Activation Of Immediate Early Genes Research Articles

The guanine nucleotide exchange factor RapGEF2 is required for ERK-dependent immediate-early gene (Egr1) activation during fear memory formation.

The MAP kinase ERK is important for neuronal plasticity underlying associative learning, yet specific molecular pathways for neuronal ERK activation are undetermined. RapGEF2 is a neuron-specific cAMP sensor that mediates ERK activation. We investigated whether it is required for cAMP-dependent ERK activation leading to other downstream neuronal signaling events occurring during associative learning, and if RapGEF2-dependent signaling impairments affect learned behavior. Camk2α-cre+/-::RapGEF2fl/fl mice with depletion of RapGEF2 in hippocampus and amygdala exhibit impairments in context- and cue-dependent fear conditioning linked to corresponding impairment in Egr1 induction in these two brain regions. Camk2α-cre+/-::RapGEF2fl/fl mice show decreased RapGEF2 expression in CA1 and dentate gyrus associated with abolition of pERK and Egr1, but not of c-Fos induction, following fear conditioning, impaired freezing to context after fear conditioning, and impaired cAMP-dependent long-term potentiation at perforant pathway and Schaffer collateral synapses in hippocampal slices ex vivo. RapGEF2 expression is largely eliminated in basolateral amygdala, also involved in fear memory, in Camk2α-cre+/-::RapGEF2fl/fl mice. Neither Egr1 nor c-fos induction in BLA after fear conditioning, nor cue-dependent fear learning, are affected by ablation of RapGEF2 in BLA. However, Egr1 induction (but not that of c-fos) in BLA is reduced after restraint stress-augmented fear conditioning, as is freezing to cue after restraint stress-augmented fear conditioning, in Camk2α-cre+/-::RapGEF2fl/fl mice. Cyclic AMP-dependent GEFs have been genetically associated as risk factors for schizophrenia, a disorder associated with cognitive deficits. Here we show a functional link between one of them, RapGEF2, and cognitive processes involved in associative learning in amygdala and hippocampus.

ERK2-topoisomerase II regulatory axis is important for gene activation in immediate early genes

The function of the mitogen-activated protein kinase signaling pathway is required for the activation of immediate early genes (IEGs), including EGR1 and FOS, for cell growth and proliferation. Recent studies have identified topoisomerase II (TOP2) as one of the important regulators of the transcriptional activation of IEGs. However, the mechanism underlying transcriptional regulation involving TOP2 in IEG activation has remained unknown. Here, we demonstrate that ERK2, but not ERK1, is important for IEG transcriptional activation and report a critical ELK1 binding sequence for ERK2 function at the EGR1 gene. Our data indicate that both ERK1 and ERK2 extensively phosphorylate the C-terminal domain of TOP2B at mutual and distinctive residues. Although both ERK1 and ERK2 enhance the catalytic rate of TOP2B required to relax positive DNA supercoiling, ERK2 delays TOP2B catalysis of negative DNA supercoiling. In addition, ERK1 may relax DNA supercoiling by itself. ERK2 catalytic inhibition or knock-down interferes with transcription and deregulates TOP2B in IEGs. Furthermore, we present the first cryo-EM structure of the human cell-purified TOP2B and etoposide together with the EGR1 transcriptional start site (–30 to +20) that has the strongest affinity to TOP2B within –423 to +332. The structure shows TOP2B-mediated breakage and dramatic bending of the DNA. Transcription is activated by etoposide, while it is inhibited by ICRF193 at EGR1 and FOS, suggesting that TOP2B-mediated DNA break to favor transcriptional activation. Taken together, this study suggests that activated ERK2 phosphorylates TOP2B to regulate TOP2-DNA interactions and favor transcriptional activation in IEGs. We propose that TOP2B association, catalysis, and dissociation on its substrate DNA are important processes for regulating transcription and that ERK2-mediated TOP2B phosphorylation may be key for the catalysis and dissociation steps.

Temporally specific gene expression and chromatin remodeling programs regulate a conserved Pdyn enhancer.

