Arc mRNA Levels Research Articles

MicroRNA-33 regulates the synaptic plasticity-related gene ARC in temporal lobe epilepsy

This study aimed to elucidate the expression patterns of miR-33 and ARC in both a rat model of temporal lobe epilepsy (TLE) and human TLE patients, to explore the role of miR-33 in epilepsy onset through its regulation of ARC expression in the hippocampus. Our findings, supported by a Dual-Luciferase reporter assay, suggest that miR-33 can bind to the 3′ UTR region of ARC. We observed that miR-33 levels were reduced at 1 hour and 60 days post-seizure, while ARC expression notably increased at these time points. In the hippocampal CA1 and CA3 regions of post-seizure rats, ARC expression significantly exceeded that of control groups. Following the transfection of HEK cells with a miR-33 mimic, there was a decrease in both ARC mRNA and protein levels, whereas the group treated with a miR-33 inhibitor displayed the opposite effect. RNA sequencing in TLE patients revealed a similar miR-33 and ARC interaction. The regulation of Arc expression by miR-33 suggests that Arc may be a target gene of miR-33 in the context of epilepsy. Our findings indicate that miR-33 downregulation could contribute to the dysregulation of Arc expression observed in TLE, potentially influencing the disease process. Further studies are required to establish the exact role of miR-33-mediated Arc regulation in the development of epilepsy.

Restoring Brain Pathways Involved in Diabetes-Associated Neurocognitive Disorders: The Potential of Dipeptidyl Peptidase 4 Inhibitors as a Therapeutic Strategy

Abstract: Diabetes, a widespread chronic metabolic disease, is projected to affect 783 million people globally by 2045. Recent studies emphasize the neuroprotective potential of dipeptidyl peptidase 4 (DPP4i) inhibitors, pointing toward a promising avenue for intervention in addressing cognitive challenges associated with diabetes. Due to limited data on the effect of DPP4i on brain pathways involved in diabetes-related neurocognitive disorders, the decision was made to conduct this study to fill existing knowledge gaps on this topic. The primary aim of our study was to evaluate the potential of DPP4 inhibitors (DPP4i) in preventing cognitive decline in mice with type 2 diabetes (T2D), placing special emphasis on gaining insight into the complex molecular mechanisms underlying this action. We examined drug efficacy in modulating neurotrophic factors, calcium levels, and the expression of key genes (HIF1α, APP, Arc) crucial for neural plasticity. Conducting cognitive assessments with the hole board and passive avoidance tests, we discerned a remarkable influence of short-term gliptin usage on the limiting progress of cognitive dysfunction in diabetic mice. The administration of DPP4 inhibitors led to heightened neurotrophin levels, increased HIF1α in the prefrontal cortex, and a significant elevation in Arc mRNA levels. Our findings reveal that DPP4 inhibitors effectively limit the progression of diabetes-related cognitive disorders. This breakthrough discovery not only opens new research avenues but also constitutes a potential starting point for creating innovative strategies for the treatment of central nervous system disorders focused on improving cognitive abilities.

The development of compulsive coping behaviour is associated with a downregulation of Arc in a Locus Coeruleus neuronal ensemble

Some compulsive disorders have been considered to stem from the loss of control over coping strategies, such as displacement. However, the cellular mechanisms involved in the acquisition of coping behaviours and their subsequent compulsive manifestation in vulnerable individuals have not been elucidated. Considering the role of the locus coeruleus (LC) noradrenaline-dependent system in stress and related excessive behaviours, we hypothesised that neuroplastic changes in the LC may be associated with the acquisition of an adjunctive polydipsic water drinking, a prototypical displacement behaviour, and the ensuing development of compulsion in vulnerable individuals. Thus, male Sprague Dawley rats were characterised for their tendency, or not, to develop compulsive polydipsic drinking in a schedule-induced polydipsia (SIP) procedure before their fresh brains were harvested. A new quantification tool for RNAscope assays revealed that the development of compulsive adjunctive behaviour was associated with a low mRNA copy number of the plasticity marker Arc in the LC which appeared to be driven by specific adaptations in an ensemble of tyrosine hydroxylase (TH)+, zif268− neurons. This ensemble was specifically engaged by the expression of compulsive adjunctive behaviour, not by stress, because its functional recruitment was not observed in individuals that no longer had access to the water bottle before sacrifice, while it consistently correlated with the levels of polydipsic water drinking only when it had become compulsive. Together these findings suggest that downregulation of Arc mRNA levels in a population of a TH+/zif268− LC neurons represents a signature of the tendency to develop compulsive coping behaviours.

