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• W2158323020 abstract "Translational control of mRNAs in dendrites is essential for certain forms of synaptic plasticity and learning and memory. CPEB is an RNA-binding protein that regulates local translation in dendrites. Here, we identify poly(A) polymerase Gld2, deadenylase PARN, and translation inhibitory factor neuroguidin (Ngd) as components of a dendritic CPEB-associated polyadenylation apparatus. Synaptic stimulation induces phosphorylation of CPEB, PARN expulsion from the ribonucleoprotein complex, and polyadenylation in dendrites. A screen for mRNAs whose polyadenylation is altered by Gld2 depletion identified >100 transcripts including one encoding NR2A, an NMDA receptor subunit. shRNA depletion studies demonstrate that Gld2 promotes and Ngd inhibits dendritic NR2A expression. Finally, shRNA-mediated depletion of Gld2 in vivo attenuates protein synthesis-dependent long-term potentiation (LTP) at hippocampal dentate gyrus synapses; conversely, Ngd depletion enhances LTP. These results identify a pivotal role for polyadenylation and the opposing effects of Gld2 and Ngd in hippocampal synaptic plasticity. Translational control of mRNAs in dendrites is essential for certain forms of synaptic plasticity and learning and memory. CPEB is an RNA-binding protein that regulates local translation in dendrites. Here, we identify poly(A) polymerase Gld2, deadenylase PARN, and translation inhibitory factor neuroguidin (Ngd) as components of a dendritic CPEB-associated polyadenylation apparatus. Synaptic stimulation induces phosphorylation of CPEB, PARN expulsion from the ribonucleoprotein complex, and polyadenylation in dendrites. A screen for mRNAs whose polyadenylation is altered by Gld2 depletion identified >100 transcripts including one encoding NR2A, an NMDA receptor subunit. shRNA depletion studies demonstrate that Gld2 promotes and Ngd inhibits dendritic NR2A expression. Finally, shRNA-mediated depletion of Gld2 in vivo attenuates protein synthesis-dependent long-term potentiation (LTP) at hippocampal dentate gyrus synapses; conversely, Ngd depletion enhances LTP. These results identify a pivotal role for polyadenylation and the opposing effects of Gld2 and Ngd in hippocampal synaptic plasticity. CPEB and the cytoplasmic polyadenylation complex are regulated by synaptic activity Synaptic stimulation induces polyadenylation in dendrites Two CPEB partners, Gld2 and Neuroguidin, enhance and repress synaptic plasticity Gld2 controls the polyadenylation of >100 mRNAs in neurons including NR2A and HuD Spatial control of mRNA translation is critical for diverse cellular functions across species (Besse and Ephrussi, 2008Besse F. Ephrussi A. Translational control of localized mRNAs: restricting protein synthesis in space and time.Nat. Rev. Mol. Cell Biol. 2008; 9: 971-980Crossref PubMed Scopus (261) Google Scholar). In the mammalian nervous system, experience-induced modifications of synaptic connections (synaptic plasticity) are thought to underlie learning and memory (Kandel, 2001Kandel E.R. The molecular biology of memory storage: a dialogue between genes and synapses.Science. 2001; 294: 1030-1038Crossref PubMed Scopus (2684) Google Scholar). These modifications require activity-dependent protein synthesis, which likely involves specific mRNA translation at or near synapses (Sutton and Schuman, 2006Sutton M.A. Schuman E.M. Dendritic protein synthesis, synaptic plasticity, and memory.Cell. 2006; 127: 49-58Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar). In the hippocampus, protein synthesis is required for multiple forms of synaptic plasticity including late-phase long term potentiation (L-LTP), neurotrophin-induced LTP, and metabotropic glutamate receptor-mediated long term depression (mGluR-LTD) (Krug et al., 1984Krug M. Lössner B. Ott T. Anisomycin blocks the late phase of long-term potentiation in the dentate gyrus of freely moving rats.Brain Res. Bull. 1984; 13: 39-42Crossref PubMed Scopus (384) Google Scholar, Frey et al., 1988Frey U. Krug M. Reymann K.G. Matthies H. Anisomycin, an inhibitor of protein synthesis, blocks late phases of LTP phenomena in the hippocampal CA1 region in vitro.Brain Res. 1988; 452: 57-65Crossref PubMed Scopus (662) Google Scholar, Kang and Schuman, 1996Kang H. Schuman E.M. A requirement for local protein synthesis in neurotrophin-induced hippocampal synaptic plasticity.Science. 