Neuronal and behavioral adaptations to novel stimuli are regulated by temporally dynamic waves of transcriptional activity, which shape neuronal function and guide enduring plasticity. Neuronal activation promotes expression of an immediate early gene (IEG) program comprised primarily of activity-dependent transcription factors, which are thought to regulate a second set of late response genes (LRGs). However, while the mechanisms governing IEG activation have been well studied, the molecular interplay between IEGs and LRGs remain poorly characterized. Here, we used transcriptomic and chromatin accessibility profiling to define activity-driven responses in rat striatal neurons. As expected, neuronal depolarization generated robust changes in gene expression, with early changes (1 hr) enriched for inducible transcription factors and later changes (4 hr) enriched for neuropeptides, synaptic proteins, and ion channels. Remarkably, while depolarization did not induce chromatin remodeling after 1 hr, we found broad increases in chromatin accessibility at thousands of sites in the genome at 4 hr after neuronal stimulation. These putative regulatory elements were found almost exclusively at non-coding regions of the genome, and harbored consensus motifs for numerous activity-dependent transcription factors such as AP-1. Furthermore, blocking protein synthesis prevented activity-dependent chromatin remodeling, suggesting that IEG proteins are required for this process. Targeted analysis of LRG loci identified a putative enhancer upstream of Pdyn (prodynorphin), a gene encoding an opioid neuropeptide implicated in motivated behavior and neuropsychiatric disease states. CRISPR-based functional assays demonstrated that this enhancer is both necessary and sufficient for Pdyn transcription. This regulatory element is also conserved at the human PDYN locus, where its activation is sufficient to drive PDYN transcription in human cells. These results suggest that IEGs participate in chromatin remodeling at enhancers and identify a conserved enhancer that may act as a therapeutic target for brain disorders involving dysregulation of Pdyn.

Temporally specific gene expression and chromatin remodeling programs regulate a conserved Pdyn enhancer

Neuronal and behavioral adaptations to novel stimuli are regulated by temporally dynamic waves of transcriptional activity, which shape neuronal function and guide enduring plasticity. Neuronal activation promotes expression of an immediate early gene (IEG) program comprised primarily of activity-dependent transcription factors, which are thought to regulate a second set of late response genes (LRGs). However, while the mechanisms governing IEG activation have been well studied, the molecular interplay between IEGs and LRGs remain poorly characterized. Here, we used transcriptomic and chromatin accessibility profiling to define activity-driven responses in rat striatal neurons. As expected, neuronal depolarization generated robust changes in gene expression, with early changes (1 hr) enriched for inducible transcription factors and later changes (4 hr) enriched for neuropeptides, synaptic proteins, and ion channels. Remarkably, while depolarization did not induce chromatin remodeling after 1 hr, we found broad increases in chromatin accessibility at thousands of sites in the genome at 4 hr after neuronal stimulation. These putative regulatory elements were found almost exclusively at non-coding regions of the genome, and harbored consensus motifs for numerous activity-dependent transcription factors such as AP-1. Furthermore, blocking protein synthesis prevented activity-dependent chromatin remodeling, suggesting that IEG proteins are required for this process. Targeted analysis of LRG loci identified a putative enhancer upstream of Pdyn (prodynorphin), a gene encoding an opioid neuropeptide implicated in motivated behavior and neuropsychiatric disease states. CRISPR-based functional assays demonstrated that this enhancer is both necessary and sufficient for Pdyn transcription. This regulatory element is also conserved at the human PDYN locus, where its activation is sufficient to drive PDYN transcription in human cells. These results suggest that IEGs participate in chromatin remodeling at enhancers and identify a conserved enhancer that may act as a therapeutic target for brain disorders involving dysregulation of Pdyn.

Neuronal activation in the geomagnetic responsive region Cluster N covaries with nocturnal migratory restlessness in white-throated sparrows (Zonotrichia albicollis).

