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Title: CREB-B acts as a key mediator of NPF/NO pathway involved in phase-related locomotor plasticity in locusts}
Article:
Abstract:
Gene expression changes in neural systems are essential for environment-induced behavioral plasticity in animals; however, neuronal signaling pathways mediating the effect of external stimuli on transcriptional changes are largely unknown. Recently, we have demonstrated that the neuropeptide F( NPF) /nitric oxide( NO) signaling pathway plays a regulatory role in phase-related locomotor plasticity in the migratory locust, Locusta migratoria. Here, we report that a conserved transcription factor, cAMP response element-binding protein B( CREB-B), is a key mediator involved in the signaling pathway from NPF2 to NOS in the migratory locust, triggering locomotor activity shift between solitarious and gregarious phases. We find that CREB-B directly activates brain NOS expression by interacting with NOS promoter region. The phosphorylation at serine 110 site of CREB-B dynamically changes in response to population density variation and is negatively controlled by NPF2. The involvement of CREB-B in NPF2-regulated locomotor plasticity is further validated by RNAi experiment and behavioral assay. Furthermore, we reveal that protein kinase A mediates the regulatory effects of NPF2 on CREB-B phosphorylation and NOS transcription. These findings highlight a precise signal cascade underlying environment-induced behavioral plasticity.
Introduction:
Animals can adjust to a changing environment by developing alternative behavioral phenotypes that improve their fitness; this phenomenon is known as “behavioral plasticity”[1–3]. Environmental stimuli acts directly on the nervous system and induces short-term changes in neural and endocrine activity, or long-term changes in gene expression, thus lead to behavioral alterations at different time scales[4, 5]. Various neural modulators with distinct profiles of molecular action are involved in this process[6–8]. In a given context, long-term behavioral plasticity is greatly shaped by transcriptional changes in key genes that are governed intricately by the interactions of neural modulators in the brain[9–11]. Transcription factors( TFs) play central roles in the regulation of behavioral plasticity through integrating neural signals and downstream transcriptional events[8, 12, 13]. Nuclear TFs primarily modify behavioral performance by binding to the regulatory region( e. g., promoter or enhancer) of their target genes[14, 15]. With distinct stimulation, TFs undergo either expression alteration or protein modification changes that affect their subcellular location, binding activity, or stability; and then result in transcriptional changes of downstream behavior-related genes[16–18]. For example, Fos family proteins can be induced rapidly or transiently in specific regions( such as nucleus accumbens and dorsal striatum) by drug abuse, thus influencing rewarding and locomotor behaviors[19]. Nuclear factor-κB( NF-κB) family members are essential for hippocampus-dependent long-term memory formation by regulating memory-associated genes[20]. Therefore, uncovering the precise signaling cascade by which TFs respond to upstream signal and regulate downstream gene transcription will provide insights into the regulatory mechanism underlying environment-induced behavioral plasticity. The migratory locust, Locusta migratoria, displays phase-related behavioral plasticity in response to population density variation[21, 22]. Gregarious locusts are highly active and attracted to their conspecifics, whereas solitarious locusts are cryptic and repelled from other individuals. The attraction index and locomotion activity substantially decrease during the isolation of gregarious locusts but are promoted by forced crowding in solitarious locusts[23]. During the time-course processes of solitarization and gregarization, gene expression profiles in the locus brain display dynamic changes[24]. The regulatory roles of several key genes have been revealed in behavioral phase transition[23, 25]. In particular, we have recently demonstrated that a neural neuropeptide F( NPF) /nitric oxide( NO) signaling pathway plays an essential role in phase-related locomotor plasticity, which results from the sequential changes in the phosphorylation and transcriptional states of nitric oxide synthase( NOS) as regulated by NPF1a and NPF2 systems, respectively[26]. NPF1a-regulated NOS phosphorylation initiates an immediate change in locomotor activity, whereas NPF2-regulated NOS transcription is responsible for long-term locomotor plasticity. However, the mechanism underlying NOS transcription changes as regulated by the up-stream TFs in response to population density variation is still unknown. In this study, we aim to identify the TF activating NOS transcription in response to population density change. Our results uncovered several novel components of the NPF/NO signaling cascade underlying phase-related locomotor plasticity in locusts.
