Arc/Arg3.1 Translation Is Controlled by Convergent N-Methyl-D-aspartate and Gs-coupled Receptor Signaling Pathways
2007; Elsevier BV; Volume: 283; Issue: 1 Linguagem: Inglês
10.1074/jbc.m702451200
ISSN1083-351X
AutoresWendy A.C. Bloomer, Hendrika M.A. VanDongen, Antonius M.J. VanDongen,
Tópico(s)Phosphodiesterase function and regulation
ResumoArc/Arg3.1 is an immediate early gene whose expression is necessary for the late-phase of long-term potentiation (LTP) and memory consolidation. Whereas pathways regulating Arc transcription have been extensively investigated, less is known about the role of post-transcriptional mechanisms in Arc expression. Fluorescence microscopy experiments in cultured hippocampal neurons revealed that Arc protein level was dramatically increased by activation of the cAMP-dependent protein kinase (PKA) pathway, which is implicated in long-term memory. A PKA-dependent increase in Arc protein level was observed after pharmacological or synaptic activation of N-methyl-d-aspartate (NMDA) receptors, which play a critical role in both LTP induction and learning. Arc protein was also up-regulated by activation of PKA through Gs-coupled dopamine and β-adrenergic receptors, which regulate the late-phase of LTP and memory. When agonists for the NMDA and Gs-coupled receptors were co-applied, they had an additive effect on Arc protein expression. Interestingly, Gs-coupled receptor stimulation was ineffective in the presence of an NMDA receptor antagonist, suggesting calcium influx through the NMDA receptor plays a gating role in this pathway. Stimulation of the cAMP/PKA pathway did not affect Arc mRNA level or protein stability, identifying translational efficacy as the main determinant of Arc protein expression level. It is concluded that efficient Arc translation requires NMDA receptor activity, whereas a further enhancement can be achieved with activation of Gs-coupled receptors. These experiments have, therefore, revealed remarkable similarities in the signaling pathways that control Arc expression and those that regulate LTP, learning, and memory. Arc/Arg3.1 is an immediate early gene whose expression is necessary for the late-phase of long-term potentiation (LTP) and memory consolidation. Whereas pathways regulating Arc transcription have been extensively investigated, less is known about the role of post-transcriptional mechanisms in Arc expression. Fluorescence microscopy experiments in cultured hippocampal neurons revealed that Arc protein level was dramatically increased by activation of the cAMP-dependent protein kinase (PKA) pathway, which is implicated in long-term memory. A PKA-dependent increase in Arc protein level was observed after pharmacological or synaptic activation of N-methyl-d-aspartate (NMDA) receptors, which play a critical role in both LTP induction and learning. Arc protein was also up-regulated by activation of PKA through Gs-coupled dopamine and β-adrenergic receptors, which regulate the late-phase of LTP and memory. When agonists for the NMDA and Gs-coupled receptors were co-applied, they had an additive effect on Arc protein expression. Interestingly, Gs-coupled receptor stimulation was ineffective in the presence of an NMDA receptor antagonist, suggesting calcium influx through the NMDA receptor plays a gating role in this pathway. Stimulation of the cAMP/PKA pathway did not affect Arc mRNA level or protein stability, identifying translational efficacy as the main determinant of Arc protein expression level. It is concluded that efficient Arc translation requires NMDA receptor activity, whereas a further enhancement can be achieved with activation of Gs-coupled receptors. These experiments have, therefore, revealed remarkable similarities in the signaling pathways that control Arc expression and those that regulate LTP, learning, and memory. Activity-regulated, cytoskeletal-associated protein Arc, also known as Arg3.1, was identified as an immediate early gene, whose mRNA is induced by patterns of synaptic activity that elicit LTP (1Link W. Konietzko U. Kauselmann G. Krug M. Schwanke B. Frey U. Kuhl D. Link W. Proc. 