CrebB-17A
CREB interaction with DREAM The calcium-binding protein DREAM (Drosophila homolog: CG5890) binds specifically to DRE sites (refering to downstream regulatory element of the prodynorphin gene where the DRE sequence is GAGTCAAGG) in the DNA and represses transcription of target genes. Derepression at DRE sites following PKA activation depends on a specific interaction between CREM and DREAM. Two leucine-charged residue-rich domains (LCD) located in the kinase-inducible domain (KID) and in the leucine zipper of CREM and two LCDs in DREAM participate in a two-site interaction that results in the loss of DREAM binding to DRE sites and derepression. Since the LCD motif located within the KID in CREM is also present in CREB, and maps in a region critical for the recruitment of CBP, whether DREAM may affect CRE-dependent transcription was investigated. In the absence of Ca2+ DREAM binds to the LCD in the KID of CREB. As a result, DREAM impairs recruitment of CBP by phospho CREB and blocks CBP-mediated transactivation at CRE sites in a Ca2+-dependent manner. Thus, Ca2+-dependent interactions between DREAM and CREB represent a novel point of cross-talk between cAMP and Ca2+ signaling pathways in the nucleus (Ledo, 2002).
Transcriptional activity of the repressor DREAM depends on its high affinity binding to DRE sites in target genes. The process is controlled by the levels of nuclear calcium, the PI3 kinase pathway and the formation of DREAM-alphaCREM heteromers. Three functional EF-hands in the DREAM protein sense the intracellular concentration of Ca2+ and EF-hand occupancy by Ca2+ blocks binding of DREAM to DRE sites. Thus, increased levels of intracellular Ca2+ following membrane depolarization or release from intracellular stores result in DREAM-mediated transcriptional derepression. Site-directed mutagenesis of two residues within any of the functional EF-hands in DREAM produces mutant proteins (EFmutDREAM) that remain bound to DNA in the presence of elevated Ca2+ concentrations. In a background of wild-type DREAM, EFmutDREAMs behave as dominant-negative mutants and block Ca2+-dependent derepression. In addition to calcium, in hematopoietic progenitors binding of DREAM to DRE sites depends on PI3 kinase activation. The residues in DREAM and the downstream kinases in the PI3 kinase pathway responsible for this are presently unknown (Ledo, 2002 and references therein).
Transcriptional derepression at DRE sites following PKA activation depends on a specific protein-protein interaction between DREAM and alphaCREM that blocks binding of DREAM to the DRE site. The transcriptional repressor alphaCREM modulates CRE-dependent gene expression and does not bind directly to DRE sites. The DREAM-alphaCREM interaction involves two leucine-charged residue rich domains (LCDs) located in DREAM at positions 47 and 155, and two LCDs in alphaCREM located in the kinase-inducible domain (KID) and in the leucine zipper (LZ). The LCD motif was first described in nuclear coactivators (NCoA-1, p/CIP) and corepressors (N-CoR, SMRT) and has been implicated in protein-protein interactions with nuclear hormone receptors and CBP. Moreover, LCDs in the N- and C-terminals of CBP mediate the interaction with nuclear receptors and p/CIP, respectively. Two classes of LCDs have been defined; the NR box and the CoRNR 'corner box' whose consensus sequence are LxxLL and L/IxxV/II, respectively, where x denotes any amino acid. Interestingly, both LCDs in alphaCREM (ILNEL and LIEEL) have an anti parallel orientation compared with the NR consensus box, and define a third type of LCD. Among the different isoforms derived from the CREM gene, only alpha and epsilonCREM displace DREAM from DRE sites. Short ICER-CREM isoforms lack the N-terminal LCD while isoforms containing the second DNA-binding domain (ßCREM) have a non-functional LCD at the C-terminal. Moreover, the spacing between the two LCDs in alpha or epsilonCREM is important since tauCREM or CREB, two proteins that contain the same or very similar LCDs in the KID and LZ domains, but have the Q2 transactivation domain in between, do not displace the binding of DREAM from DRE sites. Accordingly, a two-site model of interaction was proposed to stabilize the DREAM-alphaCREM interaction that prevents DREAM binding to the DRE. Site-directed mutagenesis of one residue in any of the LCDs of DREAM blocks the two-site interaction with alphaCREM and produces DREAM mutants insensitive to PKA-dependent derepression (Ledo, 2002 and references therein).