Reactivation of cocaine contextual memory engages mechanistic target of rapamycin/S6 kinase 1 signaling.

Mechanistic target of rapamycin (mTOR) C1 and its downstream effectors have been implicated in synaptic plasticity and memory. Our prior work demonstrated that reactivation of cocaine memory engages a signaling pathway consisting of Akt, glycogen synthase kinase-3β (GSK3β), and mTORC1. The present study sought to identify other components of mTORC1 signaling involved in the reconsolidation of cocaine contextual memory, including eukaryotic translation initiation factor 4E (eIF4E)-eIF4G interactions, p70 S6 kinase polypeptide 1 (p70S6K, S6K1) activity, and activity-regulated cytoskeleton (Arc) expression. Cocaine contextual memory was established in adult CD-1 mice using conditioned place preference. After cocaine place preference was established, mice were briefly re-exposed to the cocaine-paired context to reactivate the cocaine memory and brains examined. Western blot analysis showed that phosphorylation of the mTORC1 target, p70S6K, in nucleus accumbens and hippocampus was enhanced 60 min following reactivation of cocaine memories. Inhibition of mTORC1 with systemic administration of rapamycin or inhibition of p70S6K with systemic PF-4708671 after reactivation of cocaine contextual memory abolished the established cocaine place preference. Immunoprecipitation assays showed that reactivation of cocaine memory did not affect eIF4E–eIF4G interactions in nucleus accumbens or hippocampus. Levels of Arc mRNA were significantly elevated 60 and 120 min after cocaine memory reactivation and returned to baseline 24 h later. These findings demonstrate that mTORC1 and p70S6K are required for reconsolidation of cocaine contextual memory.

Long-Term Environmental Enrichment Relieves Dysfunctional Cognition and Synaptic Protein Levels Induced by Prenatal Inflammation in Older CD-1 Mice.

A mounting body of evidence suggests that prenatal inflammation may enhance the rate of age-associated cognitive decline and may involve aberrant amounts of synaptic proteins in the hippocampus, including synaptotagmin-1 (Syt1) and activity-regulated cytoskeleton-associated protein (Arc). However, little is known about the specific impact of adolescent environmental enrichment (EE) on age-associated cognitive decline and the changes in synaptic proteins caused by prenatal inflammation. In this study, CD-1 mice in late pregnancy were given intraperitoneal doses of lipopolysaccharide (LPS, 50 μg/kg) or normal saline. Offspring arising from LPS dams were divided into a LPS group and a LPS plus EE (LPS-E) group. The LPS-E mice were exposed to EE from 2 months of age until the end of the experiment (3 or 15 months old). The Morris water maze (MWM) was used to assess the spatial learning and memory capacities of experimental mice, while western blotting and RNA-scope were used to determine the expression levels of Arc and Syt1 in the hippocampus at the protein and mRNA levels, respectively. Analysis revealed that at 15 months of age, the control mice experienced a reduction in cognitive ability and elevated expression levels of Arc and Syt1 genes when compared to control mice at 3 months of age. The LPS-E group exhibited better cognition and lower protein and mRNA levels of Arc and Syt1 than mice in the LPS group of the same age. However, the enriched environment mitigated but did not counteract, the effects of prenatal inflammation on cognitive and synaptic proteins when tested at either 3 or 15 months of age. Our findings revealed that long-term environmental enrichment improved the expression levels of synaptic proteins in CD-1 mice and that this effect was linked to the dysfunctional cognition caused by prenatal inflammation; this process may also be involved in the reduction of hippocampal Arc and Syt1 gene expression.

Molecular Mechanisms Underlying the Retrieval and Extinction of Morphine Withdrawal-Associated Memories in the Basolateral Amygdala and Dentate Gyrus.