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The signaling pathways controlling Arc transcription include several receptors (for NMDA, 2The abbreviations used are:NMDAN-methyl-d-aspartateqRT-PCRquantitative real-time PCRFPSfluorescence/pixel/sECFPenhanced cyan fluorescent proteinEYFPenhanced yellow fluorescent proteinhnRNPheterogeneous nuclear ribonucleoproteinmiRmicroRNAPKAcAMP-dependent protein kinaseRTroom temperatureCaMcalmodulinACadenylyl cyclaseUTRuntranslated region8-CPT8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic-monophosphatePBSphosphate-buffered salineCMVcytomegalovirusAPV2-amino-5-phosphonovaleric acid dopamine, serotonin, brain-derived neurotrophic factor, insulin, adrenaline, and acetylcholine), second messengers (Ca2+ and cAMP), protein kinases (cAMP-dependent protein kinases A and C (PKA and PKC), Src, extracellular signal-regulated kinase), and transcription factors (Egr1 and Egr3) (6Ying S.W. Futter M. Rosenblum K. Webber M.J. Hunt S.P. Bliss T.V. Bramham C.R. J. 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Neurosci. 2005; 21: 1703-1711Crossref PubMed Scopus (40) Google Scholar, 20Waltereit R. Dammermann B. Wulff P. Scafidi J. Staubli U. Kauselmann G. Bundman M. Kuhl D. J. Neurosci. 2001; 21: 5484-5493Crossref PubMed Google Scholar, 21Li L. Carter J. Gao X. Whitehead J. Tourtellotte W.G. Mol. Cell. Biol. 2005; 25: 10286-10300Crossref PubMed Scopus (165) Google Scholar). N-methyl-d-aspartate quantitative real-time PCR fluorescence/pixel/s enhanced cyan fluorescent protein enhanced yellow fluorescent protein heterogeneous nuclear ribonucleoprotein microRNA cAMP-dependent protein kinase room temperature calmodulin adenylyl cyclase untranslated region 8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic-monophosphate phosphate-buffered saline cytomegalovirus 2-amino-5-phosphonovaleric acid Post-transcriptional regulation of Arc protein expression has not been studied in as much detail. Brain-derived neurotrophic factor and reelin-integrin receptor signaling increase Arc protein synthesis in synaptoneurosome preparations (22Yin Y. Edelman G.M. Vanderklish P.W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2368-2373Crossref PubMed Scopus (226) Google Scholar, 23Dong E. Caruncho H. Liu W.S. Smalheiser N.R. Grayson D.R. Costa E. Guidotti A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5479-5484Crossref PubMed Scopus (99) Google Scholar). In addition, β-adrenergic receptor activation increases Arc protein expression and enhances memory without an increase in the fluorescence in situ hybridization signal of Arc mRNA (15McIntyre C.K. Miyashita T. Setlow B. Marjon K.D. Steward O. Guzowski J.F. McGaugh J.L. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10718-10723Crossref PubMed Scopus (211) Google Scholar). These results suggest that Arc protein expression can be regulated post-transcriptionally. The level at which this may occur is unknown and could include regulation of mRNA stability, translational efficacy, protein degradation, or a combination of the above. To further study the post-transcriptional regulation of Arc protein expression, both Arc mRNA and protein levels were determined in cultured hippocampal neurons using quantitative real-time PCR (qRT-PCR) and fluorescence microscopy. Stimulation of the cAMP/PKA pathway by either NMDA receptor or Gs-coupled receptor activation resulted in a strong up-regulation of Arc protein. Activation of the NMDA receptor results in calcium influx, which can activate PKA through calcium-calmodulin (Ca2+-CaM)-dependent adenylyl cyclases (ACs) type I and VIII, whereas D1/D5 dopamine and β-adrenergic receptor receptors activate ACs through the heterotrimeric GTP-binding protein Gs. The effects of activating NMDA and Gs-coupled receptors on Arc protein levels were additive, whereas NMDA receptor function was required for the dopamine and β-adrenergic effects. The PKA-induced increase in Arc protein level occurred without a change in Arc mRNA level or Arc protein stability but was prevented by a translation inhibitor. Together, these results show that NMDA and Gs-coupled receptor signaling pathways converge to regulate Arc translation. Cell Culture—Hippocampal rat brain tissue (E18) obtained from Brainbits, Inc. (Springfield, IL) was cultured as previously described (24Van de Ven T.J. VanDongen H.M.A. VanDongen A.M.J. J. Neurosci. 