopiazonic acid (CPA, 30 M) was applied to ACC slices 20 min before and during the DHPG (100 M, 15 min) treatment. We found that the phosphorylation of CaMKIV induced by DHPG was partially blocked by cyclopiazonic acid (223 Ϯ 10 and 152 Ϯ 12% that of the control levels for DHPG and CPA treatment, respectively, p Ͻ 0.05, compared with DHPG treatment, n ϭ 6, Fig. 3A).
Stimulating Group I mGluRs can facilitate L-type voltage-dependent Ca 2ϩ channels (L-VDCCs) in different cell types (39 -42). DHPG treatment may induce Ca 2ϩ influx through L-VDCCs in striatal neurons (40). In this study we found that application of L-VDCC blocker nifedipine (Nif, 25 M) 20 min before and during the DHPG treatment also partially blocked the phosphorylation of CaMKIV caused by DHPG (217 Ϯ 13 and 150 Ϯ 10% of the control levels for DHPG and nifedipine treatment, respectively, p Ͻ 0.05, compared with DHPG treatment, n ϭ 6, Fig. 3B). In addition, coapplication of cyclopiazonic acid with nifedipine almost completely blocked the phosphorylation of CaMKIV induced by DHPG treatment (220 Ϯ 14 and 103 Ϯ 13% that of the control levels for DHPG and CPA plus nifedipine treatment, respectively, p Ͻ 0.01, compared with DHPG treatment, n ϭ 6, Fig. 3C). By contrast, cyclopiazonic acid, nifedipine, or coapplication of cyclopiazonic acid and nifedipine did not affect the basal phosphorylation levels of CaMKIV in ACC slices (p Ͼ 0.05, compared with control, n ϭ 4, Fig. 3D). These data indicate that both Ca 2ϩ release from intracellular stores and Ca 2ϩ influx through L-VDCCs are involved in the activation of CaMKIV by Group I mGluRs in ACC neurons.
To further confirm the role of calcium in the activation of CaMKIV by Group I mGluRs, we then tested the effect of raising intracellular Ca 2ϩ by application of calcium ionophore A23187 (54,55)in ACC slices. We found that application of calcium ionophore A23187 (5 M, 15 min, and 30 min) could induce the phosphorylation of CaMKIV in ACC slices (169 Ϯ 8 and 157 Ϯ 9% of the control levels for A23187 at 15 and 30 min, respectively; p Ͻ 0.01, compared with control, n ϭ 4, Fig. 3E).
Coapplication of calcium ionophore (5 M, 15 min) and DHPG (100 M, 15 min) enhanced the phosphorylation of CaMKIV caused by DHPG (216 Ϯ 11 and 262 Ϯ 12% that of the control levels for DHPG or A23187 treatment, respectively, p Ͻ 0.05, compared with DHPG treatment, n ϭ 4, Fig. 3F). These data provide additional evidence for the role of calcium in the activation of CaMKIV by Group I mGluRs in ACC neurons.
Raising Intracellular Calcium or CaMKIV Enhances Regulation of FMRP by Group I mGluRs in the ACC Neurons-Previous studies of the regulation of FMRP by mGluRs were mainly carried out in cultured neurons and hippocampal slices (9, 13, 30, 56 -58) (see Table 1). Our recent study has found that the activation of Group I mGluRs by DHPG increased the expression of FMRP in ACC slices from adult mice, and calcium signaling is critical for the regulation of FMRP by Group I mGluRs (31).
Because raising intracellular Ca 2ϩ by application of calcium ionophore A23187 could activate CaMKIV and enhance the phosphorylation of CaMKIV by stimulating Group I mGluRs, we then tested the effect of calcium ionophore A23187 on FMRP in ACC neurons. We found that application of calcium ionophore A23187 (5 M, 15 and 30 min) did not affect the expression FMRP in ACC slices (p Ͼ 0.05, compared with control, n ϭ 5, Fig. 4A). These data indicate that simply raising intracellular calcium cannot up-regulate FMRP, although it can activate CaMKIV. However, coapplication of calcium ionophore A23187 (5 M, 30 min) and DHPG (100 M, 30 min) enhanced the up-regulation of FMRP caused by DHPG (197 Ϯ 10 and 246 Ϯ 13% that of the control levels for DHPG or A23187 treatment, respectively, p Ͻ 0.05, compared with DHPG treatment, n ϭ 5, Fig. 4B). Taken together these results suggest that calcium signaling may specifically contribute to the regulation of FMRP by Group I mGluRs.
