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Serum response factor (SRF) is a representative transcription factor that plays crucial roles in various biological phenomena by regulating immediate early genes (IEGs) and genes related to cell morphology and motility, among others. Over the years, the signal transduction pathways activating SRF have been clarified and SRF-target genes have been identified. In this overview, we initially briefly summarize the basic biology of SRF and its cofactors, ternary complex factor (TCF) and megakaryoblastic leukemia (MKL)/myocardin-related transcription factor (MRTF). Progress in the generation of nervous system-specific knockout (KO) or genetically modified mice as well as genetic analyses over the last few decades has not only identified novel SRF-target genes but also highlighted the neurochemical importance of SRF and its cofactors. Therefore, here we next present the phenotypes of mice with nervous system-specific KO of SRF or its cofactors by depicting recent findings associated with brain development, plasticity, epilepsy, stress response, and drug addiction, all of which result from function or dysfunction of the SRF axis. Last, we develop a hypothesis regarding the possible involvement of SRF and its cofactors in human neurological disorders including neurodegenerative, psychiatric, and neurodevelopmental diseases. This overview should deepen our understanding, highlight promising future directions for developing novel therapeutic strategies, and lead to illumination of the mechanisms underlying higher brain functions based on neuronal structure and function. [ABSTRACT FROM AUTHOR]

Enokipodins are antimicrobial sesquiterpenes produced by Flammulina velutipes in a mycelial culture medium. To date, enokipodin production has not been reported in other members of the genus Flammulina. Hence, in this study, the production of enokipodins A, B, C, and D by F. velutipes and F. rossica was investigated. Some strains of F. rossica were confirmed to produce at least one of the four enokipodins in the culture medium. However, some strains of F. velutipes did not produce any of the enokipodins. In an antibacterial assay using liquid medium, enokipodin B showed the strongest growth inhibitory activity against Bacillus subtilis among the four types of enokipodins. Enokipodin B inhibited the spore germination of some plant pathogenic fungi. Enokipodins B and D exerted moderate anti-proliferative activity against some cancer cell lines, and enokipodins A and C inhibited the proliferation of the malarial parasite, Plasmodium falciparum. Productivity of enokipodins of some strains of F. velutipes and F. rossica and antibacterial activity of culture filtrate extract against B. subtilis. [ABSTRACT FROM AUTHOR]

Suppressor of cancer cell invasion (SCAI) acts as a transcriptional repressor of serum response factor (SRF)‐mediated gene expression by binding to megakaryoblastic leukemia (MKL)/myocardin‐related transcription factor (MRTF), which is an SRF transcriptional coactivator. Growing evidence suggests that SCAI is a negative regulator of neuronal morphology, whereas MKL2/MRTFB is a positive regulator. The mRNA expression of SCAI is downregulated during brain development, suggesting that a reduction in SCAI contributes to the reduced suppression of SRF‐mediated gene induction, thus increasing dendritic complexity and developing neuronal circuits. In the present study, we hypothesized that brain‐derived neurotrophic factor (BDNF), which is important for neuronal plasticity and development, might alter SCAI mRNA levels. We therefore investigated the effects of BDNF on SCAI mRNA levels in primary cultured cortical neurons. Furthermore, because alternative splicing generates several SCAI variants in the brain, we measured SCAI variant mRNA after BDNF stimulation. Both SCAI variant 1 and total SCAI mRNA expression levels were downregulated by BDNF. Moreover, the extracellular signal‐regulated protein kinase/mitogen‐activated protein kinase (ERK/MAPK) pathway was involved in the BDNF‐mediated decrease in SCAI mRNA expression. Our findings provide insights into the molecular mechanism underlying a neurotrophic factor switch for the repressive transcriptional complex that includes SCAI. [ABSTRACT FROM AUTHOR]

We investigated the population genetics and fine-scale genetic structure of Rhizopogon roseolus . A total of 173 R. roseolus sporocarps were collected from two stands in the Tottori sand dune. We developed and applied five novel polymorphic microsatellite (SSR; simple sequence repeat) markers for sporocarp genotyping. In total, we identified 110 genets, most of which were small in size. Spatial autocorrelation analyses revealed a significantly positive genetic structure in short-distance classes. The inbreeding coefficient value was significant in both stands ( F IS = 0.18), while the F ST value ( F ST = 0.020) indicated little genetic differentiation between the two populations. The majority of alleles were distributed in both stands with similar frequencies. These results suggest that short-distance spore dispersal plays a dominant role in generating new genets, and eventually increases the frequency of inbreeding in the Tottori sand dune, whereas rare gene flow between the two stands, possibly associated with spore dispersal by mycophagous animals, could reduce genetic differentiation. [ABSTRACT FROM AUTHOR]