Discussion:
Our study shows that the transcription factor CREB-B acts as a key mediator of the NPF/NO signaling pathway in regulating phase-related locomotor plasticity in migratory locusts. The essential role of CREB-B in locomotor plasticity is achieved through its direct control of NOS transcription under NPF2 regulation, and PKA transmits the effects of NPF2 on p-CREB-B. By combined using genome walking and dual-luciferase assays, we characterized the promoter sequence of NOS gene. The core promoter sequence of NOS lies at 150 bp to 120 bp upstream region of the NOS ORF that is required for basic NOS transcription. In this region, there might exist other negative regulatory elements because we found that transcriptional activity of NOS promoter decreases when more than-140 bp DNA sequence is included in pGL4. 1 vector, implying a complex regulatory networks in NOS promoter[33]. Apparently, gene transcription is a complex process that not only involves different regulators, but also depends on the sequence characteristics of promoter region[33, 34]. In fact, we bioinformatically predicted multiple CREB binding sites in the promoter region of NOS. And all three locust CREB members have active effects in HEK293T cells, although there are some differences in extent probably due to the differential expression levels of three CREB proteins or their distinct transactivation efficiency. In addition, the lower transcription activation effect of CREB3 observed in longer NOS promoter might be due to some inhibitory regulators, or more negative regulatory sequence were involved. Further in vivo studies show that only CREB-B can directly regulate NOS transcription by interacting with CREB R4, whereas the other two CREBs, CREB-A and CREB3, do not have significant regulatory effects on NOS transcription in the locust brain. CREB-B and its target NOS are extensively expressed in the neurons upon PI which is a brain region that involves in the regulation of locomotor rhythms in insects[35]. The similar localization of CREB-B and NOS provided additional evidence for their interaction and may support their roles in locomotor modulation in the locust. To our best knowledge, this result provides the first evidence showing the regulatory roles of CREB in dynamic NOS transcription in insects. A number of studies have reported that CREB family members display diverse biological functions depending on their cell- or tissue- specific distributions in insects. For example, in Apis mellifera, CREB is localized in mushroom bodies and is associated with age-dependent labor division[36]. Moreover, CREB has different regulatory effects on the feeding behavior of Drosophila when it expresses in neuronal and peripheral tissues[37]. Therefore, three locust CREB proteins may have distinct distributions and functions. Actually, similar phenomena have also been reported in mammal species[38]. Many reports suggested that NOS transcription is essential for both neural and behavioral plasticity by controlling NO content under various physiological or external stimuli[39, 40]. The transcriptional regulatory mechanisms of three NOS isoforms in vertebrates such as neural NOS( nNOS, NOS1), inducible NOS( iNOS, NOS2), and epithelial NOS( eNOS, NOS3) have been well studied. eNOS is regulated by FOXO in human umbilical vein endothelial cells[41], whereas iNOS transcription is controlled by both STAT1 and NF-κB in human fibroblasts[42]. For nNOS, multiple TFs have been proposed, such as NF-κB, SP, and ZNF family members[43–45]. To date, only one NOS isoform in insects has been characterized[46]. The phylogenetic analysis suggests that insect NOS and vertebrate NOSs evolve from the same ancestor. It seems that three vertebrate NOSs duplicate from a single gene after evolutionarily divergence with insect NOS( S15 Fig). Although the importance of NO signaling has been reported in many insects[47, 48], the regulatory mechanism underlying NO synthesis is rarely uncovered. This study provides insights into the inducibility of NOS expression in response to environmental stimuli in animals. Our results suggested that the phosphorylation level of CREB-B, but not the transcription level, is involved in the regulation of NOS transcription in response to the changes of population density in the locusts. Except for transcription alteration, several kinds of post-translational modifications, including phosphorylation, acetylation, and ubiquitination, have also been reported to control the transcriptional activation of CREB[31, 49, 50]. Phosphorylation, especially at Ser133 in the kinase-inducible domain( KID), is the most conserved indicator for CREB activation in mammals[51]. Our results showed that among the three antibodies that we tested; only the antibody against Ser133 phosphorylation of mammalian CREB1( homolog for locust CREB-B) can detect the predicted band in the locust brain. We also observed that the phosphorylation level