1996; 273: 1402-1406Crossref PubMed Scopus (754) Google Scholar, Huber et al., 2000Huber K.M. Kayser M.S. Bear M.F. Role for rapid dendritic protein synthesis in hippocampal mGluR-dependent long-term depression.Science. 2000; 288: 1254-1257Crossref PubMed Scopus (757) Google Scholar). In the latter two cases, protein synthesis was required in the dendrite-rich stratum radiatum even after it had been severed from the cell body layer. These studies point to the importance of activity-dependent dendritic translation for synaptic plasticity (Costa-Mattioli et al., 2009Costa-Mattioli M. Sossin W.S. Klann E. Sonenberg N. Translational control of long-lasting synaptic plasticity and memory.Neuron. 2009; 61: 10-26Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar, Richter and Klann, 2009Richter J.D. Klann E. Making synaptic plasticity and memory last: mechanisms of translational regulation.Genes Dev. 2009; 23: 1-11Crossref PubMed Scopus (280) Google Scholar, Sutton and Schuman, 2006Sutton M.A. Schuman E.M. Dendritic protein synthesis, synaptic plasticity, and memory.Cell. 2006; 127: 49-58Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar). Indeed, dendrites harbor mRNAs (Poon et al., 2006Poon M.M. Choi S.H. Jamieson C.A. Geschwind D.H. Martin K.C. Identification of process-localized mRNAs from cultured rodent hippocampal neurons.J. Neurosci. 2006; 26: 13390-13399Crossref PubMed Scopus (172) Google Scholar), ribosomes (Steward and Levy, 1982Steward O. Levy W.B. Preferential localization of polyribosomes under the base of dendritic spines in granule cells of the dentate gyrus.J. Neurosci. 1982; 2: 284-291Crossref PubMed Google Scholar), micro-RNAs, and RISC (Schratt, 2009Schratt G. microRNAs at the synapse.Nat. Rev. Neurosci. 2009; 10: 842-849Crossref PubMed Scopus (392) Google Scholar), supporting the notion of synaptic activity-induced local protein synthesis. One protein involved in neuronal mRNA translation is CPEB (Wu et al., 1998Wu L. Wells D. Tay J. Mendis D. Abbott M.A. Barnitt A. Quinlan E. Heynen A. Fallon J.R. Richter J.D. CPEB-mediated cytoplasmic polyadenylation and the regulation of experience-dependent translation of alpha-CaMKII mRNA at synapses.Neuron. 1998; 21: 1129-1139Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar), which binds the 3′ untranslated region (UTR) cytoplasmic polyadenylation element (CPE) and modulates poly(A) tail length. In Xenopus oocytes, CPEB associates with several factors including: (i) cleavage and polyadenylation specificity factor (CPSF), which binds the hexanucleotide AAUAAA; (ii) Gld2, a poly(A) polymerase; (iii) PARN, a deadenylating enzyme; (iv) maskin, which interacts with the cap-binding factor eIF4E; and (v) symplekin, a scaffold protein upon which the ribonucleoprotein (RNP) complex is assembled (Richter, 2007Richter J.D. CPEB: a life in translation.Trends Biochem. Sci. 2007; 32: 279-285Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). When tethered to mRNAs by CPEB, PARN activity is dominant to that of Gld2, leading to poly(A) tail shortening of CPE-containing mRNAs (Barnard et al., 2004Barnard D.C. Ryan K. Manley J.L. Richter J.D. Symplekin and xGLD-2 are required for CPEB-mediated cytoplasmic polyadenylation.Cell. 2004; 119: 641-651Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, Kim and Richter, 2006Kim J.H. Richter J.D. Opposing polymerase-deadenylase activities regulate cytoplasmic polyadenylation.Mol. Cell. 2006; 24: 173-183Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). Upon stimulation of oocytes to re-enter meiosis, Aurora A phosphorylates CPEB, leading to expulsion of PARN from the RNP complex and polyadenylation by Gld2. The poly(A) tail serves as a platform for poly(A) binding protein, which binds eIF4G and helps it displace maskin from eIF4E and recruit the 40S ribosomal subunit to the mRNA (Kim and Richter, 2006Kim J.H. Richter J.D. Opposing polymerase-deadenylase activities regulate cytoplasmic polyadenylation.Mol. Cell. 2006; 24: 173-183Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, Stebbins-Boaz et al., 