Cyclic AMP-dependent gene expression is controlled at the transcriptional level by several bZIP transcription factors, including CREB, CREM and ATF proteins. They bind to CRE sites in target genes as homo- or heterodimers. Dimerization is achieved by the LZ located next to the basic DNA-binding domain in the C-terminal of the protein. Transcriptional activity by these dimers follows after phosphorylation in their KIDs and the recruitment of the transcriptional cofactor CREB-binding protein, CBP. Importantly, the LCD within the KID domain in alphaCREM, common to all CREM isoforms and almost identical in CREB, is located within a region important for the interaction with the CREB-interacting domain of CBP (amino acids 455-679) known as the KIX domain. In this study, it has been shown that there exists a Ca2+-dependent protein-protein interaction between DREAM and CREB through the LCD located in the KID domain of CREB. As a result of this interaction, DREAM prevents the recruitment of CBP and represses CRE-dependent transcription (Ledo, 2002).
Miscellaneous CREB interactions Deregulated expression of v-abl and BCR/abl genes has been associated with myeloproliferative
syndromes and myelodysplasia, both of which can progress to acute leukemia. These studies
identify the localization of the oncogenic form of the abl gene product encoded by the Abelson
murine leukemia virus in the nuclei of myeloid cells and the association of the v-Abl protein with
the transcriptional regulator cyclic AMP response element-binding protein. The specific domains within each of the proteins responsible for this interaction
have been mapped. Complex formation is a prerequisite for transcriptional potentiation of
CREB. Transient overexpression of the homologous cellular protein c-Abl also results in the
activation of promoters containing an intact CRE. These observations identify a novel function
for v-Abl, that of a transcriptional activator that physically interacts with a transcription factor
(Birchenall-Roberts. 1995).
An examination was made of the molecular basis for the synergistic regulation of the minimal TCR alpha enhancer by multiple
proteins was examined. Reconstitution of TCR alpha enhancer function in nonlymphoid cells requires expression of the
lymphoid-specific proteins LEF-1, Ets-1 and PEBP2 alpha (CBF alpha), and a specific arrangement of their binding sites in
the enhancer. Ets-1 cooperates with PEBP2 alpha to bind adjacent sites at one end of the enhancer, forming a
ternary complex that is unstable by itself. Stable occupancy of the Ets-1- and PEBP2 alpha-binding sites in a DNase I
protection assay was found to depend on both a specific helical phasing relationship with a nonadjacent ATF/CREB-binding
site at the other end of the enhancer and on LEF-1. The HMG domain of LEF-1 bends the DNA helix
in the center of the TCR alpha enhancer. The HMG domain of the distantly related SRY protein, which also
bends DNA, can partially replace LEF-1 in stimulating enhancer function in transfection assays. Taken together with the
observation that Ets-1 and members of the ATF/CREB family have the potential to associate in vitro, these data suggest that
LEF-1 can coordinate the assembly of a specific higher-order enhancer complex by facilitating interactions between proteins
bound at nonadjacent sites (Giese, 1995).
Transcriptional activation by CREB and CREM requires phosphorylation of a serine residue within the activation domain (Ser 133 in CREB; Ser 117 in CREM),
which as a result interacts with the coactivator CBP. The activator CREM is highly expressed in male germ cells and is required for post-meiotic gene expression.
Using a two-hybrid screen, a testis-derived complementary DNA has been isolated encoding a protein that has been termed ACT (for activator of CREM in testis), a
LIM-only protein that specifically associates with CREM. ACT is expressed coordinately with CREM in a tissue- and developmentally regulated manner. It
strongly stimulates CREM transcriptional activity in yeast and mammalian cells and contains an intrinsic activation function. As ACT bypasses the classical
requirements for activation, namely phosphorylation of Ser 117 and interaction with CBP, it represents a new route for transcriptional activation by CREM and
CREB. ACT may define a previously undiscovered class of tissue-specific coactivators whose function could be specific for distinct cellular differentiation
programs (Fimia, 1999).