Neurotrophin-3 (NT-3), a neurotrophin member, plays crucial roles in neuronal development, function and plasticity. Previous studies have demonstrated that NT-3 gene transcription is driven by alternative promoters A and B, located upstream of exons 1A (EIA) and 1B (EIB), respectively. However, the transcription factors and DNA elements that drive NT-3 gene transcription remain to be identified. Here, we analysed the promoter region of the NT-3 gene and found that an NT-3 transcript containing EIB is predominantly expressed in cortical neurons which preferentially utilize promoter B, and two tandemly repeated GC-boxes, located between −100 and −60 base pairs within promoter B, are required for the transcription. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that both specificity protein (Sp)3 and Sp4 were able to bind to the Sp1 binding sequences within the GC boxes. Expression of dominant-negative Sp3 and Sp4 small interfering RNA in cortical neurons reduced the activity of the NT-3 gene promoter. Over-expression of Sp1 family members, especially Sp4, resulted in an increase of the NT-3 gene promoter. These findings indicate that the NT-3 gene is a target gene for Sp4 that is abundantly expressed in the brain. [ABSTRACT FROM AUTHOR]

Abstract: Expanded polyglutamines (polyQ) have been demonstrated to impair the CREB-dependent transcription in established cell lines. Since activity-dependent transcription in neurons, which plays an important role in forming neuronal plasticity, is largely controlled by CREB, it is important to study whether polyQ interferes with the activity-dependent transcriptional activation of genes in neurons. In cultured rat cortical neurons, over-expression of truncated dentatorubral-pallidoluysian atrophy proteins containing expanded polyQ, which form aggregation bodies in nucleus, reduced the calcium (Ca2+) signal-mediated transcriptional activation of brain-derived neurotrophic factor, c-fos, and pituitary adenylate cyclase-activating polypeptide gene promoters in a dose-dependent manner. The interference with the transcriptional activation was dependent upon the presence of polyQ, the strength of which was increased as the length of polyQ stretches was expanded. Thus, polyQ interferes with the activity-dependent transcription in a polyQ-length-dependent manner, which may correspond to the severity of polyglutamine diseases. [Copyright &y& Elsevier]

Although it is well established that RhoA signaling pathways play key roles in regulating neuronal morphology, their involvement in other aspects of neuronal function has received little attention. Recent studies have elucidated a novel intracellular signaling pathway used by RhoA to elicit activation of serum response factor (SRF)-mediated transcription. In this pathway, activation of RhoA triggers nuclear translocation of the SRF co-activator, megakaryocytic acute leukemia (MAL). In assessing whether RhoA regulates transcription in neurons via this pathway, we have found that a constitutively active form of Tech (transcript-enriched in cortex and hippocampus), a RhoA guanine nucleotide exchange factor (GEF) that is expressed in forebrain neurons, stimulates SRF reporter activity in extracts of primary cortical cultures and induces nuclear translocation of MAL in cortical neurons. Both of these responses appear to be mediated by Tech's activation of RhoA as they are not mimicked by a mutant Tech construct lacking RhoA GEF activity and are blocked by C3 transferase, a selective inhibitor of RhoA. Furthermore, Tech-induced increases in SRF activity are suppressed by a dominant negative MAL construct. These findings demonstrate that RhoA signaling pathways are able to regulate transcription in neurons by triggering translocation of the SRF co-activator MAL. [ABSTRACT FROM AUTHOR]

Although it has been established that an activity-dependent gene transcription is induced by the calcium (Ca2+) signals in neurons, it is unclear how the specific mRNA moieties are transiently accumulated in response to synaptic transmission which evokes multiple intracellular signals including Ca2+ and cAMP ones. The expression of pituitary adenylate cyclase activating polypeptide (PACAP), a neuropeptide, is controlled by Ca2+ signals evoked via membrane depolarization in neurons, and, in cultured rat cortical neuronal cells, we found that the Ca2+ signal-mediated activation of the PACAP gene promoter was critically controlled by a single cAMP-response element (CRE) located at around -200, to which the CRE-binding protein predominantly bound. The Ca2+ signal-induced expression of PACAP mRNA was enhanced by forskolin, which evokes cAMP signals. In support, the PACAP gene promoter was synergistically enhanced by Ca2+ and cAMP signals through the CRE, accompanying a prolonged activation of extracellular signal-related protein kinase ½ and CRE-binding protein. On the other hand, sole administration of forskolin markedly reduced the cellular content of PACAP mRNA, which was restored by the addition of Ca2+ signals. We found that the stability of PACAP mRNA was increased in response to Ca2+ signals but not that of activity-regulated cytoskeleton-associated protein (Arc) mRNA, indicating an activity-dependent stabilization of specific mRNA species in neurons, which can antagonize the regulation mediated by cAMP signals. Thus, the transcriptional activation and mRNA stabilization are coordinately regulated by Ca2+ and cAMP signals for the cumulative expression of PACAP mRNA in neurons. [ABSTRACT FROM AUTHOR]

Neural-restrictive silencer element (NRSE)/repressor element-1 (RE1) regulates neuron-specific gene expression by binding the transcriptional factor REST/NRSF which functions as a silencer in nonneuronal cells. In neuronal cells, a truncated, neuronal-specific REST/NRSF isoform, REST4, has been found but little is known about its function. To address this, we investigated the effect of REST/NRSF and REST4 on the activity-dependent activation of BDNF gene promoter I (BDNFp-I) using cultured rat cortical neurons. REST/NRSF markedly repressed the transcriptional activation of BDNFp-I, whereas the effect of REST4 was weak, depending upon the NRSE/RE1 sequence. In addition, REST4 enhanced the basal transcriptional activity of BDNFp-I. Coexpression of REST4 with REST/NRSF competitively inhibited the silencing effect of REST/NRSF on the activation of BDNFp-I. Although REST4 itself has a weak repressive effect on activation of the BDNF gene via NRSE/RE1, it can compete the silencing effect of REST/NRSF, suggesting a primary role for REST4 in preventing the neuron-specific gene from being inactivated by REST/NRSF and allowing gene activation in response to a variety of neuronal stimuli. [Copyright &y& Elsevier]

