Brain-derived neurotrophic factor (BDNF) downregulates mRNA levels of suppressor of cancer cell invasion (SCAI) variants in cortical neurons.

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.

Endogenous SOLOIST/MRTFB i4, a Neuronal Isoform of MKL2/MRTFB, Positively and Negatively Regulates SRF Target Immediate Early Genes in Neuro-2a Cells.

Megakaryoblastic leukemia 2 (MKL2)/myocardin-related transcription factor-B (MRTFB) is a serum response factor (SRF) cofactor that is enriched in the brain and controls SRF target genes and neuronal morphology. There are at least four isoforms of MKL2/MRTFB. Among these, MKL2/MRTFB isoform 1 and spliced neuronal long isoform of SRF transcriptional coactivator (SOLOIST)/MRTFB isoform 4 (MRTFB i4) are highly expressed in neurons. Although, when overexpressed in neurons, isoform 1 and SOLOIST/MRTFB i4 have opposing effects on dendritic morphology and differentially regulate SRF target genes, it is unknown how endogenous SOLOIST/MRTFB i4 regulates gene expression. Using isoform-specific knockdown, we investigated the role of endogenous SOLOST/MRTFB i4 in regulating the expression of other MKL2/MRTFB isoforms and SRF-target genes in Neuro-2a cells. Knockdown of SOLOIST/MRTFB i4 downregulated SOLOIST/MRTFB i4, while it upregulated isoform 1 without affecting isoform 3. Knockdown of SOLOIST/MRTFB i4 downregulated the SRF target immediate early genes egr1 and Arc, while it upregulated c-fos. Double knockdown of isoform 1 and SOLOIST/MRTFB i4 inhibited c-fos expression. Taken together, our findings in Neuro-2a cells suggest that endogenous SOLOIST/MRTFB i4 positively regulates egr1 and Arc expression. In addition, endogenous SOLOIST/MRTFB i4 may negatively regulate c-fos expression, possibly by downregulating isoform 1 in Neuro-2a cells.

SRF and SRF Cofactor mRNA Expression Is Differentially Regulated by BDNF Stimulation in Cortical Neurons.

Serum response factor (SRF) is a transcription factor that plays essential roles in multiple brain functions in concert with SRF cofactors such as ternary complex factor (TCF) and megakaryoblastic leukemia (MKL)/myocardin-related transcription factor (MRTF), which comprises MKL1/MRTFA and MKL2/MRTFB. Here, we stimulated primary cultured rat cortical neurons with brain-derived neurotrophic factor (BDNF) and investigated the levels of SRF and SRF cofactor mRNA expression. We found that SRF mRNA was transiently induced by BDNF, whereas the levels of SRF cofactors were differentially regulated: mRNA expression of Elk1, a TCF family member, and MKL1/MRTFA were unchanged, while in contrast, mRNA expression of MKL2/MRTFB was transiently decreased. Inhibitor experiments revealed that BDNF-mediated alteration in mRNA levels detected in this study was mainly due to the extracellular signal-regulated protein kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway. Collectively, BDNF mediates the reciprocal regulation of SRF and MKL2/MRTFB at the mRNA expression level through ERK/MAPK, which may fine-tune the transcription of SRF target genes in cortical neurons. Accumulating evidence regarding the alteration of SRF and SRF cofactor levels detected in several neurological disorders suggests that the findings of this study might also provide novel insights into valuable therapeutic strategies for the treatment of brain diseases.

SRF in Neurochemistry: Overview of Recent Advances in Research on the Nervous System.

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.

Expression of SOLOIST/MRTFB i4, a novel neuronal isoform of the mouse serum response factor coactivator myocardin-related transcription factor-B, negatively regulates dendritic complexity in cortical neurons.

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, ß-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.

Differential localization and roles of splice variants of rat suppressor of cancer cell invasion (SCAI) in neuronal cells.

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.

Neuron-enriched phosphatase and actin regulator 3 (Phactr3)/ nuclear scaffold-associated PP1-inhibiting protein (Scapinin) regulates dendritic morphology via its protein phosphatase 1-binding domain.

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.

Productivity and bioactivity of enokipodins A-D of Flammulina rossica and Flammulina velutipes.

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.

