[Strategy for the development of small-molecule antidepressant targeting PAC1 receptor].

Major depressive disorder (MDD) is a psychiatric disorder that affects more than 300 million people worldwide and has a serious impact on society. Conventional antidepressants targeting monoamines in the brain based on the monoamine hypothesis are known to take a prolonged time to be effective or less effective in 30% of MDD patients. Hence, there is a need to develop antidepressants that are effective against treatment-resistant depression and have a new mechanism different from the monoamine hypothesis. An increasing number of research groups including us have been establishing that pituitary adenylate cyclase-activating polypeptide (PACAP) and one of its receptors, PAC1 receptor, are closely related to the etiology of stress-related diseases such as MDD. Therefore, it is strongly suggested that the PAC1 receptor is a promising target in the treatment of psychiatric disorders. We developed a novel, non-peptidic, small-molecule, high-affinity PAC1 receptor antagonists and conducted behavioral pharmacology experiments in mice to characterize a novel PAC1 receptor antagonist as a new option for MDD therapy. The results show that our novel PAC1 receptor antagonist has the potential to be a new antidepressant with a high safety profile. In this review, we would like to present the background of developing our novel PAC1 receptor antagonist and its effects on mouse models of acute stress.

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.

OMICS Analyses Unraveling Related Gene and Protein-Driven Molecular Mechanisms Underlying PACAP 38-Induced Neurite Outgrowth in PC12 Cells.

The study aimed to understand mechanism/s of neuronal outgrowth in the rat adrenal-derived pheochromocytoma cell line (PC12) under pituitary adenylate cyclase-activating polypeptide (PACAP) treatment. Neurite projection elongation was suggested to be mediated via Pac1 receptor-mediated dephosphorylation of CRMP2, where GSK-3ß, CDK5, and Rho/ROCK dephosphorylated CRMP2 within 3 h after addition of PACAP, but the dephosphorylation of CRMP2 by PACAP remained unclear. Thus, we attempted to identify the early factors in PACAP-induced neurite projection elongation via omics-based transcriptomic (whole genome DNA microarray) and proteomic (TMT-labeled liquid chromatography-tandem mass spectrometry) analyses of gene and protein expression profiles from 5-120 min after PACAP addition. The results revealed a number of key regulators involved in neurite outgrowth, including known ones, called 'Initial Early Factors', e.g., genes Inhba, Fst, Nr4a1,2,3, FAT4, Axin2, and proteins Mis12, Cdk13, Bcl91, CDC42, including categories of 'serotonergic synapse, neuropeptide and neurogenesis, and axon guidance'. cAMP signaling and PI3K-Akt signaling pathways and a calcium signaling pathway might be involved in CRMP2 dephosphorylation. Cross-referencing previous research, we tried to map these molecular components onto potential pathways, and we may provide important new information on molecular mechanisms of neuronal differentiation induced by PACAP. Gene and protein expression data are publicly available at NCBI GSE223333 and ProteomeXchange, identifier PXD039992.

GSTA4 Governs Melanoma Immune Resistance and Metastasis.

IMPLICATIONS: Considering the importance of GSTA4 in controlling IFNγ responsiveness and the metastatic potential of other melanoma cells, our results highlight a novel mechanism whereby cancer cells escape from host immunity and gain metastatic ability by acquiring resistance to oxidative stress responses through the upregulation of GSTA4.

Spinal Astrocyte-Neuron Lactate Shuttle Contributes to the Pituitary Adenylate Cyclase-Activating Polypeptide/PAC1 Receptor-Induced Nociceptive Behaviors in Mice.

We have previously shown that spinal pituitary adenylate cyclase-activating polypeptide (PACAP)/PACAP type 1 (PAC1) receptor signaling triggered long-lasting nociceptive behaviors through astroglial activation in mice. Since astrocyte-neuron lactate shuttle (ANLS) could be essential for long-term synaptic facilitation, we aimed to elucidate a possible involvement of spinal ANLS in the development of the PACAP/PAC1 receptor-induced nociceptive behaviors. A single intrathecal administration of PACAP induced short-term spontaneous aversive behaviors, followed by long-lasting mechanical allodynia in mice. These nociceptive behaviors were inhibited by 1,4-dideoxy-1,4-imino-d-arabinitol (DAB), an inhibitor of glycogenolysis, and this inhibition was reversed by simultaneous L-lactate application. In the cultured spinal astrocytes, the PACAP-evoked glycogenolysis and L-lactate secretion were inhibited by DAB. In addition, a protein kinase C (PKC) inhibitor attenuated the PACAP-induced nociceptive behaviors as well as the PACAP-evoked glycogenolysis and L-lactate secretion. Finally, an inhibitor for the monocarboxylate transporters blocked the L-lactate secretion from the spinal astrocytes and inhibited the PACAP- and spinal nerve ligation-induced nociceptive behaviors. These results suggested that spinal PAC1 receptor-PKC-ANLS signaling contributed to the PACAP-induced nociceptive behaviors. This signaling system could be involved in the peripheral nerve injury-induced pain-like behaviors.