of CREB-B remarkably responded to the population density change. This result is similar to those from other studies; for example, the reduced CREB activity by protracted social isolation in rats[52] and the age-dependent increase in CREB phosphorylation in honeybees[36]. These findings indicate the CREB phosphorylation serves as a common molecular signal in response to the changes in internal or environmental states. Among the candidate kinases that catalyze the phos-Ser133 of CREB[31, 32], only PKA has been demonstrated to be required for p-CREB-B in activating NOS transcription in the locust brain in our studies. However, we cannot exclude the potential roles of other several kinases on CREB phosphorylation in other physiological processes or tissues. The PKA-stimulated CREB phosphorylation has been accepted as a critical step for both neural and behavioral plasticity[53, 54]. The importance of PKA has been reported in the behavioral phase transition of desert locusts, Schistocerca gregaria[55]. Moreover, PKA has been recognized as a conserved down-stream signal transducer of both dopamine and serotonin[56, 57]. The two neurotransmitters induce behavioral gregarization and solitariness in the migratory locust, respectively[25, 58]. The involvement of PKA in NPF2/CREB-B axis was further validated by a series of pharmacological experiments of manipulating PKA activity. The importance of PKA in NPY-induced behavioral processes has also been revealed in other species. For example, the NPY signaling displays strongly suppressive effects on PKA-sensitized stress response in Drosophila[59]. In addition, the cAMP/PKA signal inhibits NPY-induced feeding behavior in rats[58]. We previously reported that the locust NPF2 peptide displays a close evolutionary relationship with NPY[26]. Thus, the NPY/PKA/CREB-B signaling cascade may represent a common mechanism underlying behavioral plasticity in animals. Surprisingly, although cAMP has been well-known as the main upstream messenger for PKA activation[53], we did not find any changes in cAMP level after manipulating either NPF1a or NPF2 peptide when we examined the whole locust brain[26]. One reasonable explanation is that the effects of NPF peptide on cAMP levels might have cell-specific patterns in the locust brain as reported in Drosophila[60]. The cAMP level may change only in a few neurons refer to NPF-NO circuit so that we cannot detect its changes in the whole brain. So, further work is needed to validate whether cAMP can link to NPF and PKA in specific regions of the locust brain during phase transition. Here, we show that NPF2 displays a long-term effect on locomotor activity by negatively regulating p-CREB-B level and subsequent NOS transcription in locust brain. Another locust NPF member, NPF1a gene, has been confirmed to be not involved in the regulation of p-CREB-B by the RNAi-induced knockdown, or the injection of either the full length or the truncated peptide. Apparently, p-CREB-B/NOS signal transduction may specifically respond to NPF2 manipulation. According to this work and our previous findings[26], we infer that NPF2, CREB-B, and NOS should have overlap expressions in the same neuron cells of PI in locust brains. The effects of NPF2 on CREB-B/NOS might through autocrine or paracrine manner by disperse from its expressing cells as reported in other species[61, 62]. In summary, this study extends our previous findings by uncovering two key signal components CREB-B and PKA in the NPF2/NO signaling pathway involved in the regulation of phase-related locomotor plasticity( See model in Fig 8). Our findings present a previously undefined regulatory mechanism on NOS transcription in insects and shed light on how neuromodulator/TF/effect gene network contributes to environment-induced behavioral plasticity.
The migratory locust , Locusta migratoria , is a worldwide agricultural pest that displays a remarkable density-dependent phase polyphenism , where being kept in a crowd triggers individuals to transit from the sedentary solitarious phase into the high-active gregarious phase . So , the migratory locust has been regarded as an excellent study model for environment-induced behavioral plasticity . Our previous finding shows that NPF2-regulated NOS transcription plays important roles in phase-related locomotor plasticity in the locust . Here , we further demonstrate that phosphorylated CREB-B directly activates NOS transcription in the pars intercerebralis , thus mediates phase-related locomotor plasticity . Further studies show that the levels of CREB-B phosphorylation is positively correlated with the crowding treatment and suppressed by NPF2 . Among several candidate kinases , protein kinase A is demonstrated to transmit the inhibitory effects of NPF2 on CREB-B phosphorylation and NOS transcription . Our study provides deep insight into the precise regulatory mechanisms underlying environment-induced behavioral plasticity .