DNA methylation is essential for epigenetic gene regulation during development. The cyclic AMP (cAMP)-responsive element (CRE) is found in the promoter of many cAMP-regulated genes and plays important roles in cAMP-regulated gene expression. Methylation occurs on the CRE site and results in transcriptional repression via a direct mechanism, that is, prevention by the methyl group of binding of the cAMP-responsive factor CREB to this site. A recent study indicated that the nucleosome is also important in repressing transcription. In this study, the regulation of transcriptional repression on methylated CRE was investigated. Focus was placed on methyl-CpG binding domain protein 2 (MBD2; see Drosophila MBD-like). MBD2 consists of two forms, MBD2a and MBD2b, the latter lacking the N-terminal extension of MBD2a. Unexpectedly, it was found that MBD2a, but not MBD2b, promotes activation of the unmethylated cAMP-responsive genes. An in vivo binding assay revealed that MBD2a selectively interacts with RNA helicase A (RHA), a component of CREB transcriptional coactivator complexes. MBD2a and RHA cooperatively enhances CREB-dependent gene expression. Interestingly, coimmunoprecipitation assays demonstrate that MBD2a binding to RHA is not associated with histone deacetylase 1. These results indicate a novel role for MBD2a in gene regulation (Fujita, 2003).
Elevations in circulating glucose and gut hormones during feeding promote pancreatic islet cell viability in part via the calcium- and cAMP-dependent activation of the transcription factor CREB. A signaling module is described that mediates the synergistic effects of these pathways on cellular gene expression by stimulating the dephosphorylation and nuclear entry of TORC2, a CREB coactivator. This module consists of the calcium-regulated phosphatase calcineurin and the Ser/Thr kinase SIK2, both of which associate with TORC2. Under resting conditions, TORC2 is sequestered in the cytoplasm via a phosphorylation-dependent interaction with 14-3-3 proteins. Triggering of the calcium and cAMP second messenger pathways by glucose and gut hormones disrupts TORC2:14-3-3 complexes via complementary effects on TORC2 dephosphorylation; calcium influx increases calcineurin activity, whereas cAMP inhibits SIK2 kinase activity. These results illustrate how a phosphatase/kinase module connects two signaling pathways in response to nutrient and hormonal cues (Screaton, 2004).
Krox-20, originally identified as a member of 'immediate-early' genes, plays a crucial role in the formation of two specific segments in the hindbrain during early development of the vertebrate nervous system. A genomic sequence of Xenopus Krox-20 (XKrox-20) has been cloned and a promoter element in the flanking sequence has been studied along with associated transcription factors, which function in early Xenopus embryos. Using the luciferase reporter assay system, it has been shown that the 5' flanking sequence is sufficient to induce luciferase activities when the reporter construct is injected into embryos at the eight-cell stage. Deletion and mutagenesis analyses of the 5' flanking sequence revealed a minimal promoter element that included two known subelements, a CArG-box (sequence of the form CC (A/T)6 GG) and cAMP response element (CRE) within a stretch of 22 bp nucleotide sequence (-72 to -51 from the transcription initiation site), both of which are essential for the promoter activity. The gel mobility shift assay indicated that these two subelements bind to some components in whole cell extracts prepared from stage 20 Xenopus embryos. Antibody supershift and competition experiments revealed that these components in cell extracts are serum response factor (SRF) and a member of CRE binding protein (CREB) family, proteins that bind the CArG-box and CRE, respectively. They appear to assemble on the minimal promoter element to produce a novel ternary complex. When mRNA of a dominant-negative version of Xenopus SRF (XSRFdeltaC) is injected into animal pole blastomeres at the eight-cell stage, expression of XKrox-20 in the nervous system as well as the minimal promoter activity is strongly suppressed. Suppression by XSRFdeltaC is counteracted by coexpressed wild-type XSRF. These results indicate that XSRF functions as an endogenous activator of XKrox-20 by forming a ternary complex with CREB on the minimal promoter element (Watanabe, 2005).
Dephosphorylation of CREB An examination of protein tyrosine phosphatases implicated in CREB dephosphorylation highlights the role of protein phosphatase 1. PP1s are a family of highly conserved serine/threonine phosphatases that consist of regulatory and catalytic subunits. PP1 and PP2A account for the majority of cellular protein phosphatase activity in mammals. Both are present in hippocampal neurons. Calcineurin (CaN or PP2B), a Ca2+/Calmodulin dependent phosphatase is barely detectable in the nucleus. Okadaic acid discriminates between the possible effects of PP1 and PP2A, and on pharmacological grounds (inhibition by OA) PP1 is the phosphatase regulating CREB dephosphorylation. Nevertheless, CaN acts as a negative regulator of CREB. CaN acts through intermediate proteins to hasten the dephosphorylation of CREB. Prolongation of the synaptic input on the time scale of minutes, in part by an activity-induced inactivation of calcineurin, greatly extends the period over which phospho-CREB levels are elevated, thus affecting induction of downstream genes (Bito, 1996).
