In the present study, effects of dexamethasone (Dex) on allergic rhinitis (AR) induced by Japanese cedar pollen (JCP) were investigated. Mice were sensitized by i.p. injections with JCP+alum on days 0, 7 and 14. From day 21, the sensitized mice were challenged by intranasal (i.n.) administrations of JCP for 4 consecutive days. Animals were also treated with Dex (i.p.) 30 min before each JCP challenge. Frequency of sneezing was counted for 20 min after each nasal challenge. On days 18 and 25, the histamine (Hist, i.n.)-induced sneezing was also counted. In mice that were sensitized and repeatedly challenged with JCP, both serum levels of IgG and IgE specific for Cry J1, a major allergen of JCP, were significantly increased. In the JCP-sensitized mice, JCP challenge caused a significant increase in sneezing, indicating that nasal allergic response was induced. The i.n. application of Hist also caused an increase in sneezing. The Hist-induced sneezing was further increased significantly on day 25, indicating that nasal hyperresponsiveness (NHR) had occurred after the repeated JCP challenges. Both the nasal allergic response and NHR induced by JCP were inhibited by pretreatments with Dex. Thus, the murine AR model used might be useful for making clear the mechanisms of the AR pathogenesis and the action of corticosteroid effects.

Acute respiratory distress syndrome (ARDS) is characterized by increased permeability of pulmonary blood vessels, leading to respiratory failure. Although VEGF is responsible for the vascular permeability, it remains unknown about the involvement of signaling for VEGFR1, a receptor for VEGF. We examined the role of VEGFR1 in pathology of ARDS. ARDS was created by an intra-tracheal injection of LPS to wild type (WT) mice and VEGFR1 tyrosine kinase deficient mice (TKKO). Compared with WT mice, TKKO mice displayed lower survival rates, increases in lung injury score, total protein concentrations, and pro-inflammatory cytokines including TNF and IL-6 in bronchial alveolar lavage fluids. Alveolar macrophages were diminished in both types of mice after LPS injection. Instead, neutrophils were extensively accumulated, and the number of neutrophils in TKKO mice was higher than that in WT mice. The same was true for macrophages recruited into the lung tissues. VEGFR1 was expressed in alveolar and recruited macrophages. These results suggested that VEGFR1 signaling attenuated LPS-induced acute lung injury by suppressing vascular permeability, cytokines production and neutrophil accumulation.

Angiotensin-converting enzyme 2 (ACE2) is the carboxypeptidase to degrade angiotensin II (Ang II) to angiotensin 1–7 (Ang 1–7) and improves the pathologies of cardiovascular disease and acute respiratory distress syndrome (ARDS) / acute lung injury. B38-CAP is a bacteria-derived ACE2-like carboxypeptidase as potent as human ACE2, recombinant B38-CAP is prepared with E. coli protein expression system more efficiently than recombinant soluble human ACE2. We have demonstrated that B38-CAP ameliorates hypertension, heart failure (Nat Commun. 2020) and SARS-CoV-2-induced lung injury in mice (Nat Commun. 2021). We show therapeutic effects of B38-CAP on abdominal sepsis- or acid aspiration-induced acute lung injury. ACE2 expression was downregulated in the lungs of mice with cecal ligation puncture (CLP)-induced sepsis or acid-induced lung injury thereby leading to upregulation of Ang II levels. Intraperitoneal injection of B38-CAP significantly decreased Ang II levels while upregulated angiotensin 1–7 levels. B38-CAP improved survival rate of the mice under sepsis. B38-CAP suppressed the pathologies of lung inflammation, improved lung dysfunction and downregulated elevated cytokine mRNA levels in the mice with acute lung injury. Thus, systemic treatment with an ACE2-like enzyme might be a potential therapeutic strategy for the patients with severe sepsis or ARDS (PLos One. 2022).

Male sex is more prone to septic death. However, its molecular mechanism remains unknown. Our studies revealed that inflammatory responses in skeletal muscle play a pivotal role in sepsis. Furthermore, recent findings suggested that septic responses in skeletal muscle is a key of the sex differences in septic death. To elucidate if the sex differences in sepsis is from gonadal and sex chromosomal differences, cecal ligation and puncture (CLP)-induced septic symptoms in Four Core Genotypes (FCGs) mice (XX gonadal males or females, and XY gonadal males or females) were investigated in this study. Our survival analysis showed that XX female mice were significantly resistant among FCGs to septic death. Furthermore, our RNA-seq analysis in skeletal muscle of septic FCGs revealed that different activity of inflammatory pathways and four inflammation related genes (Ifi205, Mmp3, Prg4, Saa3) were overexpressed specifically in XX females. In vitro analysis using C2C12 myotubes revealed that estradiol-treatment but not testosterone-treatment enhance mRNA expressions of these genes. Our study suggests the involvement of interactive effects of gonadal and sex chromosomal differences in sex differences in sepsis. Four genes were identified as candidate genes involved in sex difference of sepsis.

Transepithelial/endothelial resistance (TER) measurement is a non-invasive method to assess the integrity of tight junctions in model cells such as the intestinal epithelium and the blood-brain barrier. This technique is essential for experiments on drug kinetics and tissue damage, but commercial devices have many limitations. Additionally, the high-grade analyzers for long-term measurements are prohibitively expensive, raising barriers to entry into this research field. 

The open source-based experimental equipment has advantages such as cost reduction and high versatility. There have been reports of TER instruments using 'open-source way' methods, but some of these have reproducibility problems. 

We propose OpTER, a reproducible, and inexpensive TER measurement method. An Arduino-based measuring circuit can be created for less than 10,000 yen. Our method enables the recording of results equivalent to those of a commercially available product. Along with homemade electrodes made of biocompatible metals, this enables continuous measurement of TER in an incubator.

The circuits and program will be available, and its simple mechanism, which can be assembled by non-experts in electrical engineering, can easily be modified to suit the researcher's objectives. This idea is a new 'OpTion' for both amateurs and professionals.

Immune Checkpoint Inhibitors (ICI) show anti-tumor activity against various types of cancer, but they also disrupt the balance of the immune system and cause autoimmune-like adverse events. ICI-related myocarditis, in particular, has a fatality rate of over 40%, making the development of preventive and therapeutic agents an urgent priority. In present study, we developed a simple and reproducible experimental model for ICI-associated myocarditis.

Myocardial myosin peptide (50 µg) was administered subcutaneously to male, 8-week-old BALB/c wild-type and PD-1KO mice at day 0 and 7. Three weeks after the initial myosin administration, the development of myocarditis was evaluated. HE staining and Masson trichrome staining showed inflammatory cell infiltration and fibrosis in myocardial tissue in myosin peptide-treated PD-1 KO mice. Next, the involvement of CD4⁺ and CD8⁺ cells was examined by immunostaining, and the infiltration of CD4⁺ and CD8