2005; 25: 9488-9496Crossref PubMed Scopus (59) Google Scholar). Briefly, cells were dissociated and plated at 50,000 cells/cm2 on poly-d-lysine-coated glass-bottomed dishes (MatTek, Ashland, MA) in a chemically defined medium of B27, glutamate, and glycine in Neurobasal medium (Invitrogen). Neurons were fed once weekly by replacing half the medium. For the Western blot and qRT-PCR experiments neurons were cultured at the same density in 6-well tissue culture plates from BD Biosciences Falcon. DNA Constructs—The Arc coding region with flanking untranslated regions (UTRs) was amplified by a PCR reaction using CHORI-230 rat BAC Clone DNA as a template (Children's Hospital Oakland-BACPAK Resources, Oakland, Ca) and was subcloned into the pGemT-easy vector (Promega, Madison WI). The region of the gene containing two introns was replaced with DNA amplified by PCR from Marathon rat brain cDNA (Clontech, Cambridge, UK) via restriction digest and ligation. A novel BsrGI restriction site was added directly before the stop codon in the Arc coding region by PCR using the Arc pGemT-easy construct without introns as template. EYFP from the Clontech vector EYFP-N1 was amplified by PCR using primers to incorporate BsrGI sites flanking the coding region. BsrGI-EYFP-BsrGI was subcloned into the Arc-pGemT-easy vector, and the Arc-EYFP DNA was subsequently subcloned into pcDNA3.1+ (Invitrogen). The Arc coding region construct was generated from DNA amplified by PCR from Marathon rat brain cDNA (Clontech) and subcloned into the pGemT-easy vector (Promega). The Arc coding region was then subcloned into EYFP-N1 (Clontech). Plasmids expressing cyan and red Fluorescent proteins (ECFP-C1 and dsRed2-C1, Clontech) were used as controls. The DNA sequence of the coding region of the monomeric form of EosFP was codon-optimized using Oligo Version 4.0 software (Molecular Biology Insights, Cascade, Co) and was synthesized and subcloned into pBluescript-SK by Epoch Biolabs (Sugarland, TX). EYFP was replaced by EosFP in the Arc-EYFPN1 plasmid by restriction digest and ligation. The sequence of the full-length Arc cDNA used in these experiments is identical to the NCBI rat Arc mRNA sequence with GenBank™ accession number NM_019361. In the Arc plasmid containing the coding region only, the Arc 5′-UTR has been replaced with the short optimal Kozak consensus sequence GCCACC, whereas the 3′-UTR was removed. Transfections and Stimulation—Neuronal cultures were transfected between day 17 and 25 as previously described (24Van de Ven T.J. VanDongen H.M.A. VanDongen A.M.J. J. Neurosci. 2005; 25: 9488-9496Crossref PubMed Scopus (59) Google Scholar). Briefly, cells were transfected with plasmid DNA using Lipofectamine 2000 (Invitrogen). For each transfection, 2 μg of DNA (1 μg of each plasmid) was added to 50 μl of Neurobasal medium, and 3 μl of Lipofectamine 2000 was added to a separate 50 μl of Neurobasal medium. After a 5-min room temperature (RT) incubation, the samples were combined and allowed to incubate for an additional 20 min at RT. Culture media was removed from the neurons, and the DNA/Lipofectamine 2000 solution was added dropwise to the cultures. After a 10-min incubation at 37 °C, the medium was replaced, and the dishes were returned to the incubator for 16 h. When stated, hippocampal neurons were stimulated with 50 μm forskolin, 1 mm 8-bromo-cAMP, 10 μm NMDA, 100 μm SKF-38393, 10 μm isoproterenol, 20 μm H89, 20 μm anisomycin, 10 μm MK-801, 10 μm APV, 10 μm 8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic-monophosphate (8-CPT), all from Sigma-Aldrich, or 10 μm MG-132 (Calbiochem) in the defined media. For the APV withdrawal experiments, neurons were cultured with conditioned media containing APV starting on day 5 in culture (day 5). Neurons were transfected on D23, and APV free media was added on D24. Immunofluorescence and Imaging—For immunofluorescence experiments we used the monoclonal C-7 Arc antibody (Santa Cruz Biotechnology, Santa Cruz, CA). C-7 is highly specific for Arc (Fig. 1A) and has been used previously for immunofluorescence (25Chowdhury S. Shepherd J.D. Okuno H. Lyford G. Petralia R.S. Plath N. Kuhl D. Huganir R. Worley P.F. Neuron. 2006; 52: 445-459Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar, 26Shepherd J.D. Rumbaugh G. Wu J. Chowdhury S. Plath N. Kuhl D. Huganir R.L. Worley P.F. Neuron. 2006; 52: 475-484Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar). Transfected neuron dishes were rinsed once with Dulbecco's phosphate-buffered saline with glucose and pyruvate (PBS) and fixed with a solution containing 4% paraformaldehyde, 4% sucrose, and 1× PBS for 15 min at 4 °C. Cells were then washed and imaged in PBS. Cells used for immunofluorescence were fixed with paraformaldehyde as above and permeabilized with ice-cold methanol for 20 min at -20 °C. After permeabilization, the cells were blocked with a solution containing 10% goat serum, 2% bovine serum albumin, and 1× PBS for 1 h at RT. Primary C-7 antibody diluted 1:100 in block was incubated overnight at 4 °C. The dishes were washed 3 times with block for 10 min each and incubated with the secondary antibody (Alexa Fluor 488-goat anti-mouse IgG, Molecular Probes-Invitrogen) diluted 1:500 in block solution for 1-2 h at RT. The dishes were washed as above with block then once with PBS for 10 min and imaged in PBS. Neurons were imaged as previously described (24Van de Ven T.J. VanDongen H.M.A. VanDongen A.M.J. J. Neurosci. 