1999Stebbins-Boaz B. Cao Q. de Moor C.H. Mendez R. Richter J.D. Maskin is a CPEB-associated factor that transiently interacts with elF-4E.Mol. Cell. 1999; 4: 1017-1027Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). In the brain, CPEB regulates synaptic plasticity and hippocampal-dependent memories (Alarcon et al., 2004Alarcon J.M. Hodgman R. Theis M. Huang Y.S. Kandel E.R. Richter J.D. Selective modulation of some forms of schaffer collateral-CA1 synaptic plasticity in mice with a disruption of the CPEB-1 gene.Learn. Mem. 2004; 11: 318-327Crossref PubMed Scopus (136) Google Scholar, Berger-Sweeney et al., 2006Berger-Sweeney J. Zearfoss N.R. Richter J.D. Reduced extinction of hippocampal-dependent memories in CPEB knockout mice.Learn. Mem. 2006; 13: 4-7Crossref PubMed Scopus (86) Google Scholar, Zearfoss et al., 2008Zearfoss N.R. Alarcon J.M. Trifilieff P. Kandel E. Richter J.D. A molecular circuit composed of CPEB-1 and c-Jun controls growth hormone-mediated synaptic plasticity in the mouse hippocampus.J. Neurosci. 2008; 28: 8502-8509Crossref PubMed Scopus (76) Google Scholar). N-methyl-D-aspartate receptor (NMDAR) activation promotes CPEB phosphorylation (Atkins et al., 2004Atkins C.M. Nozaki N. Shigeri Y. Soderling T.R. Cytoplasmic polyadenylation element binding protein-dependent protein synthesis is regulated by calcium/calmodulin-dependent protein kinase II.J. Neurosci. 2004; 24: 5193-5201Crossref PubMed Scopus (133) Google Scholar, Huang et al., 2002Huang Y.S. Jung M.Y. Sarkissian M. Richter J.D. N-methyl-D-aspartate receptor signaling results in Aurora kinase-catalyzed CPEB phosphorylation and alpha CaMKII mRNA polyadenylation at synapses.EMBO J. 2002; 21: 2139-2148Crossref PubMed Scopus (214) Google Scholar), triggering mRNA-specific polyadenylation and translation (Huang et al., 2002Huang Y.S. Jung M.Y. Sarkissian M. Richter J.D. N-methyl-D-aspartate receptor signaling results in Aurora kinase-catalyzed CPEB phosphorylation and alpha CaMKII mRNA polyadenylation at synapses.EMBO J. 2002; 21: 2139-2148Crossref PubMed Scopus (214) Google Scholar, McEvoy et al., 2007McEvoy M. Cao G. Montero Llopis P. Kundel M. Jones K. Hofler C. Shin C. Wells D.G. Cytoplasmic polyadenylation element binding protein 1-mediated mRNA translation in Purkinje neurons is required for cerebellar long-term depression and motor coordination.J. Neurosci. 2007; 27: 6400-6411Crossref PubMed Scopus (39) Google Scholar, Wu et al., 1998Wu L. Wells D. Tay J. Mendis D. Abbott M.A. Barnitt A. Quinlan E. Heynen A. Fallon J.R. Richter J.D. CPEB-mediated cytoplasmic polyadenylation and the regulation of experience-dependent translation of alpha-CaMKII mRNA at synapses.Neuron. 1998; 21: 1129-1139Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar). Although CPEB stimulates polyadenylation in neurons, the mechanism by which it does so and whether polyadenylation occurs in dendrites are unknown. CPEB can repress translation without influencing polyadenylation (Groisman et al., 2006Groisman I. Ivshina M. Marin V. Kennedy N.J. Davis R.J. Richter J.D. Control of cellular senescence by CPEB.Genes Dev. 2006; 20: 2701-2712Crossref PubMed Scopus (73) Google Scholar) and modulate alternative splicing (Lin et al., 2010Lin C.L. Evans V. Shen S. Xing Y. Richter J.D. The nuclear experience of CPEB: implications for RNA processing and translational control.RNA. 2010; 16 (Published online December 29, 2009): 338-348https://doi.org/10.1261/rna.1779810Crossref PubMed Scopus (49) Google Scholar), indicating that cytoplasmic 3′ end processing does not necessarily affect plasticity. Finally, maskin is not detected in mammals, implicating other factors in CPEB-mediated translation. Regarding this, mammalian neurons contain neuroguidin (Ngd), a CPEB and eIF4E-binding protein that may function in a manner analogous to maskin (Jung et al., 2006Jung M.Y. Lorenz L. Richter J.D. Translational control by neuroguidin, a eukaryotic initiation factor 4E and CPEB binding protein.Mol. Cell. Biol. 2006; 26: 4277-4287Crossref PubMed Scopus (84) Google Scholar). The nexus of observations showing that CPEB is synapto-dendritic, that it modulates plasticity, and that local protein synthesis is necessary for LTP and LTD suggests that cytoplasmic polyadenylation