To explore the role of CaMKIV in the up-regulation of FMRP by stimulating Group I mGluRs, we have taken the advantage of another line of transgenic mice for CaMKIV. In these transgenic mice, the expression of CaMKIV was selectively up-regulated in the forebrain. The transgenic mice could develop normally and did not exhibit any abnormalities in brain structures. However, overexpression of CaMKIV enhanced the animal ability to form long term memory and rescued memory loss with aging (35).
By Western blot we found that the expression of CaMKIV was upregulated in ACC slices from these transgenic mice (162 Ϯ 8% of the WT levels, p Ͻ 0.01, compared with WT mice, n ϭ 5, Fig. 4C). We next tested the effect of DHPG (100 M, 30 min) treatment in ACC slices from mice overexpressing CaMKIV. There was no difference in the basal levels of FMRP in ACC slices between WT and CaMKIV overexpression mice (p Ͼ 0.05, compared with WT mice, n ϭ 5, Fig. 3D). DHPG treatment could increase expression of FMRP in ACC slice; the increase of FMRP was further enhanced in ACC slices from mice overexpressing CaMKIV compared with WT mice (192 Ϯ 9 and 252 Ϯ 11% that of the WT control levels for WT and CaMKIV overexpression mice, respectively; p Ͻ 0.05, compared with WT mice, n ϭ 5, Fig.  4E). The data indicate that overexpression of CaMKIV can enhance the up-regulation of FMRP by Group I mGluRs in ACC neurons.
Pharmacological Inhibition of CaMKIV in ACC Neurons-To rule out the possibility that the findings from transgenic mice might be caused by developmental changes due to genetic manipulation, we next tested the effect of CaMK inhibitors KN93 and KN62 (59,60). We found that application of KN93 (10 M) or KN62 (10 M) for 30 min did not affect the basal levels of FMRP in ACC slices (p Ͼ 0.05, compared with control, n ϭ 4, Fig. 5A). However, application of KN93 (10 M) or KN62(10 M) 20 min before and during the DHPG treatment could partially block the increase of FMRP caused by DHPG (198 Ϯ 12, 149 Ϯ 8, and 146 Ϯ 10% that of the control levels for DHPG, KN93, and KN62 treatment, respectively, p Ͻ 0.05, compared with DHPG treatment, n ϭ 6, Fig. 5B). The data indicate that CaMKs are involved in the regulation of FMRP by Group I mGluRs in ACC neurons.  JULY 10, 2009 • VOLUME 284 • NUMBER 28

JOURNAL OF BIOLOGICAL CHEMISTRY 18957
To address the isoform-specific roles of CaMKIV, we then tested the effect of CaMK inhibitor in ACC slices from CaMKIV KO mice. We found that the increase of FMRP caused by DHPG was partially blocked in ACC slices from CaMKIV KO mice compared with that of WT mice (p Ͻ 0.05, n ϭ 4, Fig.  5C). However, application of KN93 (10 M) 20 min before and during the DHPG treatment did not cause further reduction in FMRP levels in ACC slices from CaMKIV KO mice (p Ͼ 0.05, n ϭ 4, Fig. 5C). These data suggested that CaMKIV plays an isoform-specific role in the regulation of FMRP Group I mGluRs in ACC neurons; other isoforms of CaMKs, such as CaMKII, may not be involved in this process.
CaMKIV and Transcriptional Expression of Fmr1 Gene by Group I mGluRs-One previous study has shown that stimulation of Group I mGluRs with DHPG induces the increase of FMRP in a protein synthesis-dependent manner in hippocampus CA1 area (13). However, we found that the regulation of FMRP by Group I mGluRs occurs at the transcriptional level in ACC (31).
To further address the role of CaMKIV in the transcriptional expression of Fmr1 gene by Group I mGluRs in cingulate cortex, we measured the levels of Fmr1 mRNA by RT-PCR. We found that there was no difference in the basal levels of Fmr1 mRNA in ACC between WT and CaMKIV overexpression mice (p Ͼ 0.05, n ϭ 4, Fig. 6A). However, the increase of Fmr1 mRNA caused by DHPG treatment was enhanced in ACC slices from mice overexpressing CaMKIV compared with WT mice (205 Ϯ 10 and 259 Ϯ 9% that of the WT control levels for WT and CaMKIV overexpression mice, respectively; p Ͻ 0.05, compared with WT mice, n ϭ 4, Fig. 6B). We next tested the effect of CaMKIV inhibitor on transcriptional expression of Fmr1 gene by Group I mGluRs. We found that application of CaMKIV inhibitor KN93 (10 M) or KN62 (10 M) for 20 min did not affect the Fmr1 mRNA in ACC slices (p Ͼ 0.05, n ϭ 4, Fig. 6C). By contrast, application of KN93 (10 M) 20 min before and during the DHPG treatment partially blocked the increase of Fmr1 mRNA caused by DHPG (203 Ϯ 12 and 152 Ϯ 9% that of the control levels for DHPG and KN93 treatment, respectively, p Ͻ 0.05, compared with DHPG treatment, n ϭ 4, Fig. 6D). These data indicate that CaMKIV is required for the transcriptional regulation of FMRP by Group I mGluRs in ACC neurons.