Cluster N is a region of the visual forebrain of nocturnally migrating songbirds that supports the geomagnetic compass of nocturnal migrants. Cluster N expresses immediate-early genes (ZENK), indicating neuronal activation. This neuronal activity has only been recorded at night during the migratory season. Night-to-night variation in Cluster N activity in relation to migratory behaviour has not been previously examined. We tested whether Cluster N is activated only when birds are motivated to migrate and presumably engage their magnetic compass. We measured immediate-early gene activation in Cluster N of white-throated sparrows (Zonotrichia albicollis) in three conditions: daytime, nighttime migratory restless, and nighttime resting. Birds in the nighttime migratory restlessness group had significantly greater numbers of ZENK-labelled cells in Cluster N compared to both the daytime and the nighttime resting groups. Additionally, the degree of migratory restlessness was positively correlated with the number of ZENK-labelled cells in the nighttime migratory restless group. Our study adds to the number of species observed to have neural activation in Cluster N and demonstrates for the first time that immediate early gene activation in Cluster N is correlated with the amount of active migratory behaviour displayed across sampled individuals. We conclude that Cluster N is facultatively regulated by the motivation to migrate, together with nocturnal activity, rather than obligatorily active during the migration season.

Evaluation of Histone Demethylase Inhibitor ML324 and Acyclovir against Cyprinid herpesvirus 3 Infection.

Cyprinid herpesvirus 3 (CyHV-3) can cause severe disease in koi and common carp (Cyprinus carpio). Currently, no effective treatment is available against CyHV-3 infection in koi. Both LSD1 and JMJD2 are histone demethylases (HD) and are critical for immediate-early (IE) gene activation essential for lytic herpesvirus replication. OG-L002 and ML324 are newly discovered specific inhibitors of LSD1 and JMJD2, respectively. Here, HD inhibitors were compared with acyclovir (ACV) against CyHV-3 infection in vitro and in vivo. ML324, at 20-50 µM, can completely block ~1 × 103 PFU CyHV-3 replication in vitro, while OG-L002 at 20 µM and 50 µM can produce 96% and 98% inhibition, respectively. Only about 94% inhibition of ~1 × 103 PFU CyHV-3 replication was observed in cells treated with ACV at 50 µM. As expected, CyHV-3 IE gene transcription of ORF139 and ORF155 was blocked within 72 h post-infection (hpi) in the presence of 20 µM ML324. No detectable cytotoxicity was observed in KF-1 or CCB cells treated for 24 h with 1 to 50 µM ML324. A significant reduction of CyHV-3 replication was observed in ~6-month-old infected koi treated with 20 µM ML324 in an immersion bath for 3-4 h at 1-, 3-, and 5-days post-infection compared to the control and ACV treatments. Under heat stress, 50-70% of 3-4-month-old koi survived CyHV-3 infection when they were treated daily with 20 µM ML324 in an immersion bath for 3-4 h within the first 5 d post-infection (dpi), compared to 11-19% and 22-27% of koi in the control and ACV treatments, respectively. Our study demonstrates that ML324 has the potential to be used against CyHV-3 infection in koi.

Temporal pattern of Fos and Jun families expression after mitogenic stimulation with FGF-2 in rat neural stem cells and fibroblasts.

Intense stimulation of most living cells triggers the activation of immediate early genes, such as Fos and Jun families. These genes are important in cellular and biochemical processes, such as mitosis and cell death. The present study focused on determining the temporal expression pattern of Fos and Jun families in fibroblasts and neural stem cells of cerebellum, hippocampus, and subventricular zone (SVZ) of rats of different ages at 0, 0.5, 1, 3, and 6 h after stimulation with fibroblast growth factor (FGF)-2. In neonates, a similar expression pattern was observed in all cells analyzed, with lower expression in basal condition, peak expression at 0.5 h after stimulation, returning to baseline values between 1 and 3 h after stimulation. On the other hand, cells from adult animals only showed Fra1 and JunD expression after stimulation. In fibroblasts and hippocampus, Fra1 reached peak expression at 0.5 h after stimulation, while in the SVZ, peak level was observed at 6 h after stimulation. JunD in fibroblasts presented two peak expressions, at 0.5 and 6 h after stimulation. Between these periods, the expression observed was at a basal level. Nevertheless, JunD expression in SVZ and hippocampus was low and without significant changes after stimulation. Differences in mRNA expression in neonate and adult animals characterize the significant differences in neurogenesis and cell response to stimulation at different stages of development. Characterizing these differences might be important for the development of cell cultures, replacement therapy, and the understanding of the physiological response profile of different cell types.

