- Open Access
ApCPEB4, a non-prion domain containing homolog of ApCPEB, is involved in the initiation of long-term facilitation
Molecular Brain volume 9, Article number: 91 (2016)
Two pharmacologically distinct types of local protein synthesis are required for synapse- specific long-term synaptic facilitation (LTF) in Aplysia: one for initiation and the other for maintenance. ApCPEB, a rapamycin sensitive prion-like molecule regulates a form of local protein synthesis that is specifically required for the maintenance of the LTF. However, the molecular component of the local protein synthesis that is required for the initiation of LTF and that is sensitive to emetine is not known. Here, we identify a homolog of ApCPEB responsible for the initiation of LTF. ApCPEB4 which we have named after its mammalian CPEB4-like homolog lacks a prion-like domain, is responsive to 5-hydroxytryptamine, and is translated (but not transcribed) in an emetine-sensitive, rapamycin-insensitive, and PKA-dependent manner. The ApCPEB4 binds to different target RNAs than does ApCPEB. Knock-down of ApCPEB4 blocked the induction of LTF, whereas overexpression of ApCPEB4 reduces the threshold of the formation of LTF. Thus, our findings suggest that the two different forms of CPEBs play distinct roles in LTF; ApCPEB is required for maintenance of LTF, whereas the ApCPEB4, which lacks a prion-like domain, is required for the initiation of LTF.
Unlike short-term memory, long-term memory requires new protein synthesis for its formation [1–7]. Protein synthesis occurs in two spatially distinct regions of the neuron: 1) in the cell body where activity-dependent transcription and subsequent translation occurs and 2) in the presynaptic terminals and in the postsynaptic dendritic spines where mRNAs are localized and translated following synaptic activation [8–10]. The second form of translation is responsible for local protein synthesis, which is important for both the initiation and the maintenance of long-term memory.
The cytoplasmic polyadenylation element binding protein (CPEB) has been identified as one key regulator of the local protein synthesis in Aplysia . The binding of CPEB to mRNAs regulates the translation of target mRNAs by regulating their polyadenylation [11–14]. ApCPEB binds to the 3′ untranslated region (3′ UTR) of mRNAs that contains conserved cytoplasmic polyadenylation element (CPE) binding site (UUUUUAU) . ApCPEB is locally activated in response to a single pulse of 5-hydroxytryptamine (5-HT) and is inhibited by rapamycin. Interestingly, ApCPEB has a prion-like domain that is important for the ability of ApCPEB to form aggregates that are self-sustaining and can maintain the increased level of ApCPEB proteins in the terminals that is critical for maintaining long-term facilitation (LTF) in Aplysia sensory-motor neuron synapse [15–17]. When the translation of the ApCPEB mRNA is blocked locally, the initiation of LTF at 24 h is intact, whereas the maintenance of LTF at 72 h is selectively and specifically impaired. One of the major mRNA targets of ApCPEB is the actin mRNA, which contains the CPE site on its 3′ untranslated region (3′UTR) and is locally translated during LTF . ApCPEB has two isoforms, one contains poly-Q prion domain and the other lacking the prion-like domain [15, 18]. The maintenance of LTF requires the form of ApCPEB, which contains the prion domain.
In this study, we identified a new CPEB protein, ApCPEB4, in Aplysia kurodai. This protein is homologous to the mammalian CPEB4. The level of expression of ApCPEB4 was increased by 5-HT in a translation-dependent manner. Unlike ApCPEB, ApCPEB4 bound to specific RNA in a CPE-independent manner and is required for the initiation but not for the maintenance of LTF. Overexpression of ApCPEB4 reduced the threshold of the LTF induction. In addition, PKA-mediated phosphorylation of ApCPEB4 was critical for the induction of LTF. Collectively, these data suggest that ApCPEB4 plays a key role in regulating the initiation of LTF, while ApCPEB is essential for the maintenance of LTF.
Cloning of ApCPEB4 from Aplysia kurodai
We obtained the ApCPEB4 fragment of Aplysia kurodai from EST database by searching through custom-made basic local alignment software. Using this fragment as a probe, we screened ~1.5 × 105 clones of an Aplysia kurodai cDNA library and isolated several clones encoding parts of ApCPEB4. Based on the sequences of these clones, we obtained the full length of ApCPEB4. The length of coding region was 2064 bp and 664 amino acids, and it also contained two RNA Recognition Motifs (Fig. 1a). Using Expasy software (http://www.expasy.org/), potential PKA phosphorylation sites were searched.
3× CPE or CPE mutant sites were obtained by PCR with specific primer sets: 3× CPE1, CPE1-D3-S (5′-CGCCCAAGCTTGCAGCTTTTTATGACACAC AGT TTTTATGATGCCACG-3′)/CPE1-EI-A (5′-GCATGAATTCGATGGATAAAAACGTGGCA CATAAAAACTGTGTGTC-3′); 3× CPE2, CPE2-D3-S (5′-CGCCCAAGCTTGCAGCTT TTA ATG ACA CAC AGT TTT AAT GAT GCC ACG-3′)/CPE2-EI-A (5′-GCA TGA ATT CGATGGATTAAAACGTGG CATCATTAAAACTGTGTGTC-3′); 3× CPE3, CPE3-D3-S (5′-CGCCCAAGCTTGCAGCTTTTATAAGGACACACAGTTTTATAAGGATGCCACG-3′)/CPE3-EI-A (5′-GCATGAATTCGATGGCTTATAAAACGTGGCATCCTTATAAAA CTGTGTGTC-3′); 3× CPEmt1, CPEmt1-D3-S (5′-CGCCCAAGCTTGCAGCTTTTTGTG ACACACAGTTTTTGTGATGCCACG-3′)/CPEmt1-EI-A (5′-GCATGAATTCGATGGACA AAAACGTGGCATCACAAAAACTGTGTGTC-3′); 3× CPEmt2, CPEmt2-D3-S (5′-CGC CCAAGCTTGCAGCTT TTTGGTGACACACAGTTTTTGGTGATGCCACG-3′)/CPEmt2-EI-A (5′-GCATGAATTCGATGGACCAAAAACGTGGCATCACCAAAAACTGTGTGTC-3′). The PCR products were separately sub-cloned into HindIII–EcoRI-digested pcDNA3.1(+) to create pcDNA3.1-3×CPEs.