Transforming growth factor (TGF)-beta1 prevents cell cycle progression by inhibiting several regulators, including cyclin A (See Drosophila Cyclin A). The TGF-beta1-induced
down-regulation of cyclin A promoter activity appears to be mediated via the activating transcription factor (ATF) site,
because mutation of this site abolishes down-regulation. Surprisingly, although TGF-beta1 treatment for 24 h markedly
decreases cyclin A promoter activity, it does not decrease the abundance of the ATF-binding proteins ATF-1 and cyclic
AMP-responsive binding protein (CREB). However, a reduction in
phosphorylated CREB and ATF-1 was observed in lung epithelial cells treated with TGF-beta1. TGF-beta1-induced
down-regulation of cyclin A promoter activity is reversed by okadaic acid (a phosphatase inhibitor) and by cotransfection
with plasmids expressing the cAMP-dependent protein kinase catalytic subunit or the simian virus small tumor antigen (Sm-t,
an inhibitor of PP2A). These data indicate that TGF-beta1 may down-regulate cyclin A promoter activity by decreasing
phosphorylation of CREB and ATF-1 (Yoshizumi, 1997).
Alternative splicing CREB The cAMP/protein kinase A signaling pathway activates the cAMP-responsive transcription factor
CREB. A unique alternative RNA splicing event occurs during the development
of germ cells in the testis, resulting in a translational switch from an mRNA encoding activator CREB
to an mRNA encoding novel inhibitor CREB (I-CREB) isoforms. Alternative splicing of an additional
exon into the CREB mRNA in mid to late pachytene spermatocytes results in the premature
termination of translation and consequent downstream reinitiation of translation producing I-CREBs.
The I-CREBs down-regulate cAMP-activated gene expression by inhibiting activator CREB from
binding to cAMP response elements. Further, the developmental stage-specific expression of I-CREBs
in germ cells of the seminiferous tubules correlates with the cyclical down-regulation of activator
CREB, suggesting that I-CREBs repress expression of the cAMP-inducible CREB gene as well as
other genes transiently induced by cAMP during the 12-day cycle of spermatogenesis (Walker, 1996).
During cellular stresses, phosphorylation of eukaryotic initiation factor-2 (eIF2) elicits gene expression designed to ameliorate the underlying cellular disturbance. Central to this stress response is the transcriptional regulator activating transcription factor, ATF4. This study investigated the mechanism regulating ATF4 expression involving the differential contribution of two upstream ORFs (uORFs) in the 5' leader of the mouse ATF4 mRNA. The 5' proximal uORF1 is a positive-acting element that facilitates ribosome scanning and reinitiation at downstream coding regions in the ATF4 mRNA. When eIF2-GTP is abundant in nonstressed cells, ribosomes scanning downstream of uORF1 reinitiate at the next coding region, uORF2, an inhibitory element that blocks ATF4 expression. During stress conditions, phosphorylation of eIF2 and the accompanying reduction in the levels of eIF2-GTP increase the time required for the scanning ribosomes to become competent to reinitiate translation. This delayed reinitiation allows for ribosomes to scan through the inhibitory uORF2 and instead reinitiate at the ATF4-coding region. Increased expression of ATF4 would contribute to the expression of genes involved in remediation of cellular stress damage. These results suggest that the mechanism of translation reinitiation involving uORFs is conserved from yeast to mammals (Vattem, 2004; full text of article).
Stress-induced eukaryotic translation initiation factor 2 (eIF2) alpha phosphorylation paradoxically increases translation of the metazoan activating transcription factor 4 (ATF4), activating the integrated stress response (ISR), a pro-survival gene expression program. Previous studies implicated the 5' end of the ATF4 mRNA, with its two conserved upstream ORFs (uORFs), in this translational regulation. This study reports on mutation analysis of the ATF4 mRNA that revealed that scanning ribosomes initiate translation efficiently at both uORFs and ribosomes that had translated uORF1 efficiently reinitiate translation at downstream AUGs. In unstressed cells, low levels of eIF2alpha phosphorylation favor early capacitation of such reinitiating ribosomes directing them to the inhibitory uORF2, which precludes subsequent translation of ATF4 and represses the ISR. In stressed cells high levels of eIF2alpha phosphorylation delays ribosome capacitation and favors reinitiation at ATF4 over the inhibitory uORF2. These features are common to regulated translation of GCN4 in yeast. The metazoan ISR thus resembles the yeast general control response both in its target genes and its mechanistic details (Lu, 2004; full text of article).