could mediate local protein synthesis and synaptic efficacy. To investigate this possibility, we focused on factors that control polyadenylation/translation and found that CPEB, symplekin, Gld2, PARN, and Ngd formed a complex in hippocampal dendrites. NMDA stimulation promoted CPEB phosphorylation and expulsion of PARN from the complex and induced a rapid increase in dendritic poly(A) that was attenuated by Gld2 depletion or inhibition of CPEB phosphorylation. A screen for neuronal mRNAs whose polyadenylation is influenced by Gld2 identified several transcripts including one for NR2A, an NMDAR subunit. Depletion of Gld2 and Ngd toggled the expression of this dendritically localized mRNA. Moreover, knockdown of Gld2 in vivo inhibited theta-burst stimulation (TBS)-induced LTP at dentate gyrus (DG) granule cell synapses, while depletion of Ngd increased the magnitude of the LTP. These and other findings indicate that the cytoplasmic polyadenylation machinery bidirectionally regulates mRNA-specific translation and plasticity at hippocampal synapses, which we suggest represents a coherent molecular mechanism that underlies essential brain function. The distribution of CPEB, Gld2, PARN, symplekin, and Ngd in cultured hippocampal neurons was examined using immunofluorescence and digital image analysis; these proteins were detected in cell bodies and distal dendrites (>75 μm from the soma). In contrast, immunoreactivity for hnRNP A1 was restricted to nuclei and cell bodies (Figures S1A and S1B). Confocal images of sectioned hippocampal material showed punctate CPEB, Gld2, PARN, and Ngd immunoreactivity within cell bodies and MAP2-positive dendrites (Figure S1C). Moreover, CPEB, Gld2, PARN, symplekin, and Ngd were detected in synaptoneurosomes isolated from mouse hippocampus (Figure S1D). We examined the interaction of these factors in mouse brain where symplekin coimmunoprecipitated with CPSF100, CPSF73, PARN, Ngd, and CPEB (Figure 1A ), and Ngd coimmunoprecipitated with CPSF100 and symplekin (Figure 1B). In HEK293T and neuroblastoma cells, symplekin coimmunoprecipitated GFP-tagged CPEB and Gld2 (Figures 1C and 1D). Tagged CPEB coimmunoprecipitated with symplekin, tagged PARN, and tagged Gld2 (Figure 1E) and tagged PARN coimmunoprecipitated with symplekin and tagged CPEB (Figure 1F). To assess whether these components were colocalized in dendrites, cultured neurons were coimmunostained for symplekin and each other factor, and 3D deconvolved images were analyzed (Figure 1G). CPEB, PARN, Gld2, and Ngd were nonrandomly colocalized with symplekin (p > 0.95), while GluR1 was not (Figure 1H). The Mander's coefficients, the fraction of total signal that is colocalized, were 0.24–0.38, demonstrating that significant levels of CPEB, PARN, Gld2, and Ngd were colocalized with symplekin in dendritic granules (Figure 1I). These proteins were also detected in dendritic spines; 3D reconstructions of phalloidin fluorescence (Figure 1J) showed that 23.1% ± 1.24% of spines contained symplekin and 80.1% ± 2.54% of symplekin-positive spines also contained CPEB, Gld2, PARN, or Ngd immunoreactivity (n = 40 cells, 1196 spines). These data indicate that the cytoplasmic polyadenylation machinery forms complexes in dendrites and at synapses. To investigate if synaptic activity induces polyadenylation in dendrites, NMDA-stimulated neurons were processed for fluorescence in situ hybridization (FISH) with oligo(dT) probes (Figure 2). Punctate poly(A) RNA was detected in the soma and dendritic arbors in a decreasing proximal-to-distal gradient (Bassell et al., 1994Bassell G.J. Singer R.H. Kosik K.S. Association of poly(A) mRNA with microtubules in cultured neurons.Neuron. 1994; 12: 571-582Abstract Full Text PDF PubMed Scopus (125) Google Scholar); oligo(dA) FISH yielded negligible signal (Figure S2A). NMDA treatment (100 nM, 30 s) increased dendritic oligo(dT) FISH intensity by 55% in distal regions as compared to control (Figure 2B), which was abrogated by the NMDAR antagonist APV (data not shown). NMDA did not affect dendritic αCaMKII mRNA levels (Figures 2B and S2B), indicating negligible transcript transport to distal dendrites during the brief stimulation. To determine if dendritic polyadenylation was sensitive to Gld2, neurons were transduced with lentiviruses expressing Gld2 shRNA or a control (Figure S5). Depletion of Gld2 reduced dendritic oligo(dT) FISH signals relative to controls (Figures 2C and 2D), indicating that this enzyme regulates dendritic polyadenylation. In oocytes, polyadenylation is activated by CPEB phosphorylation (Hodgman et al., 2001Hodgman R. Tay J. Mendez R. Richter J.D. CPEB phosphorylation and cytoplasmic polyadenylation are catalyzed by the kinase IAK1/Eg2 in maturing mouse oocytes.Development. 