A kinase assay was carried out at 30 °C for 30 min in a final volume of 25 μl of reaction buffer (50 mM Tris-Cl, 10 mM MgCl2, pH 7.5) containing 1 μg substrate, 200 μM ATP, 1 mCi [γ32P]ATP and 5 units of PKA catalytic subunit (NEB). Reactions were stopped by adding SDS-sample buffer and boiling at 100 °C for 5 min. Then, [32 P] phosphate incorporation was analyzed by SDS-PAGE and a phosphoimager. To confirm the specificity of phosphorylation by PKA, either 40 μM KT5720 (AG Science) or dimethyl sulfoxide (DMSO) (Sigma) was added to the reaction mixture.
To examine whether ApCPEB4 is an endogenous substrate of Aplysia PKA, the crude tissue extract from Aplysia pedal-pleural ganglia was prepared as previously described . The reaction was carried out at 18 °C for 20 min containing GST-agarose bead binding 1 μg of GST-ApCPEB4, 10 μg of tissue extract and 1 mCi [γ32P]ATP in extraction buffer. To confirm the specificity of phosphorylation, the crude tissue extracts were incubated with inhibitors of specific kinases, 40 μM KT5720 (PKA inhibitor) , 20 μM PD98059 (MEK inhibitor) or 10 μM chelerythrin (PKC inhibitor), for 10 min. A GST-pull down assay was performed as previously described . The [32 P] phosphate incorporation was analyzed by SDS-PAGE and a phosphoimager.
Recombinant protein purification and antibody production
For the antibody production, the N-terminal 400 bp of ApCPEB4 was amplified by PCR and ligated into pRSETa (Invitrogen), a His-tag vector. The His-ApCPEB4-N protein expression was induced by 2 mM IPTG for 3 h at 37 °C and purified by a Ni-NTA purification system (Invitrogen). Polyclonal anti-ApCPEB4 antibodies were raised in mice using this purified protein. The peptide competition assay was performed by western blot using the ApCPEB4 antibodies incubated with either 25 μg of purified His-ApCPEB4-N or 25 μg of BSA as a control at 4 °C overnight.
RT-PCR, western blot, and immunocytochemistry
To examine the expression of ApCPEB4, an RT-PCR was performed using the total RNAs from various Aplysia tissues or HEK293T cells using gene-specific primers. For loading control, PCR was performed against S4 for Aplysia. For the induction control, PCR was performed against Aplysia CCAAT-enhancer-binding proteins (ApC/EBP). A western blot was performed in the pleural ganglia, buccal muscle, and gill extracts. Anti-ApCPEB4, and anti-actin antibodies were used to detect each protein within the same loaded sample. To examine the induction level of ApCPEB4 in response to 5-HT, pleural-pedal ganglia were prepared in a sylgard plate and then applied with 5 pulses of 5-HT (20 μM for 5 min at 20 min interval). Pleural ganglia were prepared 30 min after final application of 5-HT. For the immunostaining of endogenous ApCPEB4, cultured neurons were washed with cold ASW twice and immediately fixed with 4 % paraformaldehyde in PBS after either the application of massed 5-HT (10 μM for 1 h) or 5 pulses 5-HT (10 μM for 5 min) at 20 min interval.. Fixed cells were washed with PBS and permeabilized with 0.2 % Triton X-100 in PBS for 10 min. After blocking with 3 % BSA (Amersham Biosciences, Piscataway, NJ) for 2 h at room temperature, primary antibodies were treated (1:500 of anti-ApCPEB4 serum) overnight at 4 °C. The cells were washed with PBS and treated with secondary antibody, Cy3-conjugated anti-mouse IgG (Amersham Biosciences, Piscataway, NJ) for 1 h at room temperature. Immunostained images were acquired by a confocal laser scanning microscope (LSM510, Carl Zeiss, Jena, Germany).
mRNA-protein pull-down assay
mRNA-protein pull-down assay was performed as described previously  with small modification. Actin 3′UTR was obtained from Aplysia ganglion cDNA, and Luciferase-1904 (Luc-1904) was obtained by oligomer annealing and subcloned into pGL3UC vector (Promega) . The biotin labeled RNA was prepared by in-vitro transcription with T7 RNA polymerase (Promega) using the nucleotide analog Bio-17-ATP and Bio-11-CTP (Enzo). Each biotinylated RNA was analyzed by agarose-gel electrophoresis and quantified by nano-drop. HEK293T cells overexpressing Flag-tagged target proteins were lysed using lysis & binding buffer containing 50 mM Tris–HCl (pH 7.6), 150 mM NaCl, 5 % glycerol, 0.1 % Triton X-100, 1 mM DTT, 0.2 mg/mL heparin, 0.2 mg/mL yeast tRNA, 0.25 % BSA, protease inhibitor cocktail (Roche), and 40 U/mL RNasin (Promega). 8 μg of biotinylated RNAs were mixed with pre-cleared 200 μg (0.2 mg/mL) of 293 T cell lysate and incubated on a rotator for 1 h at 4 °C. 30 μl of NeutraAvidin Agarose Resin (Thermo) was added to each tube, and the mixture was further incubated for 2 h. Beads were washed five times with washing buffer containing 50 mM Tris–HCl (pH 7.6), 150 mM NaCl, 5 % glycerol, 0.1 % Triton X-100, 1 mM DTT and 40 U/mL RNasin. Western blots were performed with mFlag-M2 antibody (1:2000, Sigma).