Studies on various forms of synaptic plasticity have shown a link between messenger RNA translation, learning and memory. Like memory, synaptic plasticity includes an early phase that depends on modification of pre-existing proteins, and a late phase that requires transcription and synthesis of new proteins. Activation of postsynaptic targets seems to trigger the transcription of plasticity-related genes. The new mRNAs are either translated in the soma or transported to synapses before translation. GCN2, a key protein kinase, regulates the initiation of translation. This study reports a unique feature of hippocampal slices from GCN2-/- mice: in CA1, a single 100-Hz train induces a strong and sustained long-term potentiation (late LTP or L-LTP), which is dependent on transcription and translation. In contrast, stimulation that elicits L-LTP in wild-type slices, such as four 100-Hz trains or forskolin, fails to evoke L-LTP in GCN2-/- slices. This aberrant synaptic plasticity is mirrored in the behaviour of GCN2-/- mice in the Morris water maze: after weak training, their spatial memory is enhanced, but it is impaired after more intense training. Activated GCN2 stimulates mRNA translation of ATF4, an antagonist of cyclic-AMP-response-element-binding protein (CREB). Thus, in the hippocampus of GCN2-/- mice, the expression of ATF4 is reduced and CREB activity is increased. This study provides genetic, physiological, behavioural and molecular evidence that GCN2 regulates synaptic plasticity, as well as learning and memory, through modulation of the ATF4/CREB pathway (Costa-Mattioli, 2005; full text of article).
The late phase of long-term potentiation (LTP) and memory (LTM) requires new gene expression, but the molecular mechanisms that underlie these processes are not fully understood. Phosphorylation of eIF2α inhibits general translation but selectively stimulates translation of ATF4, a repressor of CREB-mediated late-LTP (L-LTP) and LTM. A pharmacogenetic bidirectional approach was used to examine the role of eIF2α phosphorylation in synaptic plasticity and behavioral learning. In eIF2α+/S51A mice, in which eIF2α phosphorylation is reduced, the threshold for eliciting L-LTP in hippocampal slices is lowered, and memory is enhanced. In contrast, only early-LTP is evoked by repeated tetanic stimulation and LTM is impaired, when eIF2α phosphorylation is increased by injecting into the hippocampus a small molecule, Sal003, which prevents the dephosphorylation of eIF2α. These findings highlight the importance of a single phosphorylation site in eIF2α as a key regulator of L-LTP and LTM formation (Costa-Mattioli, 2007).
Repeated synaptic activation results in sustained potentiation of synaptic transmission (LTP), a putative cellular model of learning and memory. Both memory and synaptic plasticity have two components. One, evoked by weak training protocols or a single-tetanic train, yields only transient phenomena, short-term memory (STM, lasting minutes to hours), and the early phase of LTP (E-LTP, lasting 1-3 hr). The second component, which follows strong training or repeated-tetanic trains, activates mechanisms that stabilize the memory and synaptic changes and results in long-term memory (LTM, lasting days, weeks or years) and the late phase of LTP (L-LTP, lasting many hours), respectively. Quite different molecular machineries, widely conserved from sea slugs to rodents, are thought to underlie these two components: modifications of pre-existing proteins are sufficient for the transient changes, whereas new gene expression (transcription and translation) is required for those that are sustained. For instance, LTM and L-LTP are suppressed by agents that block mRNA and protein synthesis and, conversely, both are induced more readily in transgenic mice in which gene expression is facilitated. Although the molecular mechanism by which gene expression is turned on is not fully understood, there is good reason to believe that the removal of constraints on gene expression is a critical step (Costa-Mattioli, 2007).