Despite their indisputable efficacy for pain management, opiate prescriptions remain highly controversial partially due to their elevated addictive potential. Relapse in drug use is one of the principal problems for addiction treatment, with drug-associated memories being among its main triggers. Consequently, the extinction of these memories has been proposed as a useful therapeutic tool. Hence, by using the conditioned place aversion (CPA) paradigm in rats, we investigated some of the molecular mechanisms that occurr during the retrieval and extinction of morphine withdrawal memories in the basolateral amygdala (BLA) and the hippocampal dentate gyrus (DG), which control emotional and episodic memories, respectively. The retrieval of aversive memories associated with the abstinence syndrome paralleled with decreased mTOR activity and increased Arc and GluN1 expressions in the DG. Additionally, Arc mRNA levels in this nucleus very strongly correlated with the CPA score exhibited by the opiate-treated rats. On the other hand, despite the unaltered mTOR phosphorylation, Arc levels augmented in the BLA. After the extinction test, Arc and GluN1 expressions were raised in both the DG and BLA of the control and morphine-treated animals. Remarkably, Homer1 expression in both areas correlated almost perfectly with the extinction showed by morphine-dependent animals. Moreover, Arc expression in the DG correlated strongly with the extinction of the CPA manifested by the group treated with the opiate. Finally, our results support the coordinated activity of some of these neuroplastic proteins for the extinction of morphine withdrawal memories in a regional-dependent manner. Present data provide evidence of differential expression and activity of synaptic molecules during the retrieval and extinction of aversive memories of opiate withdrawal in the amygdalar and hippocampal regions that will likely permit the development of therapeutic strategies able to minimize relapses induced by morphine withdrawal-associated aversive memories.

Social isolation in rats: Effects on animal welfare and molecular markers for neuroplasticity.

Early life stress compromises brain development and can contribute to the development of mental illnesses. A common animal model used to study different facets of psychiatric disorders is social isolation from early life on. In rats, this isolation can induce long-lasting alterations in molecular expression and in behavior. Since social isolation models severe psychiatric symptoms, it is to be expected that it affects the overall wellbeing of the animals. As also promoted by the 3Rs principle, though, it is pivotal to decrease the burden of laboratory animals by limiting the number of subjects (reduce, replace) and by improving the animals’ wellbeing (refine). The aim of this study was therefore to test possible refinement strategies such as resocialization and mere adult social isolation. We examined whether the alternatives still triggered the necessary phenotype while minimizing the stress load on the animals. Interestingly, we did not find reduced wellbeing-associated burrowing performance in isolated rats. The hyperactive phenotype seen in socially isolated animals was observed for rats undergoing the adult-only isolation, but resocializing ameliorated the locomotor abnormality. Isolation strongly affected markers of neuroplasticity in the prefrontal cortex independent of timing: mRNA levels of Arc, Bdnf and the pool of Bdnf transcripts with the 3’ long UTR were reduced in all groups. Bdnf splice variant IV expression was reduced in lifelong-isolated animals. Some of these deficits normalized after resocialization; likewise, exon VI Bdnf mRNA levels were reduced only in animals persistently isolated. Conversely, social deprivation did not affect the expression of Gad67 and Pvb, two GABAergic markers, whereas changes occurred in the expression of dopamine d1 and d2 receptors. As adult isolation was sufficient to trigger the hyperactive phenotype and impaired neuroplasticity in the prefrontal cortex, it could be a candidate for a refinement strategy for certain research questions. To fully grade the severity of post-weaning social isolation and the alternatives, adult isolation and resocialization, a more profound and multimodal assessment approach is necessary.

Cotinine and 6-Hydroxy-L-Nicotine Reverses Memory Deficits and Reduces Oxidative Stress in Aβ25-35-Induced Rat Model of Alzheimer's Disease.