Magnetic fields have the potential to impact biological systems and trigger various biological responses. However, the mechanisms underlying dynamic magnetic fields (DMFs), which are created by rotating magnets based on Arago's disk principle and include eddy currents, Lorentz forces, and magnetic fields, are not well understood. This study aims to investigate the effects of DMFs on gene expression in primary cultured cortical cells. To simulate DMFs, rotating magnets were used to confirm magnetic flux density, current density, and Lorentz force. Primary cortical cell cultures were then exposed to moderate‐intensity DMFs with a frequency of 40 Hz for 1 h per day over 3 days. The expression of neuroplasticity‐related immediate early genes, including proto‐oncogene c‐fos, activity‐regulated cytoskeleton‐associated protein (Arc) and brain‐derived neurotrophic factor (BDNF) were quantified. The simulation of DMFs generated a periodic magnetic flux density of up to 131 mT, a periodic current density of up to 1.69 A/m2, and a periodic Lorentz force of up to 0.88 nN in the dish. Exposure to DMFs increased c‐fos mRNA expression in primary cortical cell cultures, but did not affect Arc and BDNF expression. The findings suggest that exposure of DMFs in the gamma frequency range, which may be regulated by voltage‐, mechanical‐, and magnetic flux‐sensitive ion channels, could enhance c‐fos expression in primary cortical cell cultures. The device using rotating magnets has the potential to facilitate the development of a cost‐effective and user‐friendly cell culture system based on the principle of Arago's disk. © 2023 Institute of Electrical Engineer of Japan and Wiley Periodicals LLC. [ABSTRACT FROM AUTHOR]

Short tau inversion recovery images from magnetic resonance imaging (MRI) revealed enthesitis of bilateral adductor magnus (Fig. A sonographic evaluation study demonstrated that patients with BS showed higher enthesitis scores than healthy control subjects, indicating that enthesitis and subclinical enthesopathy may be important articular manifestations in patients with BS [[3]]. A 51-year-old Japanese man had been diagnosed with Behçet's syndrome (BS) 5 years earlier based on recurrent oral and genital ulcers, acne-like lesions, erythema nodosum, and positive results for human leukocyte antigen (HLA) B51, a split antigen of the HLA-B5 broad antigen. [Extracted from the article]

Mice lacking the gene encoding pituitary adenylate cyclase-activating polypeptide ( PACAP) or its specific receptor, PAC1, show abnormal behaviors related to schizophrenia. However, the regulation of PACAP expression in neurons remains unclear. Here, we report that Pacap mRNA levels are regulated transcriptionally and post-transcriptionally by cAMP and Ca2+ signals in cultured rat cortical cells. Pacap mRNA levels decreased proportionately with the intensity of cAMP signaling, and this decrease was accelerated by N-methyl-D-aspartate (NMDA) receptor blockade, suggesting that cAMP signaling enhances the degradation of Pacap mRNA, whereas NMDA receptor-mediated signals inhibit its degradation. However, depolarization (which produced a robust increase in Ca2+ signals) together with cAMP signaling resulted in a synergistic induction of Pacap mRNA through calcineurin and its substrate, cAMP-response element-binding protein ( CREB)-regulated transcription coactivator 1. These results strongly support the concept that while cAMP signaling can accelerate the degradation of Pacap mRNA, it can also synergistically enhance Ca2+ signaling-induced transcriptional activation of Pacap. Taken together, our findings suggest that a balance between Ca2+ and cAMP signals regulates PACAP levels in neurons and that a perturbation of this balance may result in psychiatric disorders, such as schizophrenia. [ABSTRACT FROM AUTHOR]

In cultures of rat cortical neurons, we found that stimulation of tyrosine receptor kinase B (TrkB) with brain-derived neurotrophic factor (BDNF) induced a biphasic expression of BDNF exon IV–IX mRNA, which became obvious 1–3 h (primary induction) and 24–72 h (delayed induction) after the stimulation, and characterized the delayed induction in relation to the mRNA expression of activity-regulated cytoskeleton-associated protein (Arc). Withdrawal of BDNF from the medium after stimulation for 3 h allowed the delayed induction, which was caused at the transcriptional level and dependent upon the initial contact between exogenously added BDNF and TrkB, the effect of which was time- and dose-dependent. The primary induction was controlled by the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) whereas the secondary induction by the calcium (Ca2+) signaling pathway. The enhanced Arc or Zif268 mRNA expression was controlled by activation of the ERK/MAPK pathway, both of which were repressed by blocking the binding of endogenously synthesized BDNF to TrkB. Thus, robust stimulation of TrkB autonomously induces delayed BDNF mRNA expression in an activity-dependent manner in rat cortical neurons, resulting in the stimulation of Arc mRNA expression through endogenously synthesized BDNF, the process being orchestrated by the Ca2+ and ERK/MAPK signaling pathways. [ABSTRACT FROM AUTHOR]