2001; 128: 2815-2822PubMed Google Scholar, Mendez et al., 2000Mendez R. Hake L.E. Andresson T. Littlepage L.E. Ruderman J.V. Richter J.D. Phosphorylation of CPE binding factor by Eg2 regulates translation of c-mos mRNA.Nature. 2000; 404: 302-307Crossref PubMed Scopus (292) Google Scholar), which induces PARN expulsion from the CPEB-containing complex (Kim and Richter, 2006Kim J.H. Richter J.D. Opposing polymerase-deadenylase activities regulate cytoplasmic polyadenylation.Mol. Cell. 2006; 24: 173-183Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). NMDAR activation elicits CPEB phosphorylation in neurons (Atkins et al., 2004Atkins C.M. Nozaki N. Shigeri Y. Soderling T.R. Cytoplasmic polyadenylation element binding protein-dependent protein synthesis is regulated by calcium/calmodulin-dependent protein kinase II.J. Neurosci. 2004; 24: 5193-5201Crossref PubMed Scopus (133) Google Scholar, Huang et al., 2002Huang Y.S. Jung M.Y. Sarkissian M. Richter J.D. N-methyl-D-aspartate receptor signaling results in Aurora kinase-catalyzed CPEB phosphorylation and alpha CaMKII mRNA polyadenylation at synapses.EMBO J. 2002; 21: 2139-2148Crossref PubMed Scopus (214) Google Scholar), but whether this occurs in dendrites is unknown. To assess this possibility, hippocampal neurons were treated with NMDA and immunostained for phospho- or total CPEB (Figure 3A ). NMDA stimulation increased mean pCPEB immunofluorescence intensities in dendrites ≥75 μm from the soma by 90%, while total dendritic CPEB levels were not significantly affected (Figure 3B). In synaptoneurosomes from cortical neurons, NMDA treatment increased synaptic pCPEB ∼2.5 fold compared to control (Figure 3C). Inhibitors of Aurora A and CaMKII, two enzymes that phosphorylate CPEB in neurons (Atkins et al., 2004Atkins C.M. Nozaki N. Shigeri Y. Soderling T.R. Cytoplasmic polyadenylation element binding protein-dependent protein synthesis is regulated by calcium/calmodulin-dependent protein kinase II.J. Neurosci. 2004; 24: 5193-5201Crossref PubMed Scopus (133) Google Scholar, Huang et al., 2002Huang Y.S. Jung M.Y. Sarkissian M. Richter J.D. N-methyl-D-aspartate receptor signaling results in Aurora kinase-catalyzed CPEB phosphorylation and alpha CaMKII mRNA polyadenylation at synapses.EMBO J. 2002; 21: 2139-2148Crossref PubMed Scopus (214) Google Scholar), occluded NMDA-induced CPEB phosphorylation in dendrites and synapses (Figures S3A and S3B). These data suggest that NMDAR activation leads to rapid CPEB phosphorylation at synapses. To determine whether NMDAR activation alters the composition of the dendritic polyadenylation complex, neurons were treated with NMDA, coimmunostained for complex components, and analyzed for 3D colocalization (Figure S3C). Although NMDA did not alter CPEB or Gld2 colocalization with symplekin, it reduced the colocalization of PARN with symplekin and CPEB with PARN (Figure 3D; total PARN levels were not affected, data not shown). Inhibitors of Aurora A and CaMKII blocked the NMDA-induced reduction of PARN colocalization with symplekin, suggesting that CPEB phosphorylation triggers the release of PARN from dendritic CPEB-containing complexes (Figure S3D). PARN expulsion was also indicated by biochemical data (Figures 3E and S3E). Membrane depolarization of neuroblastoma cells increased CPEB phosphorylation and decreased coimmunoprecipitation of PARN with wild-type (WT), but not phospho-mutant CPEB (CPEB-AA). Thus, activity-induced CPEB phosphorylation disrupts the interaction between PARN and CPEB-containing complexes in neurons. To determine whether CPEB phosphorylation regulates dendritic polyadenylation, we evaluated oligo(dT) FISH intensity in neurons expressing CPEB-WT or CPEB-AA (Figure 3F). The dendritic expression