The nicotinic derivatives, cotinine (COT), and 6-hydroxy-L-nicotine (6HLN), showed promising cognitive-improving effects without exhibiting the nicotine’s side-effects. Here, we investigated the impact of COT and 6HLN on memory impairment and the oxidative stress in the Aβ25-35-induced rat model of Alzheimer’s disease (AD). COT and 6HLN were chronically administered to Aβ25-35-treated rats, and their memory performances were assessed using in vivo tasks (Y-maze, novel object recognition, and radial arm maze). By using in silico tools, we attempted to associate the behavioral outcomes with the calculated binding potential of these nicotinic compounds in the allosteric sites of α7 and α4β2 subtypes of the nicotinic acetylcholine receptors (nAChRs). The oxidative status and acetylcholinesterase (AChE) activity were determined from the hippocampal tissues. RT-qPCR assessed bdnf, arc, and il-1β mRNA levels. Our data revealed that COT and 6HLN could bind to α7 and α4β2 nAChRs with similar or even higher affinity than nicotine. Consequently, the treatment exhibited a pro-cognitive, antioxidant, and anti-AChE profile in the Aβ25-35-induced rat model of AD. Finally, RT-qPCR analysis revealed that COT and 6HLN positively modulated the bdnf, arc, and il-1β genes expression. Therefore, these nicotinic derivatives that act on the cholinergic system might represent a promising choice to ameliorate AD conditions.

Apoptosis repressor with caspase recruitment domain promotes cell proliferation and phenotypic modulation through 14–3-3ε/YAP signaling in vascular smooth muscle cells

AimsIn response to vascular injury, vascular smooth muscle cells (VSMC) may change from a contractile phenotype to a proliferative phenotype and consequently become conducive to neointima formation. Apoptosis repressor with caspase recruitment domain (ARC) was initially discovered as an endogenous apoptosis inhibitor, but whether ARC plays a role in VSMCs and whether it can participate in the regulation of atherosclerosis are unknown. Methods and resultsProtein and mRNA levels of ARC in tissues and cells were detected by western blot and quantitative real-time PCR. Immunofluorescence staining was used to detect the protein location, and immunohistochemistry was used to detect protein expression in tissues. VSMC proliferation was analysed using Cell Counting Kit-8 (CCK-8) and EdU assays, while migration was assessed by Transwell assay. Mechanistically, the direct binding between two proteins was verified by immunoprecipitation. We found that ARC expression was stimulated in VSMCs during cell proliferation. Our results also showed that ARC promoted cell proliferation and induced phenotypic modulation of VSMCs in vitro and vivo. Mechanistic studies demonstrated that ARC increased the nuclear localization of Yes associated protein (YAP) by binding to 14-3-3ε and that ARC played a role in promoting cell proliferation and phenotypic modulation. Additionally, the transcription factor p53 negatively regulated ARC expression at the transcriptional level during cell proliferation and phenotypic modulation. ConclusionsOur findings define a novel role for ARC in the phenotypic transition of proliferating VSMCs, which may provide a new strategy for regulating neointimal formation.

Effects of Prenatal Exposure to Inflammation Coupled With Stress Exposure During Adolescence on Cognition and Synaptic Protein Levels in Aged CD-1 Mice.

Age-associated impairment of spatial learning and memory (AISLM) presents substantial challenges to our health and society. Increasing evidence has indicated that embryonic exposure to inflammation accelerates the AISLM, and this can be attributable, at least partly, to changed synaptic plasticity associated with the activities of various proteins. However, it is still uncertain whether social psychological factors affect this AISLM and/or the expression of synaptic protein-associated genes. Synaptotagmin-1 (Syt1) and activity-regulated cytoskeleton-associated protein (Arc) are two synaptic proteins closely related to cognitive functions. In this study, pregnant CD-1 mice received daily intraperitoneal injections of lipopolysaccharide (LPS) (50 μg/kg) or normal saline at days 15–17 of gestation, and half of the offspring of each group were then subjected to stress for 28 days in adolescence. The Morris water maze (MWM) test was used to separately evaluate spatial learning and memory at 3 and 15 months of age, while western blotting and RNAscope assays were used to measure the protein and mRNA levels of Arc and Syt1 in the hippocampus. The results showed that, at 15 months of age, control mice had worse cognitive ability and higher protein and mRNA levels of Arc and Syt1 than their younger counterparts. Embryonic exposure to inflammation or exposure to stress in adolescence aggravated the AISLM, as well as the age-related increase in Arc and Syt1 expression. Moreover, the hippocampal protein and mRNA levels of Arc and Syt1 were significantly correlated with the performance in the learning and memory periods of the MWM test, especially in the mice that had suffered adverse insults in early life. Our findings indicated that prenatal exposure to inflammation or stress exposure in adolescence exacerbated the AISLM and age-related upregulation of Arc and Syt1 expression, and these effects were linked to cognitive impairments in CD-1 mice exposed to adverse factors in early life.