Megakaryoblastic leukemia 2 (MKL2)/myocardin‐related transcription factor‐B (MRTFB), a serum response factor (SRF) coactivator, is an important regulator of gene expression and neuronal morphology. Here, we show that different mouse MRTFB splice isoforms, including a novel fourth MRTFB isoform named spliced neuronal long isoform of SRF transcriptional coactivator (SOLOIST)/MRTFB isoform 4 (MRTFB i4), play distinct roles in this process. SOLOIST/MRTFB i4 has a short exon that encodes 21 amino acid residues ahead of the first RPXXXEL (RPEL) motif in MRTFB isoform 3. Quantitative PCR revealed that SOLOIST/MRTFB i4 and isoform 1 were enriched in the forebrain and neurons, and up‐regulated during brain development. Conversely, isoform 3 was detected in various tissues, including both neurons and astrocytes, and was down‐regulated in the developing brain. Reporter assays supported the SRF‐coactivator function of SOLOIST/MRTFB i4 as well as isoform 1. Acute expression of MRTFB isoform 1, but not isoform 3 or SOLOIST/MRTFB i4, in neuronal cells within 24 hr drastically increased endogenous immediate early gene [c‐fos, egr1, and activity‐regulated cytoskeleton‐associated protein] expression, but not endogenous actinin α1, β‐actin, gelsolin, or srf gene expression measured by qPCR. Over‐expression of SOLOIST/MRTFB i4 reduced the dendritic complexity of cortical neurons, whereas over‐expression of isoform 1 increased this complexity. Co‐expression of isoform 1 and SOLOIST/MRTFB i4 in cortical neurons revealed that isoform 1 competitively counteracted down‐regulation by SOLOIST/MRTFB i4. Our findings indicate that MRTFB isoforms have unique expression patterns and differential effects on gene expression and dendritic complexity, which contribute to shaping neuronal circuits, at least in part. [ABSTRACT FROM AUTHOR]

Suppressor of cancer cell invasion (SCAI) is a suppressor of myocardin-related transcription factor (MRTF)-mediated transcription and cancer cell invasion. However, roles of SCAI in the brain and neuronal cells are not fully resolved. In this study, we initially investigated the distribution of Scai mRNA in the developing rat brain and in neurons. We found that, although Scai mRNA levels decreased during brain development, it was highly expressed in several brain regions and in neurons but not astrocytes. Subsequently, in addition to Scai variant 1, we identified novel rat Scai variants 2 and 3 and characterized their functions in Neuro-2a cells. The novel Scai variants 2 and 3 contain unique exons that possess stop codons and therefore encode shorter proteins compared with the full-length Scai variant 1. SCAI variants 2 and 3 possess a nuclear localization signal, but do not have an MRTF-binding site. Immunostaining of green fluorescent protein (GFP)-tagged SCAI variants revealed a nuclear localization of variant 1, whereas localization of variants 2 and 3 was throughout the cytoplasm and nucleus, suggesting that other nuclear localization signals, which act in Neuro-2a cells, exist in SCAI. All three SCAI variants suppressed the neuron-like morphological change of Neuro-2a cells induced by a Rho effector, constitutively active mDia; however, the suppressive effects of variants 2 and 3 were weaker than that of full-length SCAI variant 1, indicating that the SCAI-mediated change toward a neuronal morphology appeared to be consistent with their nuclear localization. These findings indicate that generation of multiple SCAI splice variants fines-tune neuronal morphology. • Expression of Scai mRNA decreases during brain development. • Expression of Scai mRNA is higher in neurons than in astrocytes. • Newly identified SCAI splice variants are shorter than full-length SCAI. • Localization of short SCAI splice variants is more cytoplasmic than full-length SCAI. • Short SCAI variants inhibit mDia-induced neurite extension less than full-length SCAI. [ABSTRACT FROM AUTHOR]

Phosphatase and actin regulator 3/nuclear scaffold-associated protein phosphatase 1-inhibiting protein (Phactr3/Scapinin) is an actin- and protein phosphatase 1 (PP1)-binding protein known to negatively regulate axon elongation. In this study, we examined the expression pattern of Phactr3/Scapinin in several tissues and investigated the effect of Phactr3/Scapinin on dendritic morphology of cortical neurons. Results showed that Phactr3/Scapinin expression was up-regulated in the developing brain and enriched in neurons and in the postsynaptic density fraction, but not in astrocytes. Overexpression of wild type or mutant Phactr3/Scapinin, which lacked actin-binding activity, resulted in increased dendritic complexity and percentage of spines with a mushroom or stubby shape, as well as a decrease in spine density. However, overexpression of mutant Phactr3/Scapinin that lacked PP1-binding activity did not. Taken together, these findings suggest that Phactr3/Scapinin expression is neuronal and might contribute to synaptic formation via distinct actin- and PP1-binding domains involved in dendritic and axonal morphology, respectively. • Phactr3/Scapinin is highly expressed in neurons, but not in astrocytes. • Phactr3/Scapinin is localized at postsynapses. • Expression of Phactr3/Scapinin increases the percentage of mature dendritic spines. • Expression of Phactr3/Scapinin up-regulates dendritic complexity. • The PP1-binding is necessary for regulation of neuronal morphology. [ABSTRACT FROM AUTHOR]