and localization of CPEB-WT and CPEB-AA were similar (data not shown). Steady-state dendritic oligo(dT) FISH intensity was reduced by both CPEB-WT (20%) and CPEB-AA (28%) compared to controls. NMDA increased dendritic oligo(dT) FISH intensity in control (56.9%) and CPEB-WT-expressing (89.0%) neurons, whereas CPEB-AA expression blocked this effect. In addition, pretreatment with Aurora A or CaMKII inhibitors occluded the NMDA-induced increase in dendritic oligo(dT) FISH intensity (Figure S3F). We infer that the dendritic mRNA polyadenylation machinery is regulated by NMDAR activity, and that CPEB phosphorylation leads to PARN extrusion from this complex resulting in polyadenylation in dendrites. The data presented above imply that Gld2 controls dendritic polyadenylation and translation of specific mRNAs. To identify them, RNA was extracted from hippocampal neurons transduced with Gld2 shRNA-expressing or control lentivirus and applied to poly(U)-Sepharose (Figure 4A ); RNAs eluting at 50°C have poly(A) tails ∼50 nucleotides, while 65°C eluates contain RNAs with longer poly(A) (Du and Richter, 2005Du L. Richter J.D. Activity-dependent polyadenylation in neurons.RNA. 2005; 11: 1340-1347Crossref PubMed Scopus (69) Google Scholar, Simon et al., 1996Simon R. Wu L. Richter J.D. Cytoplasmic polyadenylation of activin receptor mRNA and the control of pattern formation in Xenopus development.Dev. Biol. 1996; 179: 239-250Crossref PubMed Scopus (45) Google Scholar). Both total RNA and 65°C RNA eluates were processed for microarrays. In total RNA samples, 25 mRNAs were significantly reduced by Gld2 knockdown. In the 65°C eluates, 124 mRNAs were reduced following Gld2 depletion. These RNA sample sets largely overlapped (Figure 4B and Table S1), indicating that at least 100 different mRNAs had undergone a loss of poly(A) following Gld2 knockdown. Twenty-seven of these mRNAs have been implicated in synaptic plasticity and/or nervous system disorders; five were selected, and the microarray data were validated by qRT-PCR (Figure 4C). HuD (RNA-binding protein) and Neto2 (kainate receptor modulator) mRNAs were reduced in the total and poly(A) RNA fractions following Gld2 knockdown, suggesting that they became unstable or their transcription was indirectly reduced upon Gld2 depletion. NR2A (NMDAR subunit) and Ago3 (Piwi protein) mRNAs were reduced only in the poly(A) RNA fraction, suggesting that their poly(A) tails were shortened by Gld2 depletion without affecting stability. Sos1 (Ras/Erk2 guanine nucleotide exchange factor) was significantly decreased only in the total RNA fraction. Although Gld2 probably influences neuron function by regulating many mRNAs, we focused on NR2A because its 3′UTR contains conserved CPEs (Figure 4D). Examination of neurons by FISH revealed that NR2A mRNA is partially dendritic. The ratio of dendrite to soma FISH fluorescence for NR2A mRNA was similar to αCaMKII and PSD95 mRNAs, which are dendritic, and significantly greater than that of β-tubulin, a nondendritic mRNA (Figure S4; Muddashetty et al., 2007Muddashetty R.S. Kelić S. Gross C. Xu M. Bassell G.J. Dysregulated metabotropic glutamate receptor-dependent translation of AMPA receptor and postsynaptic density-95 mRNAs at synapses in a mouse model of fragile X syndrome.J. Neurosci. 2007; 27: 5338-5348Crossref PubMed Scopus (338) Google Scholar). Upon Gld2 knockdown in hippocampal neurons, HuD and NR2A protein levels decreased by 27% and 31%, respectively (neither tubulin nor HuR was significantly affected) (Figure 5A ). Conversely, depletion of Ngd, a translational repressor that inhibits eIF4F assembly (Jung et al., 2006Jung M.Y. Lorenz L. Richter J.D. Translational control by neuroguidin, a eukaryotic initiation factor 4E and CPEB binding protein.Mol. Cell. Biol. 2006; 26: 4277-4287Crossref PubMed Scopus (84) Google Scholar), resulted in a 40% increase in NR2A (HuD was not measured; note that although tubulin was unaffected, the NMDAR subunit NR1 increased by ∼20%) (Figure 5B). To determine if this regulation occurs in dendrites, we performed immunocytochemistry for NR2A following depletion of Gld2 or Ngd. While NR1 signal was not significantly altered, depletion of Gld2 reduced dendritic NR2A by ∼20%, and depletion of Ngd