CaMKIV and CREB Activation by Stimulating Group I mGluRs-Phosphorylated CREB (pCREB) binds to the cAMP response element (CRE) site in the gene promoter and activates gene transcription (61)(62)(63). It has been reported that the FMR1 gene promoter contains the CRE site (64,65). Our recent study  Anterior cingulate cotex slices Group I mGluR agonist DHPG Transcription-dependent up-regulation of FMRP Wang et al. (31) and this study found that DHPG treatment could increase the pCREB levels in ACC slices and suggests that the regulation of FMRP by Group I mGluRs in ACC neurons likely occurs through CREB activation (31). CaMKIV activates the transcription factor CREB by phosphorylating CREB at the regulatory Ser-133 residue (34,48,62). Up-regulation of CaMKIV enhanced learning-induced CREB activity in the CA1 region of hippocampus (35). To further investigate whether CaMKIV is involved in the phosphorylation of CREB caused by stimulating Group I mGluRs, we then tested the phosphorylation of CREB induced by DHPG (100  M, 15 min) in ACC slices from mice overexpressing CaMKIV. We found that the basal levels of pCREB were not changed in ACC slices from mice overexpressing CaMKIV (p Ͼ 0.05, compared with WT, n ϭ 4, Fig. 7A). However, the phosphorylation of CREB induced by DHPG treatment was enhanced in ACC slices from mice overexpressing CaMKIV as compared with WT mice (221 Ϯ 12 and 293 Ϯ 14% that of the WT control levels for WT and CaMKIV overexpression mice, respectively; p Ͻ 0.05, compared with WT mice, n ϭ 4, Fig. 7B). These results indicate that CaMKIV contributes to the phosphorylation of CREB induced by stimulating Group I mGluRs in ACC neurons. Consistently, application of CaMKIV inhibitor KN93 (10 M) or KN62 (10 M) for 20 min before and during DHPG (100 M, 15 min) treatment partially blocked the increase of pCREB caused by DHPG in ACC slices (223 Ϯ 11, 155 Ϯ 9, and 156 Ϯ 13% that of the control levels for DHPG, KN93, and KN62 treatment, respectively, p Ͻ 0.05, compared with DHPG treatment, n ϭ 6, Fig. 7D). By contrast, application of KN93 (10 M) or KN62 (10 M) for 20 min did not affect the basal levels of pCREB in ACC slices (p Ͼ 0.05, compared with control, n ϭ 4, Fig. 7C). These results indicate that CaMKIV is the key molecule that phosphorylates CREB during Group I mGluR activation in ACC neurons.

DISCUSSION
Our previous studies suggest that FMRP is required for the physiological function of ACC (21,25), and the mGluRs in ACC may contribute to the activity-dependent synaptic plasticity and fear memory (27,28). Recently, we provided the direct biochemical evidence that activation of Group I mGluRs up-regulates FMRP in the ACC neurons of adult mice. The up-regulation of FMRP by Group I mGluRs occurs at the transcriptional level. Activation of Group I mGluRs induced the phosphorylation of CREB in ACC neurons (31). In this study we provided novel evidence that stimulating Group I mGluRs activates CaMKIV in ACC neurons. CaMKIV contributes to the up-regulation of FMRP and the phosphorylation of CREB induced by stimulating Group I mGluRs. Our results indicate that CaMKIV acts as a key intracellular messenger for signaling pathways between Group I mGluRs and FMRP in cingulate cortex.
Group I mGluRs, Ca 2ϩ , and CaMKIV Activation-The Ca 2ϩ signaling pathways are believed to play a pivotal role in synaptic plasticity (45,62,63,66). CaMKIV, a key Ca 2ϩ signaling molecule in Ca 2ϩ signaling pathways, can be activated by the increase of intracellular Ca 2ϩ (32-34, 45, 67). Stimulation of Group I mGluRs can cause the increase of intracellular Ca 2ϩ and initiates the Ca 2ϩ signaling pathways (31,40,42). In this study, we found that stimulating Group I mGluRs could induce the phosphorylation of CaMKIV at threonine residues, which is known to be critical for its kinase activity (44,46,47,68). Both mGluR1 and mGluR5 are involved in the activation of CaMKIV by Group I mGluRs. These data provide novel evidence that CaMKIV can be activated by stimulating Group I mGluRs.