Cell cultures and microinjection
Primary culture of Aplysia sensory neurons and coculture of sensory-to-motor neurons were made as described previously [24–26]. Briefly, Abdominal and central ganglia were dissected from Aplysia kurodai (50-100 g) and incubated at 34 °C for 1.5 ~ 2.5 h in 1 % protease (type IX, Sigma) dissolved in isotonic L15/ASW (1:1) media (ASW: 460 mM NaCl, 10 mM KCl, 11 mM CaCl2, 55 mM MgCl2, and 10 mM HEPES, pH 7.6). After a thorough washing with ASW several times to remove residual protease, the ganglia were incubated at 18 °C for at least 3 h in L15/ASW to allow for recovery from heat shock. LFS motor neurons were dissected from the abdominal ganglia and cultured in a solution of 50 % Aplysia hemolymph in isotonic L15 media. The next day, pleural sensory neurons were isolated from the pleural ganglia and cocultured with LFS motor neurons and maintained at 18 °C in an incubator for 3 days to allow time for the formation and stabilization of synaptic connections. Microinjection of DNAs and double-strand RNAs into Aplysia neurons was done by air pressure as described elsewhere [27, 28].
The LFS motor neuron was impaled with a glass microelectrode filled with 2 M K-acetate, 0.5 M KCl, 10 mM K-HEPES (10–15 MΩ), and the membrane potential was held at −80 mV. The excitatory postsynaptic potential (EPSP) in the motor neuron was evoked by stimulating the sensory neurons with a brief depolarizing stimulus using an extracellular electrode. The initial EPSP value was measured 24 h after microinjection. The cultures then received one pulse or five pulses of 10 μM 5-HT for 5 min at 15-min interval to induce LTF. The amount of synaptic facilitation was calculated as a percentage change in EPSP amplitude recorded after the 5-HT treatment compared with its initial value before treatment.
Cloning of ApCPEB4-like protein, a homologue of mammalian CPEB4
As an initial step in investigating the role of other CPEBs in Aplysia, we obtained an expressed sequence tag (EST) clone homologous to the conserved RNA recognition motif (RRM) of mammalian CPEB2-4 family from the Aplysia kurodai EST database . Using this EST clone as a probe, we carried out a library screening and cloned a full-length cDNA of a novel Aplysia CPEB (Fig. 1a). We named the clone ApCPEB4 as it is 99 % identical to CPEB4-like gene in the genomic database of A. californica (NCBI accession #, XP005089812). ApCPEB4 has a unique N-terminus and two conserved RRM on the C-terminus [15, 30] (Fig. 1a). Unlike the long form of ApCPEB, which was cloned previously , ApCPEB4 does not have a prion poly-Q domain. ApCPEB4 has a potential PKA phosphorylation site (RRST, consensus sequence (RRX(S/T)) outside the RRM domains (Fig. 1a). Even though the sequence was not identical, the overall phylogenetic analysis of the phosphorylation site and the RRM domain of ApCPEB4 revealed that ApCPEB4 is homologous to mammalian CPEB2-4 and Drosophila Orb2 (Fig. 1b and c). The amino acid sequences of the ApCPEB4 RRM domain are 83.0 % identical to mouse CPEB2, 82.0 % to mouse CPEB3, 80.7 % to mouse CPEB4, 77.4 % to Orb2, 34.4 % to mouse CPEB1, 32.7 to Orb1 and 31.0 % to ApCPEB, respectively. These analyses suggest that ApCPEB4 is homologous to the members of the mammalian CPEB2-4 family. Interestingly, the ApCPEB4 3′ untranslated region (UTR) (~1 kb) contains the nuclear polyadenylation hexanucleotide sequence (Fig. 1d).
We next examined the expression of ApCPEB4 in various Aplysia tissues by Reverse Transcription-Polymerase Chain reaction analysis (RT-PCR). ApCPEB4 was expressed in the extracts of central nervous system (CNS) and other tissues including gill and ovotestis (Fig. 1e). Western blot analysis detected significant bands with the size of ~100 kDa and ~70 kDa in both purified proteins and protein extracts from Aplysia pleural ganglia, respectively (Fig. 1f). Taken together, these data indicate that ApCPEB4 is another neuronal CPEB protein that belongs to CPEB family in Aplysia.
ApCPEB4 is synthesized in response to 5-HT signaling
We next asked whether the expression of ApCPEB4 is regulated in response to 5-HT. We found that the level of ApCPEB4 protein in the ganglia extracts was significantly increased by either spaced (5 times pulses of 5 min each) (Fig. 1g) or massed (2 h) application of 5-HT onto the intact pleural-to-pedal ganglia, both of which are known to induce long-term facilitation (Additional file 1: Figure S1). The increase in protein level was not transcription-dependent, because ApCPEB4 RNA transcript was not increased by 5-HT treatment (Fig. 1h).