Restoration of DNA integrity and the cell cycle by electric stimulation in planarian tissues damaged by ionizing radiation.

Exposure to high levels of ionizing γ radiation leads to irreversible DNA damage and cell death. Here, we establish that exogenous application of electric stimulation enables cellular plasticity and the re-establishment of stem cell activity in tissues damaged by ionizing radiation. We show that subthreshold direct current stimulation (DCS) rapidly restores pluripotent stem cell populations previously eliminated by lethally γ-irradiated tissues of the planarian flatworm Schmidtea mediterranea. Our findings reveal that DCS enhances DNA repair, transcriptional activity, and cell cycle entry in post-mitotic cells. These responses involve rapid increases in cytosolic Ca2+ concentration through the activation of L-type Cav channels and intracellular Ca2+ stores, leading to the activation of immediate early genes and ectopic expression of stem cell markers in post-mitotic cells. Overall, we show the potential of electric current stimulation to reverse the damaging effects of high-dose γ radiation in adult tissues. Furthermore, our results provide mechanistic insights describing how electric stimulation effectively translates into molecular responses capable of regulating fundamental cellular functions without the need for genetic or pharmacological intervention.

Bclaf1 promotes hematopoietic stem cell repopulating capacity and self-renewal

Abstract Hematopoietic stem cells (HSC) balance differentiation and self-renewal to promote maintenance of mature blood cell lineages. The mechanisms that regulate HSC expansion during fetal hematopoietic development and adult HSC maintenance remain significant knowledge gaps. BCLAF1 (Bcl2-associated factor 1), a transcriptional regulator, is highly expressed in hematopoietic cells. We recently defined its function as a modulator of ETS-family transcription factor activity in developing B cells. Our objective in this study is to determine the molecular mechanism of BCLAF1 in regulation of fetal and adult HSCs. We used mouse models with selective deletion of Bclaf1 in hematopoietic cells to test whether BCLAF1 regulates HSC development and function. We find that loss of Bclaf1 results in significantly reduced numbers of fetal HSCs. The reduced HSC numbers persist in adult mice; but there is no additional decline or development of bone marrow failure with aging. In competitive transplant experiments, Bclaf1-deficient fetal and adult HSCs have defective repopulation and self-renewal capacity resulting in reduced reconstitution of all hematopoietic populations. Bclaf1-deficient HSCs do not have abnormalities in emergence, proliferation, cell death, or homing activity. Using single cell RNA-sequencing of fetal HSCs, we find that loss of BCLAF1 results in reduction of a subpopulation of HSCs and upregulation of the early response genes Jun, Fos, and Egr1. Collectively, these findings suggest BCLAF1 restrains activity of immediate early genes to preserve HSC self-renewal capacity and promote HSC expansion during both fetal development and repopulation of hematopoiesis after HSC transplant. Supported by grants from Children's Discovery Institute, St. Louis Children's Hospital Foundation, Siteman Cancer Center, and NIH (T32 AI007163).

Novel objects alter immediate early gene expression globally for ZENK and regionally for c-Fos in neophobic and non-neophobic house sparrows

Neophobia – an animal’s reluctance to approach novel objects, try new foods, or explore unfamiliar environments – affects whether animals can adapt to new environments and exploit novel resources. However, despite its importance, the neurobiological mechanisms underlying this personality trait are poorly understood. In this study, we examined regional brain activity using the expression of two immediate early genes (IEGs), ZENK and c-Fos, in response to novel objects or control conditions in captive house sparrows (Passer domesticus, n = 22). When exposed to novel objects, we predicted that we would see differential IEG activity in brain regions involved in regulating stress and emotion (hippocampus, medial ventral arcopallium, lateral septum), reward and learning (striatum), and executive function (NCL) between neophobic and non-neophobic individuals. To classify birds by phenotype, we used behavior trials that tested willingness to approach a food dish in the presence of several different novel objects, habituation to one novel object, and willingness to try several different novel foods. We then exposed birds to a new novel object or a control condition and assessed protein expression of two IEGs in neophobic vs non-neophobic individuals after this final exposure. An analysis of average sparrow feeding times in the presence of novel objects showed a bimodal distribution of neophobia behavior. There was also high repeatability of individual novel object responses, and average responses to all three trial types (novel object, novel food, and habituation to a novel object) were significantly correlated. Although we saw no differences between neophobic and non-neophobic birds in IEG expression in response to novel objects in any of the 6 brain regions examined, there was a significant global decrease in ZENK expression and a significant increase in c-Fos expression in the medial ventral arcopallium and the caudal hippocampus in response to novel objects compared to controls, suggesting that these two regions may be important in novelty detection and threat perception. Additionally, there was no object effect in the rostral hippocampus, which supports the hypothesis that the avian hippocampus may have a rostrocaudal functional gradient similar to the septotemporal gradient in mammals.