In diverse phyla, the transcription factor ATF4 is a repressor of cAMP responsive element binding protein (CREB)-mediated gene expression, which is required for L-LTP and LTM. The expression of ATF4 is regulated at the level of translation. Phosphorylation of the α subunit of the translation initiation factor eIF2 suppresses general translation, but selectively stimulates the translation of ATF4 mRNA. Neuronal activity-dependent modulation of eIF2α phosphorylation is likely to be important for sustained changes in synaptic transmission as induction of L-LTP in hippocampal slices, by either tetanic stimulation or treatment with forskolin or BDNF, is correlated with decreased eIF2α phosphorylation. In mice lacking the eIF2α kinase, GCN2, the reduction in phosphorylated eIF2α is associated with altered synaptic plasticity and memory (Costa-Mattioli, 2005). To investigate the role of eIF2α phosphorylation in long-term plasticity and behavioral memory, eIF2α heterozygous mice (eIF2α+/S51A) were used in which the phosphorylation site is mutated. In eIF2α+/S51A mice L-LTP and LTM formation are facilitated, as determined by several behavioral tasks. Moreover, a small molecule inhibitor of eIF2α dephosphorylation, Sal003, blocks L-LTP and memory storage, thus further demonstrating that eIF2α phosphorylation is a critical step in L-LTP and memory formation (Costa-Mattioli, 2007).
These results provide new insight into the mechanism of mnemonic processes by showing that changes at a single phosphorylation site of a key translation initiation factor bidirectionally modulate synaptic plasticity and memory storage. Specifically, it was found that reduced phosphorylation of eIF2α in eIF2α+/S51A mice is associated with enhanced synaptic plasticity, learning, and memory. Conversely, when dephosphorylation of eIF2α is blocked by Sal003, both L-LTP induction (but not maintenance) and long-term memory are impaired. Thus, this study provides genetic, chemical, physiological, behavioural, and molecular evidence that eIF2α dephosphorylation is essential for the induction of L-LTP and LTM (Costa-Mattioli, 2007).
Other lines of evidence support the hypothesis that dephosphorylation of eIF2α is critical for the induction of gene expression leading to L-LTP and LTM: (1) eIF2α phosphorylation promotes the translation of ATF4 mRNA, which encodes an inhibitor of CREB-driven gene expression, long-term synaptic and memory storage in different phyla; (2) eIF2α phosphorylation regulates protein synthesis, which is required for long-lasting synaptic plasticity and memory consolidation; (3) eIF2α phosphorylation is decreased by procedures that induce L-LTP and memory formation; (4) both L-LTP and LTM are more readily induced in mice lacking GCN2, the major kinase responsible for the phosphorylation of eIF2α in the brain (Costa-Mattioli, 2007).
How does eIF2α phosphorylation mediate the switch from short-term to long-term synaptic changes and memory? The proposed model is based on the regulation of gene expression by eIF2α phosphorylation. Under basal conditions, when eIF2α is partly phosphorylated by GCN2, ATF4 acts as a brake on the expression of CREB-dependent genes and protein synthesis is diminished. By reducing eIF2α phosphorylation, repeated training or tetanic stimulation lowers the level of ATF4 and removes the inhibitory constraint and also increases protein synthesis. Both mechanisms lead to the expression of genes required for long-term synaptic plasticity and memory. As a result of these changes in gene expression, the threshold for eliciting L-LTP and LTM is lowered. For instance, in eIF2α+/S51A and GCN2−/− mice, in which both eIF2α phosphorylation and ATF4 levels are reduced, the threshold for eliciting L-LTP and LTM is lowered. This model is supported by the increase in ATF4 expression upon treatment with Sal003, which leads to an impairment of L-LTP and LTM. The critical role of ATF4 in these processes is emphasized by the preservation of L-LTP in Sal003-treated ATF4−/− slices. Thus, these data provide direct genetic evidence that the impairment of L-LTP caused by Sal003 is dependent on ATF4's repressor action (Costa-Mattioli, 2007).
Additional evidence that ATF4 regulates L-LTP and LTM comes from the study of Chen (2003), who reported that in a transgenic mouse expressing a dominant-negative inhibitor (EGFP-AZIP) that targets both C/EBP proteins and ATF4, long-term synaptic plasticity and memory were facilitated under weak training protocol. This scheme is also consistent with previous studies on the ATF4 homolog in Aplysia (ApCREB2). After injection of anti-ApCREB2 antibodies into Aplysia sensory neurons, a single pulse of serotonin (5-HT), which normally induces short-term facilitation, evoked a gene expression-dependent facilitation that lasted more than one day. Thus, activation of gene expression is critical for long-term synaptic plasticity and memory formation (Costa-Mattioli, 2007).