The expression of immediate early genes (IEGs) is thought to be an essential molecular basis of neuronal plasticity for higher brain function. Many IEGs contain serum response element in their transcriptional regulatory regions and their expression is controlled by serum response factor (SRF). SRF is known to play a role in concert with transcriptional cofactors. However, little is known about how SRF cofactors regulate IEG expression during the process of neuronal plasticity. We hypothesized that one of the SRF‐regulated neuronal IEGs, activity‐regulated cytoskeleton‐associated protein (Arc; also termed Arg3.1), is regulated by an SRF coactivator, megakaryoblastic leukemia (MKL). To test this hypothesis, we initially investigated which binding site of the transcription factor or SRF cofactor contributes to brain‐derived neurotrophic factor (BDNF)‐induced Arc gene transcription in cultured cortical neurons using transfection and reporter assays. We found that BDNF caused robust induction of Arc gene transcription through a cAMP response element, binding site of myocyte enhancer factor 2, and binding site of SRF in an Arc enhancer, the synaptic activity‐responsive element (SARE). Regardless of the requirement for the SRF‐binding site, the binding site of a ternary complex factor, another SRF cofactor, did not affect BDNF‐mediated Arc gene transcription. In contrast, chromatin immunoprecipitation revealed occupation of MKL at the SARE. Furthermore, knockdown of MKL2, but not MKL1, significantly decreased BDNF‐mediated activation of the SARE. Taken together, these findings suggest a novel mechanism by which MKL2 controls the Arc SARE in response to BDNF stimulation. The activity‐regulated cytoskeleton‐associated protein (Arc) gene is a serum response factor (SRF) target, but little is known about its transcriptional regulation by SRF cofactors. Here, we demonstrate that SRF cofactor megakaryoblastic leukemia 2 (MKL2) binds to the Arc gene enhancer, a synaptic activity‐responsive element (SARE), and mediates brain‐derived neurotrophic factor (BDNF)‐induced Arc SARE activation. [ABSTRACT FROM AUTHOR]

CCG-1423, a chemical inhibitor of Rho signaling, blocks serum response factor (SRF)/megakaryoblastic leukemia 1 (MKL1)-mediated gene expression by inhibiting the nuclear accumulation of MKL1. Several studies have suggested that CCG-1423 interacts not only with MKL1, which has a critical role in the regulation of neuronal morphology, but also with phosphatase and actin regulator 1 (Phactr1), which is localized at synapses. However, the effect of CCG-1423 on neuronal cells, especially on neuronal morphology, remains to be determined. In this study, we focused on the effect of CCG-1423 on axonal elongation, dendritic length, dendritic complexity and dendritic spine morphology. Incubation of cortical neuron cultures with up to 10 μM CCG-1423 for 72 h did not significantly affect cell viability. CCG-1423 inhibited axonal elongation and blocked the increase of dendritic length and complexity, but did not affect dendritic spine morphology. Here, we demonstrated for the first time that CCG-1423 affects neurite elongation, except for dendritic spines, without affecting neuronal cell viability. This study provides a better understanding of the effects of CCG-1423 on neurons, which may be useful for the assessment of the potential clinical application of CCG-1423 and its derivatives. [ABSTRACT FROM AUTHOR]

The activity-regulated expression of immediate early genes (IEGs) contributes to long-lasting neuronal functions underlying long-term memory. However, their response properties following neuronal activity are unique and remain poorly understood. To address this knowledge gap, here we further investigated the response properties of two representative IEGs, c-fos and brain-derived neurotrophic factor ( Bdnf ). Treatment of cultured cortical cells with KCl produces a depolarization process that results in the increase of intracellular calcium concentration in a KCl concentration-dependent manner. Consistent with this increase, c-fos expression was induced in a KCl concentration-dependent manner. In contrast, however, Bdnf expression was optimally activated by both 25 and 50 mM concentration of KCl. Similar results were observed when the cells were treated with okadaic acid, which inhibits protein phosphatases and elicits the hyper-phosphorylation of signaling molecules. Thus, Bdnf expression is strictly regulated by a neuronal activity threshold in an all or nothing manner, whereas c-fos expression is activated in a neuronal activity-dependent manner. Our findings also suggest that these differential responses might be due to the presence or absence of a TATA box. [ABSTRACT FROM AUTHOR]

How the expression of immediate early genes (IEGs) is controlled in response to neurotransmissions is unknown. Using cultured rat cortical cells, we investigated the expression of IEGs regulated by Ca 2+ and/or cAMP signals. The expression of c-fos was transiently induced by treatment of cells with high potassium (high K + ), which evoked depolarization, or forskolin, an adenylate cyclase activator. c-fos expression was persistently and synergistically induced by simultaneous treatment with high K + and forskolin via cAMP-response element (CRE). Microarray analysis indicated the expression profiles of IEGs caused by depolarization in the presence or absence of forskolin. When a novel index was included to investigate the profile of IEGs, we found that high K + -induced expression of IEGs was stimulatory or negatively changed in the presence of forskolin, suggesting distinct convergent effects of Ca 2+ and cAMP signals on the expression of IEGs. [ABSTRACT FROM AUTHOR]