Analysis of the early response to spinal cord injury identified a key role for mTORC1 signaling in the activation of neural stem progenitor cells

Xenopus laevis are able to regenerate the spinal cord during larvae stages through the activation of neural stem progenitor cells (NSPCs). Here we use high-resolution expression profiling to characterize the early transcriptome changes induced after spinal cord injury, aiming to identify the signals that trigger NSPC proliferation. The analysis delineates a pathway that starts with a rapid and transitory activation of immediate early genes, followed by migration processes and immune response genes, the pervasive increase of NSPC-specific ribosome biogenesis factors, and genes involved in stem cell proliferation. Western blot and immunofluorescence analysis showed that mTORC1 is rapidly and transiently activated after SCI, and its pharmacological inhibition impairs spinal cord regeneration and proliferation of NSPC through the downregulation of genes involved in the G1/S transition of cell cycle, with a strong effect on PCNA. We propose that the mTOR signaling pathway is a key player in the activation of NPSCs during the early steps of spinal cord regeneration.

Short-term transcriptomic response to plasma membrane injury

Plasma membrane repair mechanisms are activated within seconds post-injury to promote rapid membrane resealing in eukaryotic cells and prevent cell death. However, less is known about the regeneration phase that follows and how cells respond to injury in the short-term. Here, we provide a genome-wide study into the mRNA expression profile of MCF-7 breast cancer cells exposed to injury by digitonin, a mild non-ionic detergent that permeabilizes the plasma membrane. We focused on the early transcriptional signature and found a time-dependent increase in the number of differentially expressed (> twofold, P < 0.05) genes (34, 114 and 236 genes at 20-, 40- and 60-min post-injury, respectively). Pathway analysis highlighted a robust and gradual three-part transcriptional response: (1) prompt activation of immediate-early response genes, (2) activation of specific MAPK cascades and (3) induction of inflammatory and immune pathways. Therefore, plasma membrane injury triggers a rapid and strong stress and immunogenic response. Our meta-analysis suggests that this is a conserved transcriptome response to plasma membrane injury across different cell and injury types. Taken together, our study shows that injury has profound effects on the transcriptome of wounded cells in the regeneration phase (subsequent to membrane resealing), which is likely to influence cellular status and has been previously overlooked.

Hippocampal and prefrontal contributions to memory retrieval: Examination of immediate early gene, NMDA receptor and environmental interactions.

Animals can use a range of strategies to recall important locations. These include simple stimulus-response strategies and more complex spatial (place) strategies, which are thought to have distinct neural substrates. The hippocampus-and NMDA receptor activation therein-is considered to be crucial for spatial, but not response strategies. The medial prefrontal cortex has also been implicated in memory retrieval; however, evidence concerning its specific role is equivocal. Both hippocampal and prefrontal regions have been associated with flexible behavioural responding (e.g. when task demands change). Here, we investigated the use of spatial and non-spatial strategies in the Morris water maze and their associated brain areas in rats using immediate early gene (IEG) imaging of Zif268 and c-Fos. Specifically, we charted the involvement of hippocampal and prefrontal subregions during retrieval of spatial and non-spatial memories. Behavioural flexibility was also examined using intact and partial cue configurations during recall. Results indicated that regions of both the hippocampus (area CA3) and prefrontal cortex (anterior cingulate cortex) were preferentially engaged in spatial memory recall compared to response learning. In addition, both spatial and non-spatial memories were dependent on NMDA receptor activation. MK801 impaired recall performance across all groups and reduced IEG activation across hippocampal and prefrontal regions. Finally, IEG results revealed divergent patterns of Zif268 and c-Fos activity and support the suggestion that Zif268 plays a functional role in the recall of long-term memories.