Dephosphorylation of eIF2α could also lead to an increase in general protein synthesis. Since an increase in translation could facilitate LTP, it might in principle contribute to the enhancement of LTP and LTM in eIF2α+/S51A mice. However, the lack of effect of Sal003 in slices from ATF4−/− mice demonstrates that eIF2α phosphorylation acts primarily through regulation of ATF4 levels. It is likely that treatment with Sal003 for short times affects ATF4 mRNA translation to a greater extent than general translation. Indeed, in yeast it is known that levels of eIF2α phosphorylation that do not inhibit general translation, are sufficient for the enhanced translation of the GCN4 mRNA, which encodes a transcription factor of the same b-ZIP family, which contains ATF4. Though a significant block in general translation initiation was observed when Sal003 was applied for a long period ~8 hr, there was only a modest decrease in general translation when slices were incubated in the presence of Sal003 for 1 hr. Taken together, these data strongly suggest that eIF2α acts as a switch for both LTP and LTM through modulation of ATF4 mRNA translation (Costa-Mattioli, 2007).
Hippocampal ATF4 levels are regulated through GCN2-mediated phosphorylation of eIF2α (Costa-Mattioli, 2005). Accordingly, L-LTP and LTM are enhanced in eIF2α+/S51A mice following weak tetanic stimulation or weak training in various behavioral tasks. However, in contrast to GCN2−/− mice, in which L-LTP and LTM are impaired in response to repeated tetani or strong behavioral training, L-LTP and LTM are enhanced in eIF2α+/S51A mice. A possible explanation for this difference is that strong training or tetanic stimulation cause GCN2 to phosphorylate other targets in the brain (in addition to eIF2α) which interfere with L-LTP and LTM formation. Clearly, there must be limits to the extent to which the threshold for LTP induction can be manipulated. Therefore, it is conceivable that a mild induction of translation would facilitate L-LTP and LTM. However, too much translation may interfere with the optimal pattern of synaptic weight changes and may even induce an excess of proteins that antagonize L-LTP and LTM (such as depotentiation-related proteins). Another difference between GCN2−/− and eIF2α+/S51A mice is that auditory fear conditioning is normal in GCN2−/− mice, whereas it is enhanced in eIF2α+/S51A mice, after both weak and strong training protocols. Thus, in the amygdala, decreased eIF2α phosphorylation enhances memory formation, but this may occur in a GCN2-independent manner. Thus, either GCN2 is not the major eIF2α kinase in the amygdala, or the pathway that turns off GCN2 activity is not activated by auditory fear conditioning (Costa-Mattioli, 2007).
In conclusion, it has been shown that the induction of L-LTP and LTM is facilitated by decreased eIF2α phosphorylation and impaired by increased eIF2α phosphorylation. Taken together, these data strongly support the notion that under physiological conditions, a decrease in eIF2α phosphorylation constitutes a critical step for the activation of gene expression that leads to the long-term synaptic changes required for memory formation. These data suggest that ATF4 is an important regulator of these processes. These findings also raise the interesting possibility that regulators of translation could serve as therapeutic targets for the improvement of memory, for instance in human disorders associated with memory loss (Costa-Mattioli, 2007).
Memory storage and memory-related synaptic plasticity rely on precise spatiotemporal regulation of gene expression. To explore the role of small regulatory RNAs in learning-related synaptic plasticity, massive parallel sequencing was carried out to profile the small RNAs of Aplysia californica. 170 distinct miRNAs were identified, 13 of which were novel and specific to Aplysia. Nine miRNAs were brain enriched, and several of these were rapidly downregulated by transient exposure to serotonin, a modulatory neurotransmitter released during learning. Further characterization of the brain-enriched miRNAs revealed that miR-124, the most abundant and well-conserved brain-specific miRNA, was exclusively present presynaptically in a sensory-motor synapse where it constrains serotonin-induced synaptic facilitation through regulation of the transcriptional factor CREB. Direct evidence is presented that a modulatory neurotransmitter important for learning can regulate the levels of small RNAs, and a role is presented for miR-124 in long-term plasticity of synapses in the mature nervous system (Rajasethupathy, 2009).