increased it by 30% (Figures 5C and 5D). Given that Gld2 and Ngd bidirectionally regulated NR2A expression, we investigated whether the cytoplasmic polyadenylation machinery and NMDAR activation directly regulated NR2A mRNA using a PCR-based poly(A) test (PAT, Figure 6A ). NMDA stimulation increased the population of NR2A mRNA with a long (∼150 nt) poly(A) tail, while Gld2 depletion reduced this NMDA-stimulated polyadenylation (Figures 6B and 6C). NMDA treatment (100 nM for 20 min) increased NR2A protein by 73%, while NR2A mRNA was not significantly altered (Figure 6D and 6E). Gld2 depletion blocked the NMDA-induced increase in NR2A (Figure 6F), suggesting that Gld2 mediates NMDA-induced NR2A mRNA translation. To determine if CPEB phosphorylation regulated dendritic NR2A protein levels, we evaluated dendritic NR2A immunofluorescence in neurons expressing CPEB-WT or CPEB-AA. NMDA treatment significantly increased dendritic NR2A protein in control transfected cells (17.9%) and CPEB-WT-expressing cells (38.0%), whereas CPEB-AA expression blocked this effect (Figure 6G). These results demonstrate that the cytoplasmic polyadenylation complex bidirectionally regulates NR2A mRNA polyadenylation and translation upon NMDAR activation and suggest that such regulation could have important consequences for hippocampal synaptic plasticity. The data presented thus far indicate that NMDAR signaling is required for CPEB phosphorylation and mRNA polyadenylation in neurons, even though the NMDA concentration used (100 nM) is below that required to elicit plasticity. To determine whether plasticity-inducing stimulation activates the CPEB complex and stimulates NR2A mRNA translation, we treated cultured hippocampal neurons with glycine (200 μM, 3 min), which elicits NMDAR-dependent LTP (Lu et al., 2001Lu W.Y. Man H.Y. Ju W. Trimble W.S. MacDonald J.F. Wang Y.T. Activation of synaptic NMDA receptors induces membrane insertion of new AMPA receptors and LTP in cultured hippocampal neurons.Neuron. 2001; 29: 243-254Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar). Glycine induced GluR1 S831 and S845 phosphorylation (Figure S6A), indicating the induction of LTP. This treatment also induced CPEB phosphorylation (Figure S6B) and polyadenylation in distal dendrites (Figure S6C). Furthermore, glycine stimulated NR2A mRNA polyadenylation when examined by PAT assay, which was attenuated by Gld2 depletion; RNA levels were unaffected (Figure S6D). Finally, glycine stimulated NR2A protein expression but not when Gld2 was depleted (Figure S6E). These data indicate that an LTP-induced signaling activates the cytoplasmic polyadenylation complex and stimulates NR2A production. CPEB knockout mice exhibit a deficit in theta burst stimulation (TBS)-induced LTP at Schaffer collateral-CA1 synapses (Alarcon et al., 2004Alarcon J.M. Hodgman R. Theis M. Huang Y.S. Kandel E.R. Richter J.D. Selective modulation of some forms of schaffer collateral-CA1 synaptic plasticity in mice with a disruption of the CPEB-1 gene.Learn. Mem. 2004; 11: 318-327Crossref PubMed Scopus (136) Google Scholar, Zearfoss et al., 2008Zearfoss N.R. Alarcon J.M. Trifilieff P. Kandel E. Richter J.D. A molecular circuit composed of CPEB-1 and c-Jun controls growth hormone-mediated synaptic plasticity in the mouse hippocampus.J. Neurosci. 2008; 28: 8502-8509Crossref PubMed Scopus (76) Google Scholar), which is consistent with the glycine-induced LTP in cultured neurons because both are probably regulated by NMDAR-dependent mechanisms (Musleh et al., 1997Musleh W. Bi X. Tocco G. Yaghoubi S. Baudry M. Glycine-induced long-term potentiation is associated with structural and functional modifications of alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid receptors.Proc. Natl. Acad. Sci. USA. 1997; 94: 9451-9456Crossref PubMed Scopus (66) Google Scholar). Furthermore, L-LTP-inducing tetanus leads to NMDAR-dependent CPEB phosphorylation in hippocampal CA1 region (Atkins et al., 2005Atkins C.M. Davare M.A. Oh M.C. Derkach V. Soderling T.R. Bidirectional regulation of cytoplasmic polyadenylation element-binding protein phosphorylation by Ca2+/calmodulin-dependent protein kinase II and protein phosphatase 1 during hippocampal long-term potentiation.J. Ne" @default.