Although the excitatory action of GABA has been shown to activate the expression of brain-derived neurotrophic factor (BDNF), its molecular mechanisms remain unclear. Using cultured rat cortical cells, we here demonstrated that GABA induced Bdnf mRNA expression mainly via L-type voltagedependent Ca2+ channels (L-VDCC) at the early stage and inhibited it at the late stage of the culture, which corresponded to the excitatory and inhibitory states of cortical cells. The excitatory GABA-induced Bdnf mRNA expression was controlled by multiple Ca2+ signaling pathways including Ca2+/ calmodulin-dependent protein kinase (CaMK), mitogen-activated protein kinase (MAPK) and calcineurin (CN). The Bdnfpromoter IV (Bdnf-pIV) was activated by GABA, mainly via cAMP-response element (CRE)/CREB, and this was prevented by the over-expression of a dominant negative CREB. The nuclear translocation of CREB-regulated transcriptional coactivator 1 (CRTC1) was selectively induced by the GABA-induced CN pathway to activate Bdnf-pIV. On the other hand, GABA-induced Gal4-CREB-dependent transcription, which was controlled by multiple Ca2+ signaling pathways, was prevented when the serine at position 133 of Gal4-CREB was mutated to alanine. Taken together, the excitatory action of GABA transcriptionally activated Bdnf expression through the combination of nuclear-localized CRTC1 and phosphorylated CREB in immature cortical cells, and may be the molecular mechanisms underlying Bdnf expression to control neuronal development. [ABSTRACT FROM AUTHOR]

The megakaryoblastic leukemia 1 (MKL1) protein functions as a transcriptional coactivator of the serum response factor. MKL1 has three RPEL motifs (RPEL1, RPEL2, and RPEL3) in its N-terminal region. MKL1 binds to monomeric G-actin through RPEL motifs, and the dissociation of MKL1 from G-actin promotes the translocation of MKL1 to the nucleus. Although structural data are available for RPEL motifs of MKL1 in complex with G-actin, the structural characteristics of RPEL motifs in the free state have been poorly defined. Here we characterized the structures of free RPEL motifs using NMR and CD spectroscopy. NMR and CD measurements showed that free RPEL motifs are largely unstructured in solution. However, NMR analysis identified transient α-helices in the regions where helices α1 and α2 are induced upon binding to G-actin. Proline mutagenesis showed that the transient α-helices are locally formed without helix-helix interactions. The helix content is higher in the order of RPEL1, RPEL2, and RPEL3. The amount of preformed structure may correlate with the binding affinity between the intrinsically disordered protein and its target molecule. [ABSTRACT FROM AUTHOR]

Highlights: [•] Relationship between BDNF and the action of antidepressants has been implicated. [•] We investigated whether type II pyrethroids has an antidepressant-like effect in mice. [•] Deltamethrin (DM) increased the expression of BDNF mRNA in the hippocampus. [•] Single and repeated administration of DM exerted antidepressant-like effects. [•] DM may be a possible source for the development of drugs to treat depression. [ABSTRACT FROM AUTHOR]

Abstract: Brain-derived neurotrophic factor (BDNF) plays a fundamental role in neuronal synaptic plasticity. A decrease of plasticity in the brain may be related to the pathogenesis of neurodegenerative or psychiatric disorders. Pyrethroid insecticides, which affect sodium channels in neurons, are widely used to control insect pests in agriculture and in the home. We previously found that deltamethrin (DM), a type II pyrethroid, increased Bdnf mRNA expression in cultured rat cortical neurons. However, the cyano group at the α-position of type II pyrethroids is likely susceptible to hydrolytic degradation and, its degraded product, hydrogen cyanide, could generate a cellular toxicity in the human body. To determine if the cyano group is required for the Bdnf exon IV-IX (Bdnf eIV-IX) mRNA expression induced by type II pyrethroids, for this study we synthesized a series of derivatives, in which the cyano group at the α-position was replaced with an ethynyl group. Then we added various substituents at the terminal position of the ethynyl group, and biologically evaluated the effects of these derivatives on Bdnf eIV-IX mRNA expression. These ethynyl derivatives induced the Bdnf eIV-IX mRNA expression in a concentration-dependent manner, at varying levels but lower levels than that evoked by DM. The mechanisms for the Bdnf induction and the morphological changes of neurons were the same whether the cyano or ethynyl group was included in the compounds. [Copyright &y& Elsevier]