2005; 25: 9488-9496Crossref PubMed Scopus (59) Google Scholar). Briefly, cells were imaged with a Nikon (Tokyo, Japan) Diaphot inverted fluorescence microscope with a 40× oil immersion objective. A Hamamatsu (Shizuka, Japan) cooled digital camera and Open-lab Version 3.1.6 software (Improvision, Lexington, MA) were used to acquire the images. For the EosFP experiments neurons were treated with UV light for 30 s using a blue filter (Chroma Technology, Rockingham, VT) before imaging. Western Blot Analysis—Neuronal cultures were dissolved in lysis buffer (radioimmune precipitation assay buffer; 50 mm Tris HCl, pH 8.0, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and 1:100 dilution of protease inhibitor mixture (Sigma-Aldrich)), scraped into 1.5 ml tubes, and sonicated (24Van de Ven T.J. VanDongen H.M.A. VanDongen A.M.J. J. Neurosci. 2005; 25: 9488-9496Crossref PubMed Scopus (59) Google Scholar). To assure equal protein loading, the concentrations of the lysates were determined with the DC Protein Assay kit according to the microplate protocol (Bio-Rad). Samples consisting of 25 μg of lysate protein were separated using SDS-PAGE on 10% Tris-glycine gels (Invitrogen). Proteins were transferred to polyvinylidene difluoride membranes (Invitrogen) and immunoblotted as follows. The polyvinylidene difluoride membranes were blocked with 5% nonfat milk in PBS for 1 h at RT. The blots were incubated with the Arc antibody diluted 1:100 in the blocking solution overnight at 4 °C and then were washed 3 times for 10 min each with PBS plus 0.1% Tween 20 (Bio-Rad) at RT. The membranes were incubated with goat anti-mouse horseradish peroxidase in block (1:1000; Santa Cruz Biotechnology) for 1 h at RT. The membranes were then washed 8 times for 15 min each with PBS plus 0.1% Tween 20, exposed with SuperSignal West Pico chemiluminescent horseradish peroxidase) substrate (Pierce), and developed on x-ray film according to the instructions. qRT-PCR—Neurons in 6-well tissue culture plates were transfected with 1 μg each of Arc-EYFP and dsRed2C1 plasmids (Clontech). 16 h post-transfection cells were treated with 50 μm forskolin or vehicle for 4 h. RNA was isolated with the Qiashredder kit according to instructions (Qiagen, Valencia, CA). RNA was harvested from a 6-well plate via a Qiagen column, subjected to a DNase treatment, and re-purified with a second Qiagen column. 1 μg of RNA was reverse-transcribed with the Bio-Rad I-script kit according to the manufacturer's instructions. A standard curve was generated by serial dilution of all the cDNAs. Reactions were set up in 96-well PCR plates (USA Scientific, Inc. Ocala, CA) with iQ SYBR Green Supermix according to the instructions (Bio-Rad) and run in the iCycler Thermal Cycler (Bio-Rad). Data Analysis—Grayscale images (12-bit resolution) of fluorescent neurons were acquired separately for the cyan (ECFP) and yellow (EYFP) channels and analyzed using ImagePro Plus Version 4.5 (Media Cybernetics, Silver Spring, MD). Outlines of fluorescent neurons in the images were detected by the ImagePro "automatic bright objects" tool, and both the area (A) and total fluorescence intensity (F) within the outline were measured. For each neuron, the average fluorescence (F/A) was divided by the exposure time (T) used to acquire the image to obtain fluorescence/pixel/s (FPS = F/A/T), which was used as a relative measure of protein expression. FPS was measured for n neurons (n = at least 20 per condition) to allow calculation of a mean and S.E. for each condition. Cyan (control) and yellow (Arc) FPS values were obtained for each neuron, and their ratio was calculated. FPS was calculated for the red EosFP signals in a similar manner. qRT-PCR results were analyzed with iCycler iQ optical system software Version 3.0a (Bio-Rad). Post-transcriptional regulation of Arc expression was investigated in cultured hippocampal neurons. First, treatments were identified that have been reported to induce endogenous Arc expression. Previous work has shown that Arc mRNA level is increased after treatment with NMDA, dopamine receptor agonist, or forskolin (13Fosnaugh J.S. Bhat R.V. Yamagata K. Worley P.F. Baraban J.M. J. Neurochem. 