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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.

Deltamethrin (DM), a type II pyrethroid, robustly increases brain-derived neurotrophic factor (Bdnf) expression and has a neurotrophic effect in primary cultures of rat cortical neurons. In this study, we investigated the effect of DM on neurite morphology in cultured rat cortical neurons. DM significantly increased neurite outgrowth, but this increase was abolished when the BDNF scavenger tropomyosin receptor kinase B (TrkB)-Fc was added 10 min before the DM treatment. In contrast, the addition of TrkB-Fc 1 h after the treatment did not affect DM-induced neurite outgrowth. Our previous research has indicated that type II, but not type I, pyrethroids have the ability to induce Bdnf mRNA expression, but neither permethrin nor cypermethrin, which are type I and type II pyrethroids, respectively, affected neurite outgrowth in the current study. These results suggest that this effect is not due to increased Bdnf expression, and the effect is unique to DM. We previously demonstrated that calcineurin plays a role in the DM-mediated induction of Bdnf expression. However, the calcineurin inhibitor FK506 did not significantly affect DM-induced neurite outgrowth. DM-induced neurite outgrowth was abolished by U0126 and rapamycin, indicating the involvement of the mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) pathways. Taken together, these findings suggest that DM activates endogenous BDNF/TrkB-mediated MAPK and mTOR pathways, thereby increasing neurite outgrowth.Key words: BDNF, Deltamethrin, MAPK, mTOR, Neurite outgrowth.

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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 voltage-dependent Ca(2+) 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 Ca(2+) signaling pathways including Ca(2+) /calmodulin-dependent protein kinase (CaMK), mitogen-activated protein kinase (MAPK) and calcineurin (CN). The Bdnf-promoter 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 Ca(2+) 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. We demonstrated that GABA induced Bdnf expression at the early stage of the culture, in which GABA exerted its excitatory action. The excitatory GABA-induced Bdnf expression was controlled by multiple Ca(2+) signaling pathways evoked via L-VDCC. Both the CREB coactivator, CRTC1 and CREB phosphorylation participated in excitatory GABA-induced Bdnf transcription. Our present study indicates the mechanism underlying the excitatory GABA-induced Bdnf expression in immature neurons and provide new insights into molecular mechanisms underlying Bdnf expression to control neuronal development., (© 2014 International Society for Neurochemistry.)

The ability of megakaryoblastic leukemia 1 (MKL1) to function as a serum response factor (SRF) coactivator is regulated through its association with G-actin. In the cytoplasm, MKL1 binds to G-actin through RPXXXEL (RPEL) motifs. However, dissociation of MKL1 from G-actin triggers its translocation into the nucleus where it stimulates SRF-mediated gene expression. Previous characterization of rat MKL1 gene products has identified several isoforms: full-length MKL1, basic, SAP, and coiled-coil domain (BSAC), MKL1-elongated derivative of yield (MELODY), and MKL1met. In this study, we have investigated whether these MKL1 isoforms, which contain different numbers of RPEL motifs, differ in their subcellular localization, transcriptional activity, and effect on dendritic number and axonal length. Immunofluorescent staining of cultured cortical neurons expressing individual FLAG-tagged MKL1 isoforms indicated that all MKL1 isoforms are present in both the cytoplasm and the nucleus. However, MKL1met, which contains two RPEL motifs, shows enhanced nuclear staining compared with the other three isoforms, full-length MKL1, basic, SAP, and coiled-coil domain, and MKL1-elongated derivative of yield, which contain three RPEL motifs. Consistent with its preferential nuclear localization, overexpression of MKL1met, but not other isoforms, increases SRF-mediated transcriptional responses and reduces the number of dendrites. In contrast to the inhibitory effect of MKL1met on dendritic number, axonal length is not affected by overexpression of any of the MKL1 isoforms. These findings suggest that the subcellular localization of MKL1 isoforms, which is mediated by the number of actin-binding RPEL motifs, regulates their effect on SRF-mediated gene expression and dendritic morphology.

Dynamic changes in neuronal morphology and transcriptional regulation play crucial roles in the neuronal network and function. Accumulating evidence suggests that the megakaryoblastic leukemia (MKL) family members, which function not only as actin-binding proteins but also as serum response factor (SRF) transcriptional coactivators, regulate neuronal morphology. However, the extracellular ligands and signaling pathways, which activate MKL-mediated morphological changes in neurons, remain unresolved. Here, we demonstrate that in addition to MKL1, MKL2, highly enriched in the forebrain, strongly contributes to the dendritic complexity, and this process is triggered by stimulation with activin, a member of the transforming growth factor β (TGF-β) superfamily. Activin promoted dendritic complexity in a SRF- and MKL-dependent manner without drastically affecting MKL localization and protein levels. In contrast, activin promoted the nuclear export of suppressor of cancer cell invasion (SCAI), which is a corepressor for SRF and MKL. Furthermore, overexpression of SCAI blocked activin-induced SRF transcriptional responses and dendritic complexity. Collectively, these results strongly suggest that activin-SCAI-MKL signaling is a novel pathway that regulates the dendritic morphology of rat cortical neurons by excluding SCAI from the nucleus and activating MKL/SRF-mediated gene expression.

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 alpha4 subunit of the GABA(A) receptor (GABA(A)Ralpha4), known to be involved in epileptogenesis, was up-regulated, with the increase in BDNF exon I-IX mRNA expression being remarkable, whereas that for GABA(A)Rgamma2, 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 Ca(2+) 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.

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: 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]