Hypothalamic orexin prevents non-alcoholic steatohepatitis and hepatocellular carcinoma in obesity.

Non-alcoholic steatohepatitis (NASH) occasionally occurs under obesity; however, factors modulating the natural history of fatty liver disease remain unknown. Since hypothalamic orexin that regulates physical activity and autonomic balance prevents obesity, we investigate its role in NASH development. Male orexin-deficient mice fed a high-fat diet (HFD) show severe obesity and progression of NASH with fibrosis in the liver. Hepatic fibrosis also develops in ovariectomized orexin-deficient females fed an HFD but not ovariectomized wild-type controls. Moreover, long-term HFD feeding causes hepatocellular carcinoma (HCC) in orexin-deficient mice. Intracerebroventricular injection of orexin A or pharmacogenetic activation of orexin neurons acutely activates hepatic mTOR-sXbp1 pathway to prevent endoplasmic reticulum (ER) stress, a NASH-causing factor. Daily supplementation of orexin A attenuates hepatic ER stress and inflammation in orexin-deficient mice fed an HFD, and autonomic ganglionic blocker suppresses the orexin actions. These results suggest that hypothalamic orexin is an essential factor for preventing NASH and associated HCC under obesity.

Small-molecule non-peptide antagonists of the PACAP receptor attenuate acute restraint stress-induced anxiety-like behaviors in mice.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a highly conserved pleiotropic neuropeptide, implicated in emotional stress responses and anxiety-related disorders. Here, we examined whether our recently developed small-molecule non-peptide PACAP receptor antagonists could ameliorate anxiety-like behaviors induced by acute restraint stress in mice. The antagonists PA-9 and its derivative PA-915 improved anxiety-like behaviors in mice subjected to restraint stress. An anxiolytic effect was observed with single acute dose, suggesting their fast-acting properties. PA-915 demonstrated a statistically significant anxiolytic effect whereas fluoxetine did not. These results indicate the potential of PAC1 antagonists as a novel treatment for anxiety.

Comparative Analysis of Serum microRNA in Diagnosed Ocular Sarcoidosis versus Idiopathic Uveitis with Ocular Manifestations of Sarcoidosis.

"Idiopathic" is the most common category of uveitis, representing cases in which a specific diagnosis has not been established despite work-up. Sarcoidosis is a systemic granulomatous disorder affecting multiple organs including the lungs, skin, kidneys, and eyes. We used microRNA (miRNA) microarrays to investigate serum miRNA profiles of patients with ocular sarcoidosis as diagnosed by specific criteria (diagnosed ocular sarcoidosis), and patients with idiopathic uveitis characterized by ocular manifestations of sarcoidosis (suspected ocular sarcoidosis). Principal component analysis (PCA) and hierarchical clustering showed that serum miRNA profiles of diagnosed ocular sarcoidosis and suspected ocular sarcoidosis were both clearly distinguishable from healthy controls. Furthermore, comparative analysis of the miRNA profiles showed highly similar patterns between diagnosed ocular sarcoidosis and suspected ocular sarcoidosis. Pathway analysis revealed common pathways were involved in the two groups, including those of WNT signaling and TGF-beta signaling. Our study demonstrated a high overlap of differentially expressed serum miRNAs in patients with diagnosed ocular sarcoidosis and suspected ocular sarcoidosis, suggesting that these groups share a similar underlying pathology and may represent possible variants of the disease. Characterization of serum miRNA profiles may provide an opportunity for earlier diagnosis and treatment, and may inform more accurate clinical prognosis in patients with an ocular sarcoidosis phenotype.

Design and synthesis of pyrido[2,3-d]pyrimidine derivatives for a novel PAC1 receptor antagonist.