Transcription-independent increase of ApCPEB4 suggests that 5-HT signaling may regulate translation of ApCPEB4 mRNA or stability of ApCPEB4 protein. We first examined whether ApCPEB4 mRNA was present and localized at the distal neurite. When the 3′UTR of ApCPEB4 was added at the end of the cDNA sequence of a reporter gene - nGFP (nuclear GFP)- the GFP signal was observed at the distal neurite (Fig. 2a). This supports the idea that the 3′UTR of ApCPEB4 is sufficient for the localization and translation of the mRNA at the distal neurite. We next cut off the cell bodies of cultured sensory neurons, and stimulated the isolated neurites for 1 h with 10 μM 5-HT. We found that ApCPEB4 immunoreactivity was increased about 2 fold in the stimulated neurites compared with neurites treated with vehicle- (vehicle, 100.0 ± 14.4 %, n = 6 versus 5-HT, 186.8 ± 17.8 %, n = 6; * p < 0.05, one-way ANOVA; F = 12.73, Tukey’s post-hoc test.) (Fig. 2b). This increase is also observed in the neurites treated with pulsed 5-HT (5 min of 10 μM 5-HT, 5 times; vehicle, 100.0 ± 46.1 %, n = 43 versus 5×5-HT, 128.8 ± 5.9 %, n = 60; two-tailed unpaired t test, p < 0.01). The up-regulation of ApCPEB4 was blocked by emetine (100 μM), a non-selective protein synthesis inhibitor (vehicle, 100.0 ± 14.4 %, n = 6; 5-HT, 186.8 ± 17.8 %, n = 6; emetine, 98.24 ± 26.9 %, n = 5; *, p < 0.05; n.s., not significant; one-way ANOVA; F = 12.73, Tukey’s post-hoc test) (Fig. 2b). Conversely, the induction of ApCPEB4 was not affected by the transcriptional inhibitor, actinomycin D (50 μM) (actD, 244.3 ± 20.7 %, n = 7; p > 0.05, one-way ANOVA; F = 12.73, Tukey’s post-hoc test.) (Fig. 2b). These results together suggest that 5-HT signaling enhances translation, but not transcription of ApCPEB4 mRNA in the stimulated neurites.
Two distinct translational mechanisms are known to be recruited during 5-HT-mediated synaptic facilitation in Aplysia: rapamycin-sensitive and -insensitive ones . Since ApCPEB4 was translated in the isolated neurites, we further tested whether this translational induction is sensitive to the rapamycin. When the rapamycin (20 nM) was added together with 5-HT on the isolated neurites, translational induction of ApCPEB4 was not blocked, indicating that translation of ApCPEB4 is rapamycin-insensitive (Fig. 2c). Rapamycin-insensitive, but emetine-sensitive local translation requires protein kinase A (PKA) activity for the initiation of synapse-specific LTF [31, 8]. Translation of ApCPEB4 was blocked by KT-5720 (PKA inhibitor, 5 μM) (Fig. 2c), raising the possibility that the translation of ApCPEB4 might be critical for the initiation of LTF (vehicle, 100.0 ± 2.5 %, n = 10 versus 5-HT, 150.4 ± 13.7 %, n = 14; ** p < 0.05; KT-5720, 86.4 ± 10.8 %, n = 13; rapamycin, 146.7 ± 9.5 %, n = 13; *, p < 0.05; ***, p < 0.001; one-way ANOVA; F = 9.23, Tukey’s post-hoc test).
RNA binding specificity of ApCPEB4
A growing body of evidence suggests that mammalian CPEB1 and CPEB2-4 family have different target RNAs. For example, CPEB1 has higher affinity to CPE site on the 3′UTR of target mRNAs, but CPEB3-4 are believed to recognize specific RNA secondary structure . We tested whether Aplysia CPEB proteins, ApCPEB and ApCPEB4, also show difference in RNA binding properties. We first generated five different target RNA constructs containing three types of three-repeated (3×) CPE sites (CPE1 (UUUUUAU), CPE2 (UUUUAUU) and CPE3 (UUUUAUAAG) or two types of 3× CPE mutant sites (CPEmt1 (UUUUUGU) and CPEmt2 (UUUUUGGU)) (Fig. 3a). ApCPEB4 did not bind to any CPE or CPE mutant site, whereas ApCPEB bound to CPE sites but not to CPE mutant sites (Fig. 3b). These results indicate that ApCPEB4 and ApCPEB have different RNA binding properties. We tested this idea further by using the CPE site of the 3′UTR of Aplysia actin, which is a target mRNA of the ApCPEB . Interestingly, ApCPEB4 did not bind to CPE site in the 3′UTR of Aplysia actin, which contains a well-known CPE site (UGUAUUUUUUAUACAAUGUU), whereas ApCPEB showed specific binding to the 3′UTR of actin (Fig. 3c). Instead, ApCPEB4 bound to 1904 U-rich sequence (AAAGAGGAUUUGUGUUUUUCAGGAC), which was designed as a target mRNA for mammalian CPEB3-4  (Fig. 3c). These results suggest that ApCPEB4 is similar to mammalian CPEB3-4 family in its RNA-binding properties. Overall, these results suggest that in its target selectivity ApCPEB4 is functionally closer to the mammalian CPEB3-4 family and is different from ApCPEB.
ApCPEB4 is critical for the initiation of LTF
Previous reports found that ApCPEB is required for the maintenance of LTF . We thus examined whether ApCPEB4 plays any specific function during LTF in Aplysia by knocking down ApCPEB4 transcripts in Aplysia sensory neurons. We generated double-stranded (ds) RNAs against N-terminal sequences of ApCPEB (dsApCPEB) and ApCPEB4 (dsApCPEB4). Each ds RNA was injected into cultured sensory neurons, and the protein level of ApCPEB4 in neurites was measured by immunocytochemistry. Baseline expression as well as 5-HT-mediated translation of ApCPEB4 was significantly blocked in neurons injected with dsApCPEB4, but not in the naïve neurons or neurons injected with dsApCPEB (Naïve: no treatment, 100.0 ± 4.9 %, n = 26 versus 5-HT treatment, 120.9 ± 5.6 %, n = 28; two-tailed unpaired t test, ** p < 0.01; dsApCPEB: no treatment, 97.1 ± 7.8 %, n = 24 versus 5-HT treatment, 119.8 ± 6.3 %, n = 21; two-tailed unpaired t test,* p < 0.05; dsApCPEB4: no treatment, 78.5 ± 5.3 %, n = 19 verse 5-HT treatment, 90.4 ± 5.5 %, n = 20; two-tailed unpaired t test, N.S. (p > 0.05)) (Fig. 4a). These data indicate that dsApCPEB4 specifically blocks both endogenous expression and 5-HT-induced expression of ApCPEB4 in Aplysia sensory neurons.