DAPK3 participates in the mRNA processing of immediate early genes in chronic lymphocytic leukaemia.

Cross‐linking of the B‐cell receptor (BCR) induces transcriptional activation of immediate early genes (IEGs) including EGR1 and DUSP2 in chronic lymphocytic leukaemia (CLL). Here, we have shown that this transcriptional activation correlated with histone H3 threonine 6 and 11 phosphorylation. Both transcription and histone post‐translational modifications are repressed by ibrutinib, a small molecule inhibitor used in CLL treatment. Moreover, we have identified the death‐associated protein kinase 3 (DAPK3), as the kinase mediating these histone phosphorylation marks in response to activation of the BCR signalling pathway with this kinase being recruited to RNA polymerase II in an anti‐IgM‐dependent manner. DAPK inhibition mimics ibrutinib‐induced repression of both IEG mRNA and histone H3 phosphorylation and has anti‐proliferative effect comparable to ibrutinib in CLL in vitro. DAPK inhibitor does not repress transcription itself but impacts on mRNA processing and has a broader anti‐tumour effect than ibrutinib, by repressing both anti‐IgM‐ and CD40L‐dependent activation.

Chromatin Remodeling and Immediate Early Gene Activation by SLFN11 in Response to Replication Stress

Schlafen 11 (SLFN11) was recently discovered as a cellular restriction factor against replication stress. Here, we show that SLFN11 increases chromatin accessibility genome wide, prominently at active promoters in response to replication stress induced by the checkpoint kinase 1 (CHK1) inhibitor prexasertib or the topoisomerase I (TOP1) inhibitor camptothecin. Concomitantly, SLFN11 selectively activates cellular stress response pathways by inducing the transcription of the immediate early genes (IEGs), including JUN, FOS, EGR1, NFKB2, and ATF3, together with the cell cycle arrest genes CDKN1A (p21WAF1) and GADD45. Both chromatin remodeling and IEG activation require the putative ATPase and helicase activity of SLFN11, whereas canonical extrinsic IEG activation is SLFN11 independent. SLFN11-dependent IEG activation by camptothecin is also observed across 55 non-isogenic NCI-60 cell lines. We conclude that SLFN11 acts as a global regulator of chromatin structure and an intrinsic IEG activator with the potential to engage the innate immune activation in response to replicative stress.

A negative elongation factor E inhibits white spot syndrome virus replication by suppressing promoter activity of the viral immediate early genes in red claw crayfish Cherax quadricarinatus

Invertebrates rely solely on the innate immune system to protect against virus infection, while the viral infection must rely on the transcriptional system of the host cell to achieve the expression of viral genes, which is naturally regulated by the host's transcriptional system. However, the mechanism of the host against viral transcription in host cells is still poorly understood in crustaceans. Previously, we found that the partial transcript sequence of a negative elongation factor E (named as CqNELF-E) was up-regulated in a differentially expressed transcriptome library of the haematopietic tissue (Hpt) cells from red claw crayfish Cherax quadricarinatus upon white spot syndrome virus (WSSV) infection, suggesting a possible role of CqNELF-E in WSSV-host interaction. In the present study, we revealed the function of CqNELF-E. The full-length cDNA sequence of CqNELF-E was identified with 1726 bp from red claw crayfish, which contained an open reading frame of 816 bp, encoding 271 amino acids. Amino acid sequencing analysis revealed that the CqNELF-E had a conserved RNA recognition motif (RRM) and a leucine zipper motif (LZM). Tissue distribution analysis showed that CqNELF-E was widely expressed in various tissues with the highest expression in muscle, relatively abundant in Hpt and the lowest presence in heart. Interestingly, the gene expression of CqNELF-E was significantly up-regulated at both 6 and 12 hpi after WSSV infection in Hpt cell cultures in red claw crayfish. In addition, the expression of both the viral immediately early gene (IE) 1 (IE1) and a late gene envelope protein VP28 were significantly increased after gene silencing of CqNELF-E in Hpt cells, indicating the potential suppression role of CqNELF-E against the viral infection. Further study revealed that the CqNELF-E had an inhibitory effect on the promoter activity of WSSV IE genes WSV051, WSV069 (IE1) and WSV083 by a dual luciferase reporter gene assay. Taken together, these results suggest that CqNELF-E plays an antiviral role, probably via inhibition on the viral transcription activity in WSSV infection in a crustacean.