1995; 64: 2377-2380Crossref PubMed Scopus (121) Google Scholar, 14Steward O. Worley P.F. Neuron. 2001; 30: 227-240Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 20Waltereit R. Dammermann B. Wulff P. Scafidi J. Staubli U. Kauselmann G. Bundman M. Kuhl D. J. Neurosci. 2001; 21: 5484-5493Crossref PubMed Google Scholar). Forskolin directly activates ACs, resulting in cAMP production and activation of both PKA and the exchange protein directly activated by cyclic-AMP (Epac). Activation of NMDA and dopamine receptors can also result in AC activation and cAMP production, mediated by Ca2+-CaM and the stimulatory G-protein Gs, respectively. Therefore, the effect of these agonists on Arc protein level was investigated in hippocampal neurons. Because forskolin can have nonspecific effects independent of its action on ACs, neurons were also stimulated with 8-Br-cAMP, a membrane-permeable analogue of cAMP. Endogenous Arc Protein Expression—The effect of the agonists on Arc protein level was first evaluated by Western analysis using a monoclonal Arc-specific antibody. Fig. 1A illustrates that this antibody recognizes Arc protein, seen as a single band at ∼50 kDa. Arc protein levels increased after a 4-h treatment with each of the following agonists: forskolin, 8-Br-cAMP, NMDA, and the D1/D5 dopamine receptor agonist SKF-38393. Pharmacological activation of PKA by either forskolin or 8-Br-cAMP resulted in a stronger induction of Arc than activation of NMDA receptors or dopamine receptors. Western analysis reflects a combined effect of the compounds on Arc protein expression for all cell types present in the hippocampal culture, including neurons and glial cells, both of which express Arc protein (27Rodriguez J.J. Davies H.A. Silva A.T. De S.I. Peddie C.J. Colyer F.M. Lancashire C.L. Fine A. Errington M.L. Bliss T.V. Stewart M.G. Eur. J. Neurosci. 2005; 21: 2384-2396Crossref PubMed Scopus (92) Google Scholar). To determine Arc expression solely in neurons, hippocampal cultures were treated with agonists, fixed, and analyzed by immunofluorescence microscopy using the Arc antibody and Alexa Fluor 488-conjugated secondary antibody. Only neurons were selected for data analysis. Arc protein displayed a punctate distribution and was found primarily in the soma, with lower levels in the distal dendrites (Fig. 1B). A time course of Arc expression was determined by stimulating cultured neurons with forskolin for different time periods over a 24-h period. Arc expression detected with the Arc antibody peaked at 4 h, reaching a 12-fold increase over unstimulated neurons (Fig. 1C). How Arc expression level was affected by the previously used agonists was also tested (Fig. 1D). Arc protein level increased ∼12-fold for forskolin, 11-fold for 8-Br-cAMP, 7-fold for NMDA, and 4-fold with SKF-38393. NMDA and dopamine signaling are both expected to activate PKA through Ca2+/CaM and Gs, respectively. Therefore, the co-application of NMDA and SKF-38393 was investigated. This resulted in an 11-fold increase in Arc protein expression (Fig. 1D), indicating the effects of NMDA and the dopamine agonist are additive. The ability of the agonists to induce Arc in the presence of the PKA antagonist H89 was also investigated (Fig. 1D). Pretreatment with H89 abolished the effect for all the agonists tested, indicating that in each case the induction of Arc protein expression depended on PKA activation. The effects of the agonists on endogenous Arc protein level may have resulted from transcriptional and/or post-transcriptional regulation. This issue was investigated next. Post-transcriptional Regulation of Arc Protein Expression—To determine whether PKA activation could result in a post-transcriptional effect on Arc protein level, a yellow fluorescent Arc fusion protein (Arc-EYFP) was expressed in cultured hippocampal neurons through transient transfections (Fig. 2). As a control, enhanced cyan fluorescent protein (ECFP) was co-transfected with Arc-EYFP, resulting in co-expression of both cDNAs in the same subset of neurons. In these experiments, a full-length Arc cDNA construct was used that contained complete 5′- and 3′-UTRs, whereas transcription was driven by the strong viral CMV promoter. Because of its high constitutive activity, the