We then examined whether ApCPEB4 is required for LTF. Depletion of ApCPEB during 5-HT exposure to 5×5HT blocks the maintenance, beyond 24 h but not the initiation, of the 5-HT-induced LTF  during the first 24 h. Interestingly, LTF measured after 24 h was significantly impaired in neurons injected with dsApCPEB4, but not in neurons injected with dsApCPEB or dsLuci (dsLuci, 98.7 ± 17.4 %, n = 11; dsApCPEB, 82.3 ± 27.2 %, n = 11; dsApCPEB4, 20.5 ± 18.5 % EPSP change, n = 12; dsLuci vs. dsApCPEB4, * p < 0.05, F = 3.83, one-way ANOVA with Tukey’s post-hoc test) (Fig. 4b), indicating that ApCPEB4 is involved in the initiation of LTF. This result suggests that the regulation of protein synthesis mediated by ApCPEB4 is critical at the initial stage of LTF formation, whereas ApCPEB is critical for the long-term maintenance of LTF.
Overexpression of ApCPEB4 reduces the threshold of LTF induction
We further examined a specific role of ApCPEB4 in induction of LTF by overexpressing it directly in sensory neurons of sensory-motor cocultures (Fig. 4c). We found that 1× 5-HT (10 μM, 5 min), which normally induces short-term facilitation (STF), induced LTF by overexpression of ApCPEB4, but not EGFP in sensory neurons (EGFP, 21.1 ± 20.4 %, n = 11; ApCPEB4 + EGFP, 111.0 ± 27.5 % EPSP change, n = 13; two-tailed unpaired t-test, ** p < 0.05) (Fig. 4d). These results suggest that the overexpression (artificial induction) of ApCPEB4 reduced the threshold of LTF induction and thus induced LTF with single 5-HT stimulus, further supporting the idea that the translational induction of ApCPEB4 is critical for the formation of LTF in Aplysia.
Phosphorylated ApCPEB4 by PKA is critical for LTF induction
Previous report showed that ApCPEB is not phosphorylated by PKA . On the other hand, ApCPEB4 possesses one conserved putative PKA phosphorylation site on the 294th threonine residue (Fig. 1a). Thus, we hypothesized that the function of ApCPEB4 might be regulated by PKA-mediated phosphorylation. We first performed an in vitro kinase assay. Purified GST-ApCPEB4 fusion proteins were phosphorylated by the catalytic subunit of PKA in vitro (Fig. 5a). The phosphorylation was reduced in the non-phosphorylatable mutant form of ApCPEB4 (ApCPEB4 T294A), in which 294th threonine was replaced by alanine (Fig. 5a). These results indicate that the 294th threonine of ApCPEB4 is a potential PKA phosphorylation site. In addition, we found that ApCPEB4 was phosphorylated by Aplysia neuronal cell lysate in a PKA-dependent manner (Fig. 5b), indicating that ApCPEB4 is a genuine substrate of endogenous PKA in Aplysia neurons.
We next asked: Is the phosphorylation of ApCPEB4 by PKA critical for the induction of LTF? If the phosphorylation of ApCPEB4 on the 294th threonine is critical, a mutant form ApCPEB4(T294A) should act as a dominant negative inhibitor. We therefore overexpressed the mutant ApCPEB4 (T294A) in Aplysia sensory neurons cocultured with motor neurons and examined the effect of its overexpression on LTF. We found that LTF was completely blocked in the synapse overexpressed with ApCPEB4 (T294A) in sensory neurons, whereas expression of ApCPEB4-WT control had no effect on LTF (ApCPEB4 (WT), 75.0 ± 29.4 %, n = 10 versus ApCPEB4 (T294A), −15.4 ± 17.0 % EPSP change, n = 5, unpaired t-test, * p < 0.05) (Fig. 5c). Taken together, these data indicate that phosphorylation of ApCPEB4 by PKA is required for the induction of LTF in Aplysia.
In this study, we cloned a novel protein ApCPEB4, which is related to ApCPEB. Whereas ApCPEB is critical for maintenance, the translational increase of ApCPEB4 was critical for the formation of LTF. Moreover, overexpression of ApCPEB4 reduced the threshold for the LTF. In addition, phosphorylation of ApCPEB4 by PKA was required for the LTF formation. Combined, our results suggest that the two different CPEBs cooperate in different stages during LTF to first initiate and then maintain long-lasting synaptic facilitation.
ApCPEB4 is essential for the initiation of LTF: different ApCPEBs regulate distinct target mRNAs during LTF
Our data revealed an involvement of ApCPEB4 in the initiation of LTF, and that the overexpression of ApCPEB4 reduces the threshold of LTF induction. This is in contrast to Aplysia CPEB, which regulates the maintenance of LTF at 72 h. Thus the two ApCPEBs play distinct roles in 5-HT-induced LTF.
How do these two ApCPEBs regulate LTF formation and maintenance differentially? One plausible explanation is the presence of the prion-like structure in the molecule. The persistence of synaptic plasticity and memory have been found to be mediated by the prion-like CPEB such as ApCPEB in Aplysia, orb2 in Drosophila, and CPEB3 in rodent [15, 22, 32]. Synaptic plasticity is mediated by the increase in the aggregation of the prion-like translational regulator ApCPEB or mammalian CPEB3. Therefore, these aggregates serve as functional prions and regulate local protein synthesis necessary for the maintenance of long-term memory. In fact, only antibodies that are specific to the aggregated form block the maintenance of long term facilitation.