Abstract: Pyrethroids, widely used insecticides with low acute toxicity in mammals, affect sodium channels in neurons. In a primary culture of rat cortical neurons, deltamethrin (DM), a type II pyrethroid, markedly enhanced the expression of brain-derived neurotrophic factor (BDNF) exon IV-IX (Bdnf eIV-IX) mRNA. In this study, we found that DM has a neurotrophic effect on cultured neurons and investigated the mechanisms responsible for it. One μM DM increased cell survival, neurite complexity and length. Neurite complexity and length were reduced not only by a blockade of cellular excitation with GABA or Ca2+ influx via L-type voltage-dependent calcium channels with nicardipine, but also by a blockade of TrkB, a specific receptor for BDNF, with TrkB/Fc. These data indicate DM has neurotrophic actions. DM-induced Bdnf eIV-IX mRNA expression through the calcineurin and ERK/MAPK pathways, the increase of which was reduced by GABAA receptor activation. Using a promoter assay, we found that Ca2+-responsive elements including a CRE are involved in the DM-induced activation of the Bdnf promoter IV (Bdnf-pIV). The intracellular concentration of Ca2+ and activation of Bdnf-pIV remained elevated for, at least, 1 and 24 h, respectively. Moreover, GABAA receptor activation or a blockade of Ca2+ influx even after starting the incubation with DM reduced the elevated activity of Bdnf-pIV. These data demonstrated that the prolonged activation of Bdnf-pIV occurred because of this continuous increase in the intracellular Ca2+ concentration. Thus, DM has neurotrophic effects on neurons, likely due to prolonged activation of Bdnf promoter in neurons. This article is part of a Special Issue entitled ‘Post-Traumatic Stress Disorder’. [Copyright &y& Elsevier]

Abstract: Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of catecholamine but its transcriptional regulation is not fully understood. Using a reporter assay with cultured rat cortical neurons, we demonstrated that the TH gene promoter was activated by brain-derived neurotrophic factor (BDNF), through its specific receptor TrkB and the ERK/MAP kinase pathway. Using a series of mutant TH gene promoters, we found that the cAMP-response element (CRE) plays a crucial role in the TH promoter activity and the Egr-1-responsive element (ERE), at least in part, is responsible for the BDNF-induced activation. Notably, the influx of Ca2+ evoked via the N-methyl-d-aspartate receptor (NMDA-R) but not via the L-type voltage-dependent Ca2+ channel (L-VDCC) selectively antagonized the activation of the gene promoter, suggesting a new link between the catecholaminergic and glutamatergic systems. The Ca2+ signals evoked via NMDA-R did not affect the phosphorylation of ERK1/2 induced by BDNF. These results suggest that the TH gene''s transcription is positively regulated by BDNF, through the CRE and ERE of the promoter, but selectively antagonized by the Ca2+ signals evoked via NMDA-R without disturbing the ERK/MAP kinase pathway, the regulation by which may underlie the development of the catecholaminergic system in the brain. [Copyright &y& Elsevier]

Abstract: Although dynorphins are widely involved in the control of not only nociceptive neurotransmission but also a variety of brain functions such as memory and emotion, no natural regulator for inducing the mRNA expression of prodynorphin (Pdyn), a precursor protein of dynorphins, is known. Using primary cultures of rat cortical neurons, we found that pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the vasoactive intestinal polypeptide (VIP)/secretin/glucagon neuropeptide family, markedly induces Pdyn mRNA expression. PACAP was much more effective than VIP, indicating a major role for PAC1 in the PACAP-induced Pdyn mRNA expression. The increase in Pdyn mRNA expression was independent of de novo protein synthesis. Administration of forskolin, an activator for adenylate cyclase/protein kinase A (PKA), but not TPA, an activator for protein kinase C (PKC), induced Pdyn mRNA expression, suggesting a major role for PKA. The involvement of PKA was supported by the inhibition of PACAP-induced Pdyn mRNA expression upon addition of H89, an inhibitor for PKA. The PACAP-induced potentiation of NMDA-R was involved in the mRNA expression of Bdnf or c-fos but not Pdyn. These results suggest PACAP to be an upstream regulator for inducing Pdyn mRNA expression through PKA. [ABSTRACT FROM AUTHOR]

Abstract: To understand epigenetic regulation of neurotrophins in Neuro-2a mouse neuroblastoma cells, we investigated the alteration of CpG methylation of brain-derived neurotrophic factor (BDNF) promoter I and neurotrophin-3 (NT-3) promoter IB and that of histone modification in Neuro-2a cells. Bisulfite genomic sequencing showed that the CpG sites of BDNF promoter I were methylated in non-treated Neuro-2a cells and demethylated following 5-aza-2′-deoxycytidine (5-aza-dC) treatment. In contrast, methylation status of the NT-3 promoter IB did not change by 5-aza-dC treatment in Neuro-2a cells. Furthermore, we demonstrated that BDNF exon I–IX mRNA was induced by trichostatin A (TSA) treatment. However, NT-3 exon IB–II mRNA was not induced by TSA treatment. Chromatin immunoprecipitation assays showed that the levels of acetylated histones H3 and H4 on BDNF promoter I were increased by TSA. These results demonstrate that DNA methylation and/or histone modification regulate BDNF gene expression, but do not regulate NT-3 gene expression in Neuro-2a cells. [Copyright &y& Elsevier]