The temporal effect of hippocampal Arc in the working memory paradigm during novelty exploration

Arc (activity-regulated cytoskeleton-associated protein) is one of the neuronal Immediate Early Genes (IEG), which is involved in the consolidation of memory and is an essential factor in the induction of Long-term Potentiation (LTP), Long-term Depression (LTD) and homeostatic synaptic plasticity. It has also been implicated in the increased familiarization of novel environments during reference memory paradigms. However, the Arc associated temporal effects in a working memory paradigm during novelty exploration are not well studied. Therefore, in the present study, we used spontaneous alternation behavior (SAB) test along with the expression analysis of Arc to study its temporal effects on the working memory paradigms. Using a modified SAB test, we found that the increase in the duration of exposure to a novel environment in the short time-scale (<min) increases the alternations showing that short-term habituation increases the alternation rate. Additionally, during repeated exposure to a novel environment, the alternation rates decrease after shorter inter-session interval. Parallelly, we observed the upregulation of Arc mRNA and protein level 30 min after the SAB test in the cortex and hippocampus of mice, which returns to near-basal level after two hours. The novel experience, associated with the enhanced expression of Arc, helps in the decrease of alternations in subsequent sessions. This change in alternations was absent if the environment was familiar. Further, the role of Arc during these SAB test was confirmed by the inhibition of hippocampal Arc protein through the stereotaxic infusion of Arc antisense oligodeoxynucleotides. We observed that the Arc is involved in the temporal decrease of spontaneous alternations during a series of exposures to a novel environment. Finally, the significance of these results has been discussed in the light of Wagner’s Sometimes Opponent Processes model, where we suggest that Arc reduces the ability for short-term habituation during repeated exposures in the working memory paradigm, and the loss of this ability is more prominent when subjected to a novel environment.

Secreted Amyloid Precursor Protein-Alpha Promotes Arc Protein Synthesis in Hippocampal Neurons

Secreted amyloid precursor protein-α (sAPPα) is a neuroprotective and memory-enhancing molecule, however, the mechanisms through which sAPPα promotes these effects are not well understood. Recently, we have shown that sAPPα enhances cell-surface expression of glutamate receptors. Activity-related cytoskeletal-associated protein Arc (Arg3.1) is an immediate early gene capable of modulating long-term potentiation, long-term depression and homeostatic plasticity through regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor localization. Accordingly, we hypothesized that sAPPα may enhance synaptic plasticity, in part, by the de novo synthesis of Arc. Using primary cortical and hippocampal neuronal cultures we found that sAPPα (1 nM, 2 h) enhances levels of Arc mRNA and protein. Arc protein levels were increased in both the neuronal somata and dendrites in a Ca2+/calmodulin-dependent protein kinase II-dependent manner. Additionally, dendritic Arc expression was dependent upon activation of mitogen-activated protein kinase and protein kinase G. The enhancement of dendritic Arc protein was significantly reduced by antagonism of N-methyl-D-aspartate (NMDA) and nicotinic acetylcholine (α7nACh) receptors, and fully eliminated by dual application of these antagonists. This effect was further corroborated in area CA1 of acute hippocampal slices. These data suggest sAPPα-regulated plasticity within hippocampal neurons is mediated by cooperation of NMDA and α7nACh receptors to engage a cascade of signal transduction molecules to enhance the transcription and translation of Arc.

The protein regulator ArgR and the sRNA derived from the 3'-UTR region of its gene, ArgX, both regulate the arginine deiminase pathway in Lactococcus lactis.