Another plausible explanation is that these two ApCPEBs have different RNA binding specificity. We found that ApCPEB but not ApCPEB4 binds to CPE sequence as well as 3′ UTR of actin in CPE-dependent manner (Fig. 3). By contrast, ApCPEB4 bound to a different U-rich sequence, the 1904 sequence, which is a synthetic binding sequence for mammalian CPEB3-4, but not a canonical CPE (Fig. 3) . In fact, mammalian CPEB1 and mammalian CPEB2-4 also have different target mRNAs to regulate translation for different stages of synaptic plasticity via CPE site-dependent and-independent manners, respectively . In contrast to our results, it has been reported that mammalian CPEB4 seems to be dispensable for hippocampus-dependent plasticity and learning and memory . However, unlike to Aplysia and Drosophila, which have two types of CPEB, mammalian has four CPEB family including CPEB1-4, which might compensate other CPEBs.
These observations, suggest that activated ApCPEB and ApCPEB4 may regulate protein synthesis of two distinct groups of mRNAs, one group of mRNAs containing CPE sites for the maintenance of LTF and another group mRNAs containing CPE-independent sites for the initiation of LTF. It would be interesting to further discriminate target mRNAs used for distinct phases of LTF that are translated by ApCPEB and ApCPEB4, respectively.
PKA-dependent activation of ApCPEB4
In Xenopus oocytes, CPEB1 is phosphorylated by the kinase Aurora A (Eg2) at a canonical LD (S/T)R site [34, 35], and the phosphorylation of CPEB1 binds to cleavage and polyadenylation specificity factor (CPSF) to induce release of PARN from the ribonucleoprotein (RNP) complex, thereby enabling Germ-line-development factor 2 (Gld2) to elongate poly(A) tailing by default . On the other hand, ApCPEB has been found not to be phosphorylated but to be increased in the amount of protein expression to enhance the affinity to CPSF . Interestingly, ApCPEB4 is regulated differentially from ApCPEB. ApCPEB4 is directly phosphorylated by PKA on its canonical LD(S/T)R site.
In Aplysia, PKA is critical for both synapse-specific and cell-wide facilitation induced by 5-HT signaling. PKA phosphorylates many components required for LTF formation in Aplysia such as cAMP response element-binding protein (CREB), synapsin, Aplysia Activating Factor (ApAF), and Cell Adhesion Molecule-Associated Protein (CAMAP) [36–40]. Although we do not provide direct evidence, our data provide further insight into the mechanism of how the long-lasting forms of synaptic plasticity can be initiated via PKA-mediated phosphorylation and local translation of ApCPEB4. ApCPEB4 might connect PKA signaling to the local protein synthesis, which is required for the induction of more sustained synaptic activation, by means of the enhanced expression of target mRNAs of ApCPEB4 to support 5-HT-induced LTF.
Possible roles of ApCPEB4 in synapse-specific LTF
As shown in Fig. 2, ApCPEB4 protein can be localized in neurites. In addition, we previously reported that ApCPEB4-EGFP could form RNA granules within the neurites in Aplysia sensory neurons . Combined, ApCPEB4 can be localized in neurites and involved in local protein synthesis.
During synapse-specific LTF, local protein synthesis is required for two distinct phases of LTF: initiation and maintenance [8, 31]. For the maintenance of synapse-specific LTF, a rapamycin-sensitive local protein synthesis is required [8, 31]. One essential molecule which is locally synthesized in a rapamycin-sensitive manner is ApCPEB. ApCPEB regulates local translation of many specific mRNAs containing CPE sites including actin mRNA to sustain the synaptic facilitation for periods up to 72 h by supporting persistent structural and functional changes of the synapses . However, for the initiation of LTF, a second, rapamycin-insensitive, emetine-sensitive component of local protein synthesis is required in synapse-specific LTF . Our data illustrate that local induction of ApCPEB4 by 5-HT treatment is rapamycin-insensitive and emetine-sensitive. In addition, we also found that one pulse of 5-HT produced LTF in ApCPEB4-overexpressing sensory neurons. It is therefore possible that overexpression of ApCPEB4 combined with one pulse of 5-HT may be sufficient to produce the retrograde signal required for LTF induction. Overall, ApCPEB4 may be a key regulator required for generating the retrograde signal in initial local protein synthesis during synapse-specific LTF. Although it is still possible that ApCPEB4 may be involved in the rapamycin-insensitive, emetine-sensitive intermediate-term facilitation (ITF) [43, 44], it would be interesting to further dissect this possibility in a synapse-specific form of LTF.
In this study, we investigated the molecular and cellular function of a novel CPEB isoform in Aplysia, ApCPEB4. ApCPEB4 was translated and increased by stimuli inducing LTF and is required for the formation of LTF. Overexpression of ApCPEB4 reduced the threshold for LTF induction, and phosphorylation of ApCPEB4 by PKA was critical for the induction of LTF. ApCPEB4 and ApCPEB have distinct RNA binding selectivity: ApCPEB4 did not bind to the CPE sequence in actin mRNAs to which ApCPEB binds, whereas ApCPEB4 bound to non-CPE U-rich RNA sequence that was a target of mammalian CPEB2-4. Taken together, these results indicate ApCPEB4 plays a key role in the initiation of LTF in Aplysia, in parallel with the key role ApCPEB has in the maintenance of LTF.
3′ untranslated region
Aplysia activating factor
Aplysia CCAAT-enhancer-binding proteins
Cell adhesion molecule-associated protein
Central nervous system
Cytoplasmic polyadenylation element
Cytoplasmic polyadenylation element binding protein
Polyadenylation specificity factor
cAMP response element-binding protein
Expression sequence tag
Germ-line-development factor 2
The poly(A) binding protein
Element contain poly A ribonuclease
Protein kinase A
RNA recognition motif
Reverse transcription-polymerase chain reaction
Sweatt JD, Kandel ER. Persistent and transcriptionally-dependent increase in protein phosphorylation in long-term facilitation of Aplysia sensory neurons. Nature. 1989;339:51–4.