Since PA-8 (5-(4-(Allyloxy)-3-methoxyphenyl)-2-amino-5,8-dihydro-3H,6H-pyrido[2,3-d]pyrimidine-4,7-dione) was recently identified as a novel small-molecule antagonist of the pituitary adenylate cyclase-activating polypeptide (PACAP) type I (PAC1) receptor, a series of pyrido[2,3-d]pyrimidine derivatives have been designed, synthesized and subsequently evaluated for antagonistic activity on the PAC1 receptor. In this study, we synthesized 21 derivatives based on the PA-8 structure. Among them, the compound 2o (2-Amino-5-(3-trifluoromethoxy-phenyl)-5,8-dihydro-3H,6H-pyrido[2,3-d]pyrimidine-4,7-dione) showed more potent antagonistic activities than PA-8. Intrathecal (i.t.) injection of 2o blocked the induction of PACAP-induced aversive behaviors and mechanical allodynia in mice, and the effects were more potent than those of PA-8. A single i.t. injection of 2o also inhibited spinal nerve ligation (SNL)-induced mechanical allodynia. Repeated intraperitoneal administration of 2o gradually reduced the SNL-induced mechanical allodynia, and this effect appeared earlier than for PA-8. In addition, 2o exhibited a favorable ADME and pharmacokinetics profiles. These results suggest that 2o may become an analgesic for the treatment of neuropathic pain.

Fosphenytoin Alleviates Herpes Simplex Virus Infection-Induced Provoked and Spontaneous Pain-Like Behaviors in Mice.

In this study, we investigated the effects of fosphenytoin (fPHT), a water-soluble prodrug of phenytoin, on the pain responses of a mouse herpes zoster (HZ) pain model. Transdermal herpes simplex virus type 1 (HSV-1) inoculation induced mechanical allodynia and hyperalgesia of the hind paw and spontaneous pain-like behaviors, such as licking the affected skin. Intravenous injection of fPHT (15 and 30 mg/kg) alleviated HSV-1-induced provoked pain (allodynia and hyperalgesia). The suppressive effects of fPHT on provoked pain were weaker than those of diclofenac and pregabalin which were used as positive controls. fPHT, diclofenac, and pregabalin significantly suppressed HSV-1-induced spontaneous pain-like behaviors. Among them, high-dose fPHT (30 mg/kg) showed the strongest suppression. Intravenous fPHT may become a viable option for an acute HZ pain, especially for spontaneous pain.

Effect of PACAP on sweat secretion by immortalized human sweat gland cells.

The process of sweating plays an important role in the human body, including thermoregulation and maintenance of the environment and health of the skin. It is known that the conditions of hyperhidrosis and anhidrosis are caused by abnormalities in sweat secretion and can result in severe skin conditions such as pruritus and erythema, which significantly reduce the patient's quality of life. However, there are many aspects of the signaling mechanisms in the process of sweating that have not been clarified, and no effective therapies or therapeutic agents have yet been discovered. Previously, it was reported that pituitary adenylate cyclase-activating polypeptide (PACAP) promotes sweating, but details of the underlying mechanism has not been clarified. We used immortalized human eccrine gland cells (NCL-SG3 cell) to investigate how sweat secretion is induced by PACAP. Intracellular Ca2+ levels were increased in these cells following their exposure to physiological concentrations of PACAP. Intracellular Ca2+ was not elevated when cells were concomitantly treated with PA-8, a specific PAC1-R antagonist, suggesting that PAC1-R is involved in the elevation of intracellular Ca2+ levels in response to PACAP treatment. Furthermore, immunocytochemistry experiments showed that aquaporin-5 was translocated from the cytoplasm to the cell membrane by PACAP. These results suggest that PACAP acts on eccrine sweat glands to promote sweat secretion by translocation of aquaporin-5 to the cell membrane in response to increased levels of intracellular Ca2+. These findings also provide a solid basis for future research initiatives to develop new therapies to treat sweating disorders.

Molecular Mechanism for PACAP 38-Induced Neurite Outgrowth in PC12 Cells.