Small regulatory RNAs (sRNAs) and their enormous potential and versatility have provided us with an astounding insight in the complexity of bacterial transcriptomes. sRNAs have been shown to be involved in a variety of cellular processes that range from stress to general metabolism. Here we report that the gene encoding the transcriptional regulator ArgR is immediately followed by the gene of the small regulatory RNA ArgX. The latter is transcribed from its own promoter. The production of ArgX is induced by increasing arginine concentrations and repressed by CcpA. Previously, ArgR was shown to act as a transcriptional repressor of the catabolic arginine deiminase pathway (arc operon) by binding in the promoter region of arcA. Here we demonstrate that ArgX downregulates arc mRNA levels. Furthermore, ArgX putatively blocks the translation of one of the genes in the operon, arcC1, a process that would redirect an intermediate in arginine degradation, carbamoyl phosphate, towards pyrimidine synthesis. Our findings exemplify, for the first time, the combinatorial power of a transcription factor and a small regulatory RNA derived from the 3’-UTR region. The regulators ArgR and ArgX share a common target, but act on transcription and on RNA level, respectively.

Influence of acupuncture plus moxibustion on changes of synaptic structure and expression of synaptic skeleton related genes and proteins in prefrontal cortex of heroin re-addicted rats

To observe the effect of acupuncture plus moxibustion on the synaptic ultrastructure and expression of synaptic skeleton related proteins in the prefrontal cortex (PFC) of heroin re-addicted rats, so as to reveal its mechanisms underlying improvement of heroin addiction. Twenty-four Wister rats (half male and half female) were randomly divi-ded into normal control, model and acupuncture groups (n＝8 in each group). The heroin re-addicted model was established by muscular injection of heroin into the hind limbs for 8 days (incremental 0.8-3.6 mg, once daily for 6 days, and twice daily for 2 days), followed by conventional breeding for 5 days (detoxification), the procedure (addition－detoxification) was repeated 3 cycles. For rats of the acupuncture group, "Baihui" (GV20) was needled with filiform needles which were retained for 30 min, and moxibustion was then applied to bilateral "Shenshu" (BL23) for 30 min. The treatment was conducted once daily during the deto-xification. On the 39th day of experiment, the bilateral prefrontal cortex tissues were sampled for examining the ultrastructure by using transmission electron microscope (TEM) after fixative solution immersion and for determining the expression of genes and proteins of activity-regulated cytoskeleton-associated protein (Arc), microtubule-asso-ciated protein-2 (MAP-2) and microtubule-associated protein Tau (Tau) with quantitative real-time PCR and Western blot, respectively. After modeling, the expression levels of Arc mRNA and protein were significantly up-regulated, and those of MAP-2 and Tau mRNA and proteins ob-viously down-regulated in the model group relevant to the normal control group (P<0.05). Following the intervention, the up-regulated Arc protein and mRNA and the down-regulated MAP-2 and Tau were obviously reversed relevant to the model group (P<0.05). Outcomes of TEM showed unclear pre- and post-membranes of the synapses, narrowing of the synaptic gap and non-uniform of the density of the thickened dense plaque after modeling, which was relatively milder in the acupuncture group. Acupuncture plus moxibustion can improve changes of synaptic ultrastructure in heroin re-addicted rats, which may be related to their effect in regulating the expression of some synaptic skeleton proteins and genes.

Nicotine and caffeine modulate haloperidol-induced changes in postsynaptic density transcripts expression: Translational insights in psychosis therapy and treatment resistance

Caffeine and nicotine are widely used by schizophrenia patients and may worsen psychosis and affect antipsychotic therapies. However, they have also been accounted as augmentation strategies in treatment-resistant schizophrenia. Despite both substances are known to modulate dopamine and glutamate transmission, little is known about the molecular changes induced by these compounds in association to antipsychotics, mostly at the level of the postsynaptic density (PSD), a site of dopamine-glutamate interplay. Here we investigated whether caffeine and nicotine, alone or combined with haloperidol, elicited significant changes in the levels of both transcripts and proteins of the PSD members Homer1 and Arc, which have been implicated in synaptic plasticity, schizophrenia pathophysiology, and antipsychotics molecular action. Homer1a mRNA expression was significantly reduced by caffeine and nicotine, alone or combined with haloperidol, compared to haloperidol. Haloperidol induced significantly higher Arc mRNA levels than both caffeine and caffeine plus haloperidol in the striatum. Arc mRNA expression was significantly higher by nicotine plus haloperidol vs. haloperidol in the cortex, while in striatum gene expression by nicotine was significantly lower than that by both haloperidol and nicotine plus haloperidol. Both Homer1a and Arc protein levels were significantly increased by caffeine, nicotine, and nicotine plus haloperidol. Homer1b mRNA expression was significantly increased by nicotine and nicotine plus haloperidol, while protein levels were unaffected. Locomotor activity was not significantly affected by caffeine, while it was reduced by nicotine. These data indicate that both caffeine and nicotine trigger relevant molecular changes in PSD sites when given in association with haloperidol.