Squire LR, Davis HP. Cerebral protein synthesis inhibition and discrimination training: effect of extent and duration of inhibition. Behav Biol. 1975;13:49–57.
Rainbow TC. Role of RNA and protein synthesis in memory formation. Neurochem Res. 1979;4:297–312.
Pedreira ME, Dimant B, Tomsic D, Quesada-Allue LA, Maldonado H. Cycloheximide inhibits context memory and long-term habituation in the crab Chasmagnathus. Pharmacol Biochem Behav. 1995;52:385–95.
Lee YS, Bailey CH, Kandel ER, Kaang BK. Transcriptional regulation of long-term memory in the marine snail Aplysia. Mol Brain. 2008;1:3.
Kandel ER. The molecular biology of memory: cAMP, PKA, CRE, CREB-1, CREB-2, and CPEB. Mol Brain. 2012;5:14.
Lee YS. Genes and signaling pathways involved in memory enhancement in mutant mice. Mol Brain. 2014;7:43.
Martin KC, Casadio A, Zhu H, Yaping E, Rose JC, Chen M, Bailey CH, Kandel ER. Synapse-specific, long-term facilitation of aplysia sensory to motor synapses: a function for local protein synthesis in memory storage. Cell. 1997;91:927–38.
Schuman EM, Dynes JL, Steward O. Synaptic regulation of translation of dendritic mRNAs. J Neurosci. 2006;26:7143–6.
Lebeau G, DesGroseillers L, Sossin W, Lacaille JC. mRNA binding protein staufen 1-dependent regulation of pyramidal cell spine morphology via NMDA receptor-mediated synaptic plasticity. Mol Brain. 2011;4:22.
Richter JD. CPEB: a life in translation. Trends Biochem Sci. 2007;32:279–85.
Wells DG, Richter JD, Fallon JR. Molecular mechanisms for activity-regulated protein synthesis in the synapto-dendritic compartment. Curr Opin Neurobiol. 2000;10:132–7.
Richter JD. Think globally, translate locally: What mitotic spindles and neuronal synapses have in common. Proc Natl Acad Sci U S A. 2001;98:7069–71.
Kim JH, Richter JD. Opposing polymerase-deadenylase activities regulate cytoplasmic polyadenylation. Mol Cell. 2006;24:173–83.
Si K, Giustetto M, Etkin A, Hsu R, Janisiewicz AM, Miniaci MC, Kim JH, Zhu H, Kandel ER. A neuronal isoform of CPEB regulates local protein synthesis and stabilizes synapse-specific long-term facilitation in aplysia. Cell. 2003;115:893–904.
Si K, Choi YB, White-Grindley E, Majumdar A, Kandel ER. Aplysia CPEB can form prion-like multimers in sensory neurons that contribute to long-term facilitation. Cell. 2010;140:421–35.
Raveendra BL, Siemer AB, Puthanveettil SV, Hendrickson WA, Kandel ER, McDermott AE. Characterization of prion-like conformational changes of the neuronal isoform of Aplysia CPEB. Nat Struct Mol Biol. 2013;20:495–501.
Liu J, Schwartz JH. The cytoplasmic polyadenylation element binding protein and polyadenylation of messenger RNA in Aplysia neurons. Brain Res. 2003;959:68–76.
Yamamoto N, Hegde AN, Chain DG, Schwartz JH. Activation and degradation of the transcription factor C/EBP during long-term facilitation in Aplysia. J Neurochem. 1999;73:2415–23.
Khabour O, Levenson J, Lyons LC, Kategaya LS, Chin J, Byrne JH, Eskin A. Coregulation of glutamate uptake and long-term sensitization in Aplysia. J Neurosci. 2004;24:8829–37.
Sander EE, van Delft S, ten Klooster JP, Reid T, van der Kammen RA, Michiels F, Collard JG. Matrix-dependent Tiam1/Rac signaling in epithelial cells promotes either cell-cell adhesion or cell migration and is regulated by phosphatidylinositol 3-kinase. J Cell Biol. 1998;143:1385–98.
Mastushita-Sakai T, White-Grindley E, Samuelson J, Seidel C, Si K. Drosophila Orb2 targets genes involved in neuronal growth, synapse formation, and protein turnover. Proc Natl Acad Sci U S A. 2010;107:11987–92.
Huang YS, Kan MC, Lin CL, Richter JD. CPEB3 and CPEB4 in neurons: analysis of RNA-binding specificity and translational control of AMPA receptor GluR2 mRNA. EMBO J. 2006;25:4865–76.
Montarolo PG, Goelet P, Castellucci VF, Morgan J, Kandel ER, Schacher S. A critical period for macromolecular synthesis in long-term heterosynaptic facilitation in Aplysia. Science. 1986;234:1249–54.
Lee JA, Kim HK, Kim KH, Han JH, Lee YS, Lim CS, Chang DJ, Kubo T, Kaang BK. Overexpression of and RNA interference with the CCAAT enhancer-binding protein on long-term facilitation of Aplysia sensory to motor synapses. Learn Mem. 2001;8:220–6.
Lee JA, Kim H, Lee YS, Kaang BK. Overexpression and RNA interference of Ap-cyclic AMP-response element binding protein-2, a repressor of long-term facilitation, in Aplysia kurodai sensory-to-motor synapses. Neurosci Lett. 2003;337:9–12.
Kaang BK, Pfaffinger PJ, Grant SG, Kandel ER, Furukawa Y. Overexpression of an Aplysia shaker K+ channel gene modifies the electrical properties and synaptic efficacy of identified Aplysia neurons. Proc Natl Acad Sci U S A. 1992;89:1133–7.