The present research investigates the molecular mechanism of neurite outgrowth (protrusion elongation) under pituitary adenylate cyclase-activating polypeptide (PACAP) 38 treatments using a rat adrenal-derived pheochromocytoma cell line-PC12. This study specifically looks into the regulation of PACAP38-induced collapsing response mediator protein 2 (CRMP2) previously identified in a mouse brain ischemia model and which could be recovered by PACAP38 treatment. Previously, DNA microarray analysis revealed that PACAP 38-mediated neuroprotection involved not only CRMP2 but also pathways related to glycogen synthase kinase-3ß (GSK-3ß) and other signaling components. Thus, to clarify whether CRMP2 acts directly on PACAP38 or through GSK-3ß as part of the mechanism of PACAP38-induced neurite outgrowth, we observed neurite outgrowth in the presence of GSK-3ß inhibitors and activators. PC12 cells were treated with PACAP38 being added to the cell culture medium at concentrations of 10-7 M, 10-8 M, and 10-9 M. Post PACAP38 treatment, immunostaining was used to confirm protrusion elongation of the PC12 cells, while RT-PCR, two-dimensional gel electrophoresis in conjunction with Western blotting, and inhibition experiments were performed to confirm the expression of the PACAP gene, its receptors, and downstream signaling components. Our data show that neurite protrusion elongation by PACAP38 (10-7 M) in PC12 cells is mediated through the PAC1-R receptor as demonstrated by its suppression by a specific inhibitor PA-8. Inhibitor experiments suggested that PACAP38-triggered neurite protrusion follows a GSK-3ß-regulated pathway, where the AKT and cAMP/ERK pathways are involved and where the inhibition of Rho/Roc could enhance neurite protrusion under PACAP38 stimulation. Although we could not yet confirm the exact role and position of CRMP2 in PACAP38-mediated PC12 cell elongation, it appears that its phosphorylation and dephosphorylation have a correlation with the neurite protrusion elongation through the interplay of CDK5, which needs to be investigated further.

Low-intensity ultrasound inhibits melanoma cell proliferation in vitro and tumor growth in vivo.

PURPOSE: To determine the effect of low-intensity ultrasound on cancer cell proliferation in vitro and tumor growth in vivo. METHODS: In vitro, several cancer cell lines were exposed to low-intensity ultrasound at 0.11 W/cm2 for 2 min. Of the cell lines screened, melanoma C32 is one of the cell lines that showed sensitivity to growth inhibition by ultrasound and was therefore used in succeeding experiments. In vivo, under the same ultrasound conditions used in vitro, C32 tumors in mice were exposed to ultrasound daily for 2 weeks, and the tumor volumes were monitored weekly using sonography. RESULTS: In vitro, C32 cell growth was inhibited, attaining 43.2% inhibition on the 3rd day. In vivo, tumor growth was significantly inhibited, with the treated tumors exhibiting 2.7-fold slowed tumor growth vs. untreated tumors at week 2. Such inhibition was not associated with increased cell death. Several genes related to the cell cycle and proliferation were among those significantly regulated. CONCLUSION: These findings highlight the potential of low-intensity ultrasound to inhibit tumor growth in a noninvasive, safe, and easy-to-administer way. In addition, this may suggest that the mechanical stress induced by ultrasound on C32 cells may have affected the intrinsic biomolecular mechanism related to the cell growth of this particular cell line. Further research is needed to identify which of the regulated genes played key roles in growth inhibition.

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.

Attenuation of relaxing response induced by pituitary adenylate cyclase-activating polypeptide in bronchial smooth muscle of experimental asthma.

Bronchomotor tone is regulated by contraction and relaxation of airway smooth muscle (ASM). A weakened ASM relaxation might be a cause of airway hyperresponsiveness (AHR), a characteristic feature of bronchial asthma. Pituitary adenylyl cyclase-activating polypeptide (PACAP) is known as a mediator that causes ASM relaxation. To date, whether or not the PACAP responsiveness is changed in asthmatic ASM is unknown. The current study examined the hypothesis that relaxation induced by PACAP is reduced in bronchial smooth muscle (BSM) of allergic asthma. The ovalbumin (OA)-sensitized mice were repeatedly challenged with aerosolized OA to induce asthmatic reaction. Twenty-four hours after the last antigen challenge, the main bronchial smooth muscle (BSM) tissues were isolated. Tension study showed a BSM hyperresponsiveness to acetylcholine in the OA-challenged mice. Both quantitative RT-PCR and immunoblot analyses revealed a significant decrease in PAC1 receptor expression in BSMs of the diseased mice. Accordingly, in the antigen-challenged group, the PACAP-induced PAC1 receptor-mediated BSM relaxation was significantly attenuated, whereas the relaxation induced by vasoactive intestinal polypeptide was not changed. These findings suggest that the relaxation induced by PACAP is impaired in BSMs of experimental asthma due to a downregulation of its binding partner PAC1 receptor. Impaired BSM responsiveness to PACAP might contribute to the AHR in asthma.