Temporal Profile of Brain Gene Expression After Prey Catching Conditioning in an Anuran Amphibian.

A key goal in modern neurobiology is to understand the mechanisms underlying learning and memory. To that end, it is essential to identify the patterns of gene expression and the temporal sequence of molecular events associated with learning and memory processes. It is also important to ascertain if and how these molecular events vary between organisms. In vertebrates, learning and memory processes are characterized by distinct phases of molecular activity involving gene transcription, structural change, and long-term maintenance of such structural change in the nervous system. Utilizing next generation sequencing techniques, we profiled the temporal expression patterns of genes in the brain of the fire-bellied toad Bombina orientalis after prey catching conditioning. The fire-bellied toad is a basal tetrapod whose neural architecture and molecular pathways may help us understand the ancestral state of learning and memory mechanisms in tetrapods. Differential gene expression following conditioning revealed activity in molecular pathways related to immediate early genes (IEG), cytoskeletal modification, axon guidance activity, and apoptotic processes. Conditioning induced early IEG activity coinciding with transcriptional activity and neuron structural modification, followed by axon guidance and cell adhesion activity, and late neuronal pruning. While some of these gene expression patterns are similar to those found in mammals submitted to conditioning, some interesting divergent expression profiles were seen, and differential expression of some well-known learning-related mammalian genes is missing altogether. These results highlight the importance of using a comparative approach in the study of the mechanisms of leaning and memory and provide molecular resources for a novel vertebrate model in the relatively poorly studied Amphibia.

Medial prefrontal and ventral hippocampal contributions to incidental context learning and memory in adolescent rats.

The Context Preexposure Facilitation Effect (CPFE) is a contextual fear conditioning (CFC) paradigm in which context learning, context-shock learning, and retrieval of contextual fear occur in three distinct phases. The medial prefrontal cortex (mPFC), dorsal hippocampus (dHPC), and ventral hippocampus (vHPC) are required for the acquisition and/or consolidation of a context representation during incidental context exposure (Heroux et al., 2017; Robinson-Drummer et al., 2016; Rudy & Matus-Amat, 2006). This exposure also induces the expression of the immediate early genes (IEGs) c-Fos, Arc, Egr-1, and Npas4 in these regions (Heroux et al., 2018, 2019). Despite these studies, it is still unclear how mPFC and vHPC contribute to incidental context learning and memory. The current study examined whether prefrontal or ventral hippocampal inactivation during context preexposure interferes with long-term context memory and IEG activity in the mPFC, vHPC, dHPC and the ventral midline thalamus (VMT, a region connected to both the mPFC and HPC). Adolescent Long-Evans rats were given intra-mPFC (Experiment 1) or intra-vHPC (Experiment 2) infusions of the GABAA receptor agonist muscimol or PBS prior to context preexposure, and then were sacrificed 30 min later and whole mPFC, dHPC, vHPC, and VMT were collected and assayed for IEG mRNA expression via qPCR. Prefrontal or ventral hippocampal inactivation during context exposure abolished subsequent post-shock and retention test freezing in behaviorally-tested littermates of the sacrificed groups. In Experiment 1, prefrontal inactivation reduced expression of c-Fos, Arc, Egr-1, and Npas4 in the mPFC, c-Fos, Arc, and Npas4 in the vHPC, and c-Fos in the VMT, to the level of behaviorally-naïve home-cage controls. Prefrontal inactivation did not alter IEG expression in the dHPC during context exposure. In Experiment 2, ventral hippocampal inactivation impaired expression of all IEGs in the mPFC, dHPC, and vHPC, with no effect in the VMT. Taken together, these results suggest that context memory processes on the preexposure day of the CPFE may depend on mPFC-vHPC circuitry not typically emphasized in studies of incidental or configural learning and memory.