Kaang BK. Parameters influencing ectopic gene expression in Aplysia neurons. Neurosci Lett. 1996;221:29–32.
Choi SL, Lee YS, Rim YS, Kim TH, Moroz LL, Kandel ER, Bhak J, Kaang BK. Differential evolutionary rates of neuronal transcriptome in Aplysia kurodai and Aplysia californica as a tool for gene mining. J Neurogenet. 2010;24:75–82.
Theis M, Si K, Kandel ER. Two previously undescribed members of the mouse CPEB family of genes and their inducible expression in the principal cell layers of the hippocampus. Proc Natl Acad Sci U S A. 2003;100:9602–7.
Casadio A, Martin KC, Giustetto M, Zhu H, Chen M, Bartsch D, Bailey CH, Kandel ER. A transient, neuron-wide form of CREB-mediated long-term facilitation can be stabilized at specific synapses by local protein synthesis. Cell. 1999;99:221–37.
Fioriti L, Myers C, Huang YY, Li X, Stephan JS, Trifilieff P, Colnaghi L, Kosmidis S, Drisaldi B, Pavlopoulos E, Kandel ER. The persistence of hippocampal-based memory requires protein synthesis mediated by the prion-like protein CPEB3. Neuron. 2015;86:1433–48.
Tsai LY, Chang YW, Lin PY, Chou HJ, Liu TJ, Lee PT, Huang WH, Tsou YL, Huang YS. CPEB4 knockout mice exhibit normal hippocampus-related synaptic plasticity and memory. PLoS One. 2013;8:e84978.
Mendez R, Hake LE, Andresson T, Littlepage LE, Ruderman JV, Richter JD. Phosphorylation of CPE binding factor by Eg2 regulates translation of c-mos mRNA. Nature. 2000;404:302–7.
Mendez R, Murthy KG, Ryan K, Manley JL, Richter JD. Phosphorylation of CPEB by Eg2 mediates the recruitment of CPSF into an active cytoplasmic polyadenylation complex. Mol Cell. 2000;6:1253–9.
Kaang BK, Kandel ER, Grant SGN. Activation of camp-responsive genes by stimuli that produce long-term facilitation in Aplysia sensory neurons. Neuron. 1993;10:427–35.
Lee JA, Lee SH, Lee C, Chang DJ, Lee Y, Kim H, Cheang YH, Ko HG, Lee YS, Jun H, et al. PKA-activated ApAF-ApC/EBP heterodimer is a key downstream effector of ApCREB and is necessary and sufficient for the consolidation of long-term facilitation. J Cell Biol. 2006;174:827–38.
Lee SH, Lim CS, Park H, Lee JA, Han JH, Kim H, Cheang YH, Lee YS, Ko HG, Jang DH, et al. Nuclear translocation of CAM-associated protein activates transcription for long-term facilitation in Aplysia. Cell. 2007;129:801–12.
Bartsch D, Ghirardi M, Casadio A, Giustetto M, Karl KA, Zhu H, Kandel ER. Enhancement of memory-related long-term facilitation by ApAF, a novel transcription factor that acts downstream from both CREB1 and CREB2. Cell. 2000;103:595–608.
Angers A, Fioravante D, Chin J, Cleary LJ, Bean AJ, Byrne JH. Serotonin stimulates phosphorylation of Aplysia synapsin and alters its subcellular distribution in sensory neurons. J Neurosci. 2002;22:5412–22.
Chae YS, Lee SH, Cheang YH, Lee N, Rim YS, Jang DJ, Kaang BK. Neuronal RNA granule contains ApCPEB1, a novel cytoplasmic polyadenylation element binding protein, in Aplysia sensory neuron. Exp Mol Med. 2010;42:30–7.
Bailey CH, Kandel ER, Si KS. The persistence of long-term memory: a molecular approach to self-sustaining changes in learning-induced synaptic growth. Neuron. 2004;44:49–57.
Yanow SK, Manseau F, Hislop J, Castellucci VF, Sossin WS. Biochemical pathways by which serotonin regulates translation in the nervous system of Aplysia. J Neurochem. 1998;70:572–83.
Jin I, Kandel ER, Hawkins RD. Whereas short-term facilitation is presynaptic, intermediate-term facilitation involves both presynaptic and postsynaptic protein kinases and protein synthesis. Learn Mem. 2011;18:96–102.
This work was supported by the National Honor Scientist Program of the Korean Ministry of Science and Technology (to B.K.K) as well as by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (to D.J.J.; 2013-R1A1A2012804). J.S., Y.H.C., S.H.L., and S.L.C. were supported by a BK21 Research Fellowship from the Korea Ministry of Education and Human Resources Development.
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The datasets supporting the conclusions of this article are included within the article.
SHL, JHS and YHJ performed most of the experiments. YSL and YHC cloned ApCPEB4 in Aplysia kurodai. DJJ, SLC, YWJ and YSC performed RNA binding experiments. SHL JAL and JHH performed the electrophysiological experiments. SHL, YHJ, JDJ and BKK designed the experiment. SHL, CSL, KS, ERK, DJJ, and BKK prepared the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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Figure S1. A representative Western blot (left) and quantification (right) of ApCPEB4 in Aplysia pleural ganglia extracts prepared from animals exposed to 5-HT in vivo for 2 h. Total extracts were prepared at indicated times and 20 μg of proteins were blotted with anti-ApCPEB4 antibodies (left, top panel). The same extracts were also stained with Coomassie blue as loading controls (left, bottom panel). (PDF 49 kb)
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Lee, S., Shim, J., Cheong, Y. et al. ApCPEB4, a non-prion domain containing homolog of ApCPEB, is involved in the initiation of long-term facilitation. Mol Brain 9, 91 (2016). https://doi.org/10.1186/s13041-016-0271-x
- Long-term facilitation