CMV promoter should be relatively insensitive to regulation by cellular signaling pathways, allowing us to evaluate post-transcriptional mechanisms that regulate Arc expression. Similar to what was seen for endogenous protein, exogenous Arc protein was found primarily in the soma, with lower levels in the dendrites (Fig. 2A). Forskolin treatment caused Arc to be sharply up-regulated (notice the difference in exposure times), whereas its distribution became more punctate both in the soma and dendrites (Fig. 2A). Expression of both Arc-EYFP and ECFP is driven by the same viral CMV promoter; therefore, they are expected to be subject to the same transcriptional regulation, and a difference in fluorescence signal should reflect post-transcriptional events. To correct for differences in general protein expression between neurons, the Arc-EYFP signal was normalized to ECFP in the same neuron. This normalization also corrects for any effect that an agonist has on general translation within a single cell. Again, the time course of the forskolin effect on Arc protein expression was determined. Arc-EYFP- and ECFP-transfected cultures were stimulated with forskolin for different time periods over 24 h. Arc expression peaked at 4 h with a 9.4-fold increase over unstimulated neurons (Fig. 2B). Next, the effect of the agonists on Arc expression level was tested. All agonists increased Arc-EYFP expression, while having a marginal effect on ECFP. Agonist treatment did not have a negative effect on neuronal health (supplemental Fig. 1). Arc-EYFP/ECFP ratios increased 9.1-fold for forskolin, 10.4-fold for 8-Br-cAMP, 4.4-fold for NMDA, and 3.6-fold with SKF-38393 stimulation (Fig. 2C). NMDA and SKF-38393 co-application increased Arc expression additively by 8.8-fold. In addition, H89 blocked all of the agonist effects (Fig. 2D). In these experiments transcription of Arc-EYFP and ECFP was driven by the constitutively active strong CMV promoter in order to minimize transcriptional effects of the treatments. Effects of the pharmacological treatments on general transcription were corrected for by normalizing for ECFP expression. The results obtained with the ECFP construct show that the CMV promoter is almost completely insensitive to the various agonist treatments. Consequently, the strong increase in Arc-EYFP levels likely resulted from a post-transcriptional mechanism. Regulation of Arc Protein Expression Does Not Require the Arc-untranslated Regions—Several post-transcriptional mechanisms control protein levels through an interaction with the 5′- and 3′-UTRs of a target mRNA. Therefore, it was investigated whether the agonist-induced increases in Arc protein levels reported above require the presence of UTRs in the Arc mRNA. Experiments were performed with an Arc construct that lacked both the 5′- and 3′-UTRs. The 3′-UTR of Arc has previously been shown to be important in dendritic targeting, although its role in Arc protein translation is unknown (28Kobayashi H. Yamamoto S. Maruo T. Murakami F. Eur.J. Neurosci. 2005; 22: 2977-2984Crossref PubMed Scopus (48) Google Scholar). A plasmid encoding Arc-EYFP without the UTRs was co-expressed with ECFP in cultured neurons (Fig. 3). Again, forskolin treatment caused Arc to be up-regulated (notice the difference in exposure times), whereas its distribution became more punctuate in the soma and dendrites (Fig. 3A). Transfected cultures were stimulated with forskolin for different time periods over 24 h. Arc expression ratios peaked at 4 h, with a 7.6-fold increase over unstimulated neurons (Fig. 3B). Transfected cultures were also stimulated with the PKA agonists (Fig. 3C). All the previously used agonists increased Arc-EYFP expression, while having a marginal effect on ECFP. Arc expression ratios increased ∼8-fold for forskolin and 8-Br-cAMP, 4-fold for NMDA, 3.2-fold for SKF-38393, and additively 7.6-fold for NMDA plus SKF-38393 stimulation. NMDA stimulation did not enhance the forskolin effect, possibly because forskolin has already saturated the signaling pathway. Again, the PKA antagonist H89 blocked the effect of all agonists tested (Fig. 3D). The two main effectors of cAMP are PKA and Epac, an activator of the small G-protein Rap1. To investigate a possible involvement of Epac in the regulation of Arc expression, we used 8-CPT, a cAMP analogue that specifically activates Epac. 8-CPT was completely ineffective, further verifying that cAMP
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