• W2158323020 created "2016-06-24" @default.
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• W2158323020 creator A5017285977 @default.
• W2158323020 creator A5042061460 @default.
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• W2158323020 creator A5055873041 @default.
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• W2158323020 date "2012-07-01" @default.
• W2158323020 modified "2023-10-16" @default.
• W2158323020 title "Bidirectional Control of mRNA Translation and Synaptic Plasticity by the Cytoplasmic Polyadenylation Complex" @default.
• W2158323020 cites W1050445 @default.
• W2158323020 cites W1524192883 @default.
• W2158323020 cites W1571022725 @default.
• W2158323020 cites W1591026223 @default.
• W2158323020 cites W1652058896 @default.
• W2158323020 cites W1966901093 @default.
• W2158323020 cites W1968311418 @default.
• W2158323020 cites W1973191527 @default.
• W2158323020 cites W1974676736 @default.
• W2158323020 cites W1979496428 @default.
• W2158323020 cites W1981085577 @default.
• W2158323020 cites W1985241009 @default.
• W2158323020 cites W1991869273 @default.
• W2158323020 cites W1994217011 @default.
• W2158323020 cites W1994261554 @default.
• W2158323020 cites W2001987749 @default.
• W2158323020 cites W2008062776 @default.
• W2158323020 cites W2018662966 @default.
• W2158323020 cites W2019364602 @default.
• W2158323020 cites W2021975497 @default.
• W2158323020 cites W2024287181 @default.
• W2158323020 cites W2026872153 @default.
• W2158323020 cites W2029443323 @default.
• W2158323020 cites W2031698314 @default.
• W2158323020 cites W2034616998 @default.
• W2158323020 cites W2035082265 @default.
• W2158323020 cites W2036379239 @default.
• W2158323020 cites W2041037918 @default.
• W2158323020 cites W2044273755 @default.
• W2158323020 cites W2045964630 @default.
• W2158323020 cites W2048358400 @default.
• W2158323020 cites W2054091111 @default.
• W2158323020 cites W2054855377 @default.
• W2158323020 cites W2057658086 @default.
• W2158323020 cites W2059040821 @default.
• W2158323020 cites W2065538503 @default.
• W2158323020 cites W2069354675 @default.
• W2158323020 cites W2069930342 @default.
• W2158323020 cites W2071950386 @default.
• W2158323020 cites W2072636314 @default.
• W2158323020 cites W2073738336 @default.
• W2158323020 cites W2078012311 @default.
• W2158323020 cites W2080252952 @default.
• W2158323020 cites W2081299044 @default.
• W2158323020 cites W2089767611 @default.
• W2158323020 cites W2090183355 @default.
• W2158323020 cites W2093469460 @default.
• W2158323020 cites W2095370567 @default.
• W2158323020 cites W2106801137 @default.
• W2158323020 cites W2117118198 @default.
• W2158323020 cites W2117391623 @default.
• W2158323020 cites W2127202199 @default.
• W2158323020 cites W2133083344 @default.
• W2158323020 cites W2140841695 @default.
• W2158323020 cites W2141209989 @default.
• W2158323020 cites W2149155691 @default.
• W2158323020 cites W2167436769 @default.
• W2158323020 cites W2171089964 @default.
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