Abstract: It is still unclear whether an active state of transcription once established in chromatin persists in neurons. Here, we focused on BDNF exon I–IX mRNA expression because of its marked induction upon the treatment of rat cortical neurons with trichostatin A, suggesting strong repression of the expression through histone deacetylase activity. Acetylation of histones H3 and H4 in promoter-I of the BDNF gene (BDNF-PI) was induced by membrane depolarization time- and dose-dependently, corresponding with the increase in mRNA expression. Following withdrawal of the depolarization, the mRNA level remained elevated for at least 6h, the persistence of which depended upon the strength of depolarization, whereas the BDNF exon IV–IX expression did not. The acetylation of histones was also maintained with BDNF-PI. Thus, BDNF exon I–IX mRNA expression remained increased after depolarization was withdrawn, suggesting that once activated, the BDNF-PI transcription persists due to chromatin remodeling. [Copyright &y& Elsevier]

Abstract: Valproic acid (VPA), a drug used to treat epilepsy and bipolar mood disorder, inhibits histone deacetylase (HDAC), which is associated with the epigenetic regulation of gene expression. Using a microarray, we comprehensively examined which genes are affected by stimulating cultured rat cortical neurons with VPA, and found that the VPA-treatment markedly altered gene expression (up-regulated; 726 genes, down-regulated; 577 genes). The mRNA expression for brain-derived neurotrophic factor (BDNF) and the α4 subunit of the GABAA receptor (GABAARα4), known to be involved in epileptogenesis, was up-regulated, with the increase in BDNF exon I–IX mRNA expression being remarkable, whereas that for GABAARγ2, GAD65 and 67, and the K+/Cl− co-transporter KCC2, which are responsible for the development of GABAergic inhibitory neurons, was down-regulated. The number of GAD67-positive neurons decreased upon VPA-treatment. Similar changes of up- and down-regulation were obtained by trichostatin A. VPA did not affect the intracellular Ca2+ concentration and the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), suggesting its direct action on HDAC. The acetylation of histones H3 and H4 was increased in the promoters of up-regulated but not down-regulated genes. Thus, VPA may disrupt a balance between excitatory and inhibitory neuronal activities through its epigenetic effect. [Copyright &y& Elsevier]

Abstract: To know the role of repressor element-1 (RE-1)-silencing transcription factor (REST) in activity-dependent gene transcription in neurons, we investigated whether the Ca2+ signal-induced transcription of brain-derived neurotrophic factor promoter-I (BDNF-PI) is repressed by RE-1 located in exon II from far downstream of BDNF promoter-II (BDNF-PII). By constructing plasmids in which the location between BDNF-PI, -PII, and -RE-1 is maintained, we found, by conducting promoter assays with cortical neurons, that the promoter activity was constitutively repressed through the actions of BDNF-RE-1 but activated by Ca2+ signals evoked via membrane depolarization, which was due to BDNF-PI but not to BDNF-PII. The over-expression of REST reduced the level of transcriptional activation through the N- and C-terminals, suggesting the recruitment of a histone deacetylase. On over-expression of REST, an increased depolarization did not allow the activation. Thus, REST remotely represses activity-dependent gene transcription, the level of which controls the magnitude of the repression. [Copyright &y& Elsevier]

The dynamic changes in dendritic morphology displayed by developing and mature neurons have stimulated interest in deciphering the signaling pathways involved. Recent studies have identified megakaryocytic acute leukemia (MAL), a serum response factor (SRF) co-activator, as a key component of a signaling pathway linking changes in the actin cytoskeleton to SRF-mediated transcription. To help define the role of this pathway in regulating dendritic morphology, we have characterized the pattern of MAL expression in the developing and adult brain, and have examined its role in regulating dendritic morphology in cultured cortical neurons. In histological studies of mouse brain, we found prominent expression of MAL in neurons in adult hippocampus and cerebral cortex. MAL immunostaining revealed localization of this protein in neuronal cell bodies and apical dendrites. During development, an increase in MAL expression occurs during the second post-natal week. Expression of dominant negative MAL constructs or MAL siRNA in cortical neurons grown in primary culture reduces the number of dendritic processes and decreases the basal level of SRF-mediated transcription. Taken together, these findings indicate that the MAL-SRF signaling pathway plays a key role in regulating dendritic morphology. [ABSTRACT FROM AUTHOR]

Brain-derived neurotrophic factor (BDNF) is a key player in synaptic plasticity, and consequently, learning and memory. Because of its fundamental role in numerous neurological functions in the central nervous system, BDNF has utility as a biomarker and drug target for neurodegenerative and neuropsychiatric disorders. Here, we generated a screening assay to mine inducers of Bdnf transcription in neuronal cells, using primary cultures of cortical cells prepared from a transgenic mouse strain, specifically, Bdnf-Luciferase transgenic (Bdnf-Luc) mice. We identified several active extracts from a library consisting of 120 herbal extracts. In particular, we focused on an active extract prepared from Ginseng Radix (GIN), and found that GIN activated endogenous Bdnf expression via cAMP-response element-binding protein-dependent transcription. Taken together, our current screening assay can be used for validating herbal extracts, food-derived agents, and chemical compounds for their ability to induce Bdnf expression in neurons. This method will be beneficial for screening of candidate drugs for ameliorating symptoms of neurological diseases associated with reduced Bdnf expression in the brain, as well as candidate inhibitors of aging-related cognitive decline. [ABSTRACT FROM AUTHOR]