Herpes simplex virus-1 (HSV-1) infection induces a potent but ineffective IFN-\u03bb production in immune cells of AD and PD patients

BackgroundThe sequential activation of immediate early (IE), early (E) and late (L) genes is required to allow productive herpes simplex virus type 1 (HSV-1) infection. Several evidences suggest that, together with inflammation, an immunological response incapable to counteract HSV-1 reactivation plays a role in the pathogenesis of Alzheimer’s (AD) and Parkinson’s (PD) diseases. IFN-lambda (IFN-λ), a cytokine endowed with a robust antiviral activity, contains HSV-1 reactivation. HSV-1-induced IFN-λ, IL-10 and IL-1β as well as the expression of viral IE, E and L genes were analyzed in vitro in peripheral blood mononuclear cells (PBMC) of AD and PD patients as well as of healthy controls (HC).MethodsPBMC of AD, PD and HC were in vitro infected with one multiplicity of infection (1 MOI) HSV-1. IE, E, and L viral genes transcription as well as IFN-λ, IL-10 and IL-1β production were analyzed.ResultsIn HSV-1-infected cells of AD and PD patients compared to HC: (1) transcription of IE (ICP0, ICP27) genes was reduced whereas that of E (UL41, UL29) and L (UL48, LAT) genes was increased; (2) IFN-λ mRNA expression was increased. IL-1β was augmented and IL-10 was reduced in unstimulated cells of AD and PD compared to HC; HSV-1 infection significantly increased IL-10 production in HC alone.ConclusionsData herein show that a proinflammatory condition is present in AD and PD, in whom attempts to obstacle viral replication via an initial, possibly more potent IFN-λ-mediated control of IE viral genes is unsuccessful.

Early Life Stress Restricts Translational Reactivity in CA3 Neurons Associated With Altered Stress Responses in Adulthood.

Early life experiences program brain structure and function and contribute to behavioral endophenotypes in adulthood. Epigenetic control of gene expression by those experiences affect discrete brain regions involved in mood, cognitive function and regulation of hypothalamic-pituitary-adrenal (HPA) axis. In rodents, acute restraint stress increases the expression of the repressive histone H3 lysine 9 tri-methylation (H3K9me3) in hippocampal fields, including the CA3 pyramidal neurons. These CA3 neurons are crucially involved in cognitive function and mood regulation as well as activation of glucocorticoid (CORT) secretion. CA3 neurons also exhibit structural and functional changes after early-life stress (ELS) as well as after chronic stress in adulthood. Using a protocol of chronic ELS induced by limited bedding and nesting material followed by acute-swim stress (AS) in adulthood, we show that mice with a history of ELS display a blunted CORT response to AS, despite exhibiting activation of immediate early genes after stress similar to that found in control mice. We find that ELS induced persistently increased expression of the repressive H3K9me3 histone mark in the CA3 subfield at baseline that was subsequently decreased following AS. In contrast, AS induced a transient increase of this mark in control mice. Using translating ribosome affinity purification (TRAP) method to isolate CA3 translating mRNAs, we found that expression of genes of the epigenetic gene family, GABA/glutamate family, and glucocorticoid receptors binding genes were decreased transiently in control mice by AS and showed a persistent reduction in ELS mice. In most cases, AS in ELS mice did not induce gene expression changes. A stringent filtering of genes affected by AS in control and ELS mice revealed a noteworthy decrease in gene expression change in ELS mice compared to control. Only 18 genes were selectively regulated by AS in ELS mice and encompassed pathways such as circadian rhythm, inflammatory response, opioid receptors, and more genes included in the glucocorticoid receptor binding family. Thus, ELS programs a restricted translational response to stress in stress-sensitive CA3 neurons leading to persistent changes in gene expression, some of which mimic the transient effects of AS in control mice, while leaving in operation the immediate early gene response to AS.