Involvement of SRF coactivator MKL2 in BDNF-mediated activation of the synaptic activity-responsive element in the Arc gene.

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.

Deltamethrin Increases Neurite Outgrowth in Cortical Neurons through Endogenous BDNF/TrkB Pathways.

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.

Distinct regulation of activity-dependent transcription of immediate early genes in cultured rat cortical neurons.

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.

Rho signaling inhibitor, CCG-1423, inhibits axonal elongation and dendritic complexity of rat cortical neurons.

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.

Balance between cAMP and Ca(2+) signals regulates expression levels of pituitary adenylate cyclase-activating polypeptide gene in neurons.

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 Ca(2+) 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 Ca(2+) 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 Ca(2+) signaling-induced transcriptional activation of Pacap. Taken together, our findings suggest that a balance between Ca(2+) and cAMP signals regulates PACAP levels in neurons and that a perturbation of this balance may result in psychiatric disorders, such as schizophrenia.

Neuromodulatory effect of Gαs- or Gαq-coupled G-protein-coupled receptor on NMDA receptor selectively activates the NMDA receptor/Ca2+/calcineurin/cAMP response element-binding protein-regulated transcriptional coactivator 1 pathway to effectively induce brain-derived neurotrophic factor expression in neurons.

Although coordinated molecular signaling through excitatory and modulatory neurotransmissions is critical for the induction of immediate early genes (IEGs), which lead to effective changes in synaptic plasticity, the intracellular mechanisms responsible remain obscure. Here we measured the expression of IEGs and used bioluminescence imaging to visualize the expression of Bdnf when GPCRs, major neuromodulator receptors, were stimulated. Stimulation of pituitary adenylate cyclase-activating polypeptide (PACAP)-specific receptor (PAC1), a Gαs/q-protein-coupled GPCR, with PACAP selectively activated the calcineurin (CN) pathway that is controlled by calcium signals evoked via NMDAR. This signaling pathway then induced the expression of Bdnf and CN-dependent IEGs through the nuclear translocation of CREB-regulated transcriptional coactivator 1 (CRTC1). Intracerebroventricular injection of PACAP and intraperitoneal administration of MK801 in mice demonstrated that functional interactions between PAC1 and NMDAR induced the expression of Bdnf in the brain. Coactivation of NMDAR and PAC1 synergistically induced the expression of Bdnf attributable to selective activation of the CN pathway. This CN pathway-controlled expression of Bdnf was also induced by stimulating other Gαs- or Gαq-coupled GPCRs, such as dopamine D1, adrenaline ß, CRF, and neurotensin receptors, either with their cognate agonists or by direct stimulation of the protein kinase A (PKA)/PKC pathway with chemical activators. Thus, the GPCR-induced expression of IEGs in coordination with NMDAR might occur via the selective activation of the CN/CRTC1/CREB pathway under simultaneous excitatory and modulatory synaptic transmissions in neurons if either the Gαs/adenylate cyclase/PKA or Gαq/PLC/PKC-mediated pathway is activated.

Class I histone deacetylase-mediated repression of the proximal promoter of the activity-regulated cytoskeleton-associated protein gene regulates its response to brain-derived neurotrophic factor.

We examined the transcriptional regulation of the activity-regulated cytoskeleton-associated protein gene (Arc), focusing on BDNF-induced Arc expression in cultured rat cortical cells. Although the synaptic activity-responsive element (SARE), located -7 kbp upstream of the Arc transcription start site, responded to NMDA, BDNF, or FGF2, the proximal region of the promoter (Arc/-1679) was activated by BDNF or FGF2, but not by NMDA, suggesting the presence of at least two distinct Arc promoter regions, distal and proximal, that respond to extracellular stimuli. Specificity protein 4 (SP4) and early growth response 1 (EGR1) controlled Arc/-1679 transcriptional activity via the region encompassing -169 to -37 of the Arc promoter. We found that trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, significantly enhanced the inductive effects of BDNF or FGF2, but not those of NMDA on Arc expression. Inhibitors of class I/IIb HDACs, SAHA, and class I HDACs, MS-275, but not of class II HDACs, MC1568, enhanced BDNF-induced Arc expression. The enhancing effect of TSA was mediated by the region from -1027 to -1000 bp, to which serum response factor (SRF) and HDAC1 bound. The binding of HDAC1 to this region was reduced by TSA. Thus, Arc expression was suppressed by class I HDAC-mediated mechanisms via chromatin modification of the proximal promoter whereas the inhibition of HDAC allowed Arc expression to be markedly enhanced in response to BDNF or FGF2. These results contribute to our understanding of the physiological role of Arc expression in neuronal functions such as memory consolidation.