Delayed Degradation and Impaired Dendritic Delivery of Intron-Lacking EGFP-Arc/Arg3.1 mRNA in EGFP-Arc Transgenic Mice.

Arc is a unique immediate early gene (IEG) whose expression is induced as synapses are modified during learning. Newly-synthesized Arc mRNA is rapidly transported throughout dendrites and localizes near recently activated synapses. Arc mRNA levels are regulated by rapid degradation, which is accelerated by synaptic activity in a translation-dependent process. One possible mechanism is nonsense-mediated mRNA decay (NMD), which depends on the presence of a splice junction in the 3′UTR. Here, we test this hypothesis using transgenic mice that express EGFP-Arc. Because the transgene was constructed from Arc cDNA, it lacks intron structures in the 3′UTR that are present in the endogenous Arc gene. NMD depends on the presence of proteins of the exon junction complex (EJC) downstream of a stop codon, so EGFP-Arc mRNA should not undergo NMD. Assessment of Arc mRNA rundown in the presence of the transcription inhibitor actinomycin-D confirmed delayed degradation of EGFP-Arc mRNA. EGFP-Arc mRNA and protein are expressed at much higher levels in transgenic mice under basal and activated conditions but EGFP-Arc mRNA does not enter dendrites efficiently. In a physiological assay in which cycloheximide (CHX) was infused after induction of Arc by seizures, there were increases in endogenous Arc mRNA levels consistent with translation-dependent Arc mRNA decay but this was not seen with EGFP-Arc mRNA. Taken together, our results indicate: (1) Arc mRNA degradation occurs via a mechanism with characteristics of NMD; (2) rapid dendritic delivery of newly synthesized Arc mRNA after induction may depend in part on prior splicing of the 3′UTR.

Stress-induced hippocampus Npas4 mRNA expression relates to specific psychophysiological patterns of stress response

Neuronal Per-Arnt-Sim (PAS) domain protein 4 (Npas4) is a key protein that intervenes in GABA synapse scaling and neurotrophicity enhancing. Since GABA and neurotrophicity are implicated in stress response and Npas4-deficient rodents exhibit behavioral alterations, an investigation was designed in rats to verify whether stress-induced spontaneous hippocampus Npas4 mRNA expression would be associated with specific patterns of stress response. The rats were exposed to one of three stressor levels: no stress (CTL, n = 15), exposure to a footshock apparatus (Sham, S, n = 40) and footshock (F, n = 80). After stress exposure the S and F rats were tested in an activity cage, and subsequently in an elevated plus maze (EPM), just prior to the sacrifice. Using cluster analysis, the animals already assigned to a stress level were also distributed into 2 subgroups depending on their Npas4 mRNA levels. The low (L) and high (H) Npas4 expression subgroups were identified in the S and F groups, the CTL group being independent of the Npas4 levels. The Npas4 effect was studied through the interaction between stress (S and F) and Npas4 level (L and H). The biological stress response was similar in H and L rats, except blood corticosterone that was slightly lower in the H rats. The H rats were more active in the actimetry cage and presented higher levels of exploration in the EPM. They also exhibited higher hippocampus activation, as assessed by the c-fos, Egr1 and Arc mRNA levels. Therefore high Npas4 expression favors stress management.