Ca 2ϩ is released from trisphosphate-sensitive intracellular stores upon Group I mGluR activation (41,53,69). We found that application of sarco/endoplasmic reticulum Ca 2ϩ /ATPase pump inhibitor cyclopiazonic acid before and during Group I mGluR activation partially blocked the phosphorylation of CaMKIV due to Group I mGluR activation. Another possible source for intracellular Ca 2ϩ is Ca 2ϩ influx through L-VDCCs. Previous studies have shown that membrane depolarization by DHPG treatment can trigger the opening of L-VDCCs (38,40,42). Here, we found that application of L-VDCC blocker nifedipine also partially blocked the phosphorylation of CaMKIV caused by Group I mGluR activation. When the sarco/endoplasmic reticulum Ca 2ϩ / ATPase pump inhibitor cyclopiazonic acid was co-applied with nifedipine to ACC slices, the phosphorylation of CaMKIV was almost completely blocked. It indicates that both Ca 2ϩ release from intracellular stores and Ca 2ϩ influx from L-VDCCs are required for the activation of CaMKIV by Group I mGluRs in ACC neurons.
CaMKIV and the Regulation of FMRP by Group I mGluRs-It is well known that CaMKIV transduces Ca 2ϩ signaling and functions as a transcriptional activator in synaptic plasticity (33,34,48,62). It is likely that CaMKIV might be involved in the signaling pathway downstream of Group I mGluRs. By using mice overexpressing CaMKIV, we confirmed that CaMKIV acts downstream of Group I mGluRs and contributes to the regulation of FMRP by Group I mGluRs in ACC neurons.
In addition, genetic deletion of CaMKIV or inhibiting CaMKs could partially block the increase of FMRP induced by stimulating Group I mGluRs in ACC slices. This result further support the conclusion that CaMKIV is required for the up-regulation of FMRP by Group I mGluRs in ACC neurons. Pharmacological inhibition of CaMKs did not affect regulation of FMRP by Group I mGluRs in ACC slices from CaMKIV KO mice. It suggests that CaMKIV may play an isoform-specific role in regulation of FMRP by Group I mGluRs in cingulate cortex.
In this study we also found that raising intracellular calcium by application of calcium ionophore A23187 did not affect the expression of FMRP, although it could activate CaMKIV and enhance the phosphorylation of CaMKIV and the up-regulation of FMRP by stimulating Group I mGluRs in ACC slices. These results suggest that intracellular calcium and CaMKIV, which can be shared by many different signaling pathways, may specifically contribute to the regulation of FMRP by Group I mGluRs.
CaMKIV and CREB Activation by Group I mGluRs-The CREB is a transcriptional factor that plays an important role in synaptic plasticity (62,70,71). The activity of CREB is regulated by its phosphorylation; pCREB binds to the CRE site within the gene and activates the gene transcription (62,63,71). Previous studies have shown that there is the CRE site in FMR1 promoter and implicated CREB in the regulation of the FMR1 gene transcription in neural cells (65,72). Our recent study found that the Group I mGluR-dependent regulation of FMRP in ACC neurons occurs at the transcriptional level, as new Fmr1 mRNA is transcribed, as shown by RT-PCR; the up-regulation of FMRP is accompanied by the phosphorylation of CREB (Ser-133) (31). These results supported that CREB acts as a transcriptional factor for Group I mGluR-dependent regulation of FMRP in the neurons.
There are several signaling pathways that lead to the activation of CREB. Ca 2ϩ and CaMKIV is implicated in various aspects of the neuronal Ca 2ϩ signaling pathways, inducing the phosphorylation of CREB and the gene expression in response to excitatory neurotransmission (32,34,35,62). The phosphorylation of CREB induced by the Group I mGluR agonist DHPG was partially blocked in ACC slices from CaMKIV KO mice, suggesting CaMKIV could be involved in the activation of CREB induced by stimulating Group I mGluRs (31). In this study we have shown that the phosphorylation of CREB induced by DHPG was enhanced in ACC slices from mice overexpressing CaMKIV. In addition, pharmacological inhibition of CaMKIV also partially blocked phosphorylation of CREB induced by DHPG. These novel findings further confirm that CaMKIV is critical for phosphorylation of CREB induced by stimulating Group I mGluRs, and CaMKIV contributes to regulation of FMRP by Group I mGluRs probably through CREB activation.
In summary, we have demonstrated that the CaMKIV is critical for regulation of FMRP by Group I mGluRs in ACC neurons by using genetic and pharmacological approaches. CaMKIV is activated by stimulating Group I mGluRs and contributes to the up-regulation of FMRP probably through the activation of CREB. Our study has provided strong evidence for CaMKIV as the key signaling molecule for regulation of FMRP by Group I mGluRs in cingulate cortex and may help to further elucidate the cellular mechanisms underlying fragile X syndrome.