Convergent effects of Ca(2+) and cAMP signals on the expression of immediate early genes in neurons.

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.

Excitatory GABA induces BDNF transcription via CRTC1 and phosphorylated CREB-related pathways in immature cortical cells.

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.

CD106 in tumor-specific exhausted CD8+ T cells mediates immunosuppression by inhibiting TCR signaling.

T cell exhaustion is a major contributor to immunosuppression in the tumor microenvironment (TME). Blockade of key regulators of T cell exhaustion, such as PD-1, can reinvigorate tumor-specific T cells and activate anti-tumor immunity in various types of cancer. Here, we identified that CD106 was specifically expressed in exhausted CD8+ T cells in the TME using single-cell RNA-sequencing. High CD106 expression in the TME in clinical samples corresponded to improved response to cancer immunotherapy. CD106 in tumor-specific T cells suppressed anti-tumor immunity both in vitro and in vivo, and loss of CD106 in CD8+ T cells suppressed tumor growth and improved response to PD-1 blockade. Mechanistically, CD106 inhibited T-cell receptor (TCR) signaling by interacting with the TCR/CD3 complex and reducing its surface expression. Together, these findings provide insights into the immunosuppressive role of CD106 expressed in tumor-specific exhausted CD8+ T cells, identifying it as a potential biomarker and therapeutic target for cancer immunotherapy.

The extract based on the Kampo formula daikenchuto (Da Jian Zhong Tang) induces Bdnf expression and has neurotrophic effects in cultured cortical neurons.

Reductions in brain-derived neurotrophic factor (BDNF) expression levels have been reported in the brains of patients with neurological disorders such as Alzheimer's disease. Therefore, upregulating BDNF and preventing its decline in the diseased brain could help ameliorate neurological dysfunctions. Accordingly, we sought to discover agents that increase Bdnf expression in neurons. Here, we screened a library of 42 Kampo extracts to identify those with the ability to induce Bdnf expression in cultured cortical neurons. Among the active extracts identified in the screen, we focused on the extract based on the Kampo formula daikenchuto. The extract of daikenchuto in the library used in this study was prepared using the mixture of Zingiberis Rhizoma Processum (ZIN), Zanthoxyli Piperiti Pericarpium (ZAN), and Ginseng Radix (GIN) without Koi. In this study, we defined DKT as the mixture of ZIN, ZAN, and GIN without Koi (DKT extract means the extract prepared from the mixture of ZIN, ZAN, and GIN without Koi). DKT extract significantly increased endogenous Bdnf expression by mediated, at least in part, via Ca2+ signaling involving L-type voltage-dependent Ca2+ channels in cultured cortical neurons. Furthermore, DKT extract significantly improved the survival of cultured cortical neurons and increased neurite complexity in immature neurons. Taken together, our findings suggest that DKT extract induces Bdnf expression and has a neurotrophic effect in neurons. Because BDNF inducers are expected to have therapeutic potential for neurological disorders, re-positioning of Kampo formulations such as daikenchuto may lead to clinical application in diseases associated with reduced BDNF in the brain.

Case of bullous pemphigoid refractory to corticosteroids by antiepileptic drug-induced CYP3A4.

The most common treatment option for patients with bullous pemphigoid is systemic corticosteroids. CYP3A4, a drug-metabolizing enzyme in the liver, metabolizes synthetic steroids to a varying degree. Although there are many CYP3A4-inducing drugs, several antiepileptic drugs, such as phenytoin and phenobarbital, strongly induce CYP3A4, thereby reducing the effects of corticosteroids. Here, we report a case of refractory bullous pemphigoid that rapidly improved after the discontinuation of phenytoin and phenobarbital. To achieve adequate pharmacological effects of corticosteroids, we must always ensure that patients who require corticosteroids for treatment are not medicated with CYP3A4-inducing agents.