Gene expression analysis of Arc mRNA as a neuronal cell activity marker in the hippocampus and amygdala in two-way active avoidance test in rats

IntroductionImmediate early genes are widely used as neuronal cell activity markers in neuroscience. The present study investigated the relationship between their expression and abnormality in context fear conditioning. MethodsThe learning test (two-way active avoidance test) was conducted in male rats administered with nonselective muscarinic antagonist scopolamine or selective dopamine D1-like receptor antagonist SCH 23390 at a dose level of 2.0 or 0.1mg/kg, respectively, for 4days. Expression levels of Arc and Fos mRNA in the hippocampus and amygdala were also evaluated on the second day of dosing by fluorescent in situ hybridization (FISH) and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). ResultsScopolamine had no effect on avoidance rate, but decreased freezing in the two-way active avoidance test. SCH 23390 decreased avoidance rate and increased freezing. In FISH and RT-qPCR assays, scopolamine decreased Arc mRNA in the hippocampus and amygdala, whereas SCH 23390 increased Arc mRNA in the hippocampus. By contrast, scopolamine and SCH 23390 did not change Fos mRNA expression compared to Arc mRNA expression. DiscussionThe results of the learning test indicated that scopolamine or SCH 23390 respectively inhibited fear or context conditioning in rats. Furthermore, alteration of the expression of Arc mRNA but not of Fos mRNA in the hippocampus and amygdala of the brain was suggested to be a sensitive neuronal cell activity marker to detect behavioral abnormality in the two-way active avoidance test.

Prenatal exposure to valproic acid increases miR-132 levels in the mouse embryonic brain

BackgroundMicroRNAs, small non-coding RNAs, are highly expressed in the mammalian brain, and the dysregulation of microRNA levels may be involved in neurodevelopmental disorders such as autism spectrum disorder (ASD). In the present study, we examined whether prenatal valproic acid (VPA) exposure affects levels of microRNAs, especially the brain specific and enriched microRNAs, in the mouse embryonic brain.ResultsPrenatal exposure to VPA at E12.5 immediately increased miR-132 levels, but not miR-9 or miR-124 levels, in the male embryonic brain. Prenatal exposure to VPA at E12.5 also increased miR-132 levels in the female embryonic brain. We further found that the prenatal exposure to VPA at E12.5 increased mRNA levels of Arc, c-Fos and brain-derived neurotrophic factor in both male and female embryonic brains, prior to miR-132 expression. In contrast, prenatal exposure to VPA at E14.5 did not affect miR-132 levels in either male or female embryonic brain. The prenatal VPA exposure at E12.5 also decreased mRNA levels of methyl-CpG-binding protein 2 and Rho GTPase-activating protein p250GAP, both of which are molecular targets of miR-132. Furthermore, RNA sequence analysis revealed that prenatal VPA exposure caused changes in several microRNA levels other than miR-132 in the embryonic whole brain.ConclusionsThese findings suggest that the alterations in neuronal activity-dependent microRNAs levels, including an increased level of miR-132, in the embryonic period, at least in part, underlie the ASD-like behaviors and cortical pathology produced by prenatal VPA exposure.

One nuclear calcium transient induced by a single burst of action potentials represents the minimum signal strength in activity-dependent transcription in hippocampal neurons

Neurons undergo dramatic changes in their gene expression profiles in response to synaptic stimulation. The coupling of neuronal excitation to gene transcription is well studied and is mediated by signaling pathways activated by cytoplasmic and nuclear calcium transients. Despite this, the minimum synaptic activity required to induce gene expression remains unknown. To address this, we used cultured hippocampal neurons and cellular compartment analysis of temporal activity by fluorescence in situ hybridization (catFISH) that allows detection of nascent transcripts in the cell nucleus. We found that a single burst of action potentials, consisting of 24.4±5.1 action potentials during a 6.7±1.9s depolarization of 19.5±2.0mV causing a 9.3±0.9s somatic calcium transient, is sufficient to activate transcription of the immediate early gene arc (also known as Arg3.1). The total arc mRNA yield produced after a single burst-induced nuclear calcium transient was very small and, compared to unstimulated control neurons, did not lead to a significant increase in arc mRNA levels measured using quantitative reverse transcriptase PCR (qRT-PCR) of cell lysates. Significantly increased arc mRNA levels became detectable in hippocampal neurons that had undergone 5–8 consecutive burst-induced nuclear calcium transients at 0.05–0.15Hz. These results indicate that a single burst-induced nuclear calcium transient can activate gene expression and that transcription is rapidly shut off after synaptic stimulation has ceased.