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.

TBX5 R264K acts as a modifier to develop dilated cardiomyopathy in mice independently of T-box pathway.

BACKGROUND: TBX5 is a transcription factor that has an important role in development of heart. TBX5 variants in the region encoding the T-box domain have been shown to cause cardiac defects, such as atrial septal defect or ventricular septal defect, while TBX5 variants have also been identified in a few cardiomyopathy patients and considered causative. We identified a TBX5 variant (c.791G>A, p.Arg264Lys), that is over-represented in cardiomyopathy patients. This variant is located outside of the T-box domain, and its pathogenicity has not been confirmed by functional analyses. OBJECTIVE: To investigate whether the TBX5 R264K is deleterious and could contribute to the pathogenesis of cardiomyopathy. METHODS AND RESULTS: We developed mice expressing Tbx5 R264K. Mice homozygous for this variant displayed compensated dilated cardiomyopathy; mild decreased fractional shortening, dilatation of the left ventricle, left ventricular wall thinning and increased heart weight without major heart structural disorders. There was no difference in activation of the ANF promotor, a transcriptional target of Tbx5, compared to wild-type. However, analysis of RNA isolated from left ventricular samples showed significant increases in the expression of Acta1 in left ventricle with concomitant increases in the protein level of ACTA1. CONCLUSIONS: Mice homozygous for Tbx5 R264K showed compensated dilated cardiomyopathy. Thus, TBX5 R264K may have a significant pathogenic role in some cardiomyopathy patients independently of T-box domain pathway.

Synthesis of a novel and potent small-molecule antagonist of PAC1 receptor for the treatment of neuropathic pain.

We recently identified novel small-molecule antagonists of the PACAP type I (PAC1) receptor using docking-based in silico screening followed by in vitro/vivo pharmacological assays. In the present study, we synthesized 18 novel derivatives based on the structure of PA-9, a recently developed antagonist of the PAC1 receptor, with a view to obtain a panel of compounds with more potent antagonistic and analgesic activities. Among them, compound 3d showed improved antagonistic activities. Intrathecal injection of 3d inhibited both pituitary adenylate cyclase-activating polypeptide (PACAP) and spinal nerve ligation-induced mechanical allodynia. The effects were more potent than PA-9. Compound 3d also showed anti-allodynic effects following oral administration. Hence, our results suggest that 3d may become an orally available analgesic in the treatment of the neuropathic pain.

Screening inducers of neuronal BDNF gene transcription using primary cortical cell cultures from BDNF-luciferase transgenic mice.

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.

Bidirectional crosstalk between neutrophils and adipocytes promotes adipose tissue inflammation.

Chronic activation of the IL-1ß system in adipose tissue on metabolic disorders is well demonstrated. However, a mechanism for its expression and activation in the tissue has remained unexplored. Here, we demonstrate that IL-1ß transcript was enriched in neutrophils of white adipose tissue (WAT) from lean mice. Mechanistically, the interaction of neutrophils with adipocytes induced IL-1ß expression via NF-κB pathway. Lipolysis of adipocytes accumulated neutrophils prior to macrophages in WAT and produced high levels of IL-1ß via an inflammasome pathway. Leukotriene B4 (LTB4) production in WAT also contributed to neutrophil accumulation. Furthermore, an LTB4-inflammasome axis contributed to the expression of chemotactic molecules involved in high-fat diet-induced macrophage infiltration into WAT. We have identified previously unappreciated roles for neutrophils in the development of adipose tissue inflammation: robust IL-1ß production and infiltration of macrophages to initiate chronic inflammation.-Watanabe, Y., Nagai, Y., Honda, H., Okamoto, N., Yanagibashi, T., Ogasawara, M., Yamamoto, S., Imamura, R., Takasaki, I., Hara, H., Sasahara, M., Arita, M., Hida, S., Taniguchi, S., Suda, T., Takatsu, K. Bidirectional crosstalk between neutrophils and adipocytes promotes adipose tissue inflammation.