miR-124 serves as a negative constraint on serotonin-induced long-term facilitation, since increased or decreased miR-124 levels in sensory neurons leads to a significant inhibition or enhancement, respectively, of synaptic facilitation. In particular, the inhibition of miR-124 confers to sensory-motor synapses a greater sensitivity for serotonin, since just one pulse of serotonin is sufficient to cause long-term facilitation. These physiology data also suggest that miR-124 inhibition is just one of many 5HT-mediated events that activate CREB to induce long-term facilitation, since the inhibition of miR-124 alone, in the absence of 5HT, does not lead to long-term facilitation. Therefore, while the observed effects of the miR-124 manipulations on LTF are of a significant magnitude, it is likely that these effects would be even greater if there were a coordinated manipulation of several miRNAs that act together in parallel pathways during synaptic plasticity. The observation that miR-124 levels affect facilitation both at 24 and 48 hr after exposure to spaced pulses of serotonin suggests that miR-124 regulation is required not only for the induction phase but that it is also critical for the maintenance phase of synaptic facilitation. Since miR-124 levels return back to baseline within 12 hr after exposure to serotonin, the initial drop in miR-124 during this time window appears to be sufficient enough to upregulate the relevant transcripts to allow for facilitation for up to 48 hr after exposure to serotonin. Indeed, the upregulation of many plasticity-related transcripts are transient and fall into this initial time window. The data also suggest that miR-124 does not significantly affect or contribute to serotonin-independent processes such as basal and constitutive synaptic activity. However, since all of the experiments were conducted on several-day-old cultures, at which point the cells and synapses are fully mature and stable, these studies leave open the possibility that miR-124 contributes to serotonin-independent processes in immature neurons such as neurite out-growth and synapse formation (Rajasethupathy, 2009).
The negative constraint that miR-124 imposes on synaptic facilitation is mediated, at least in part, by its direct regulation of CREB. The fact that miR-124 inhibition significantly and specifically increases CREB1 levels, along with immediate downstream genes such as UCH, C/EBP, and KHC, that miR-124 serotonin kinetics parallels the CREB1 serotonin kinetics, and that miR-124 inhibition can provide the switch necessary to convert short-term facilitation into long-term facilitation all strongly support the conclusion that miR-124 can tightly control CREB and CREB-mediated signaling during plasticity. CREB has been extensively studied over the years for its regulation by kinase-dependent posttranslational modifications, such as phosphorylation by PKA and MAPK. The present study, however, is one of the first to address posttranscriptional regulation of CREB. While this additional level of regulation might appear redundant, for example by paralleling the function of CREB2, it is likely that miR-124 inhibition allows for more rapid and transient control over CREB expression, as well as the opportunity for CREB to be drawn into various distinct downstream pathways once activated. It was also noticed that CREB, in turn, may be able to regulate miR-124 expression levels since there are several putative CREB binding sites in the presumed promoter region upstream of the Aplysia mir-124 gene. Although Aplysia and mammalian systems have clear differences in the complexities of their CNS, and also even in the types of neurotransmitters used during long-term memory processes, the underlying calcium-induced signaling pathways (including cAMP, PKA, MAPK, and CREB) and their functions are very much shared. It is therefore very likely that miR-124 is activity-regulated in the mammalian hippocampus and regulates CREB in much the same way as observed in this study, especially in light of the fact that the mammalian CREB1 UTR bears a conserved miR-124 target site as predicted by targetscan, which was recently confirmed as a site directly bound by Argonaute in mouse brain (Rajasethupathy, 2009).
In summary, this study has identified a comprehensive set of brain-enriched miRNAs in Aplysia, many of which can be regulated by the neuromodulator serotonin, signifying potential roles in learning-related synaptic plasticity. Specifically, it was demonstrated that brain-specific miR-124 responds to serotonin by derepressing CREB and enhancing serotonin-dependent long-term facilitation. This initial study compels the exploration of how neuromodulators act through small RNAs during various forms of plasticity and whether some act locally at synapses. This study also provides evidence that some 5HT-regulated Aplysia miRNAs regulate plasticity-related genes involved in local protein synthesis at the synapse. The likelihood of a coordinated set of miRNAs combinatorially regulating events at the synapse makes possible a new and rich layer of computational complexity that could be responsible for the emergence of discrete and long-lasting states of activity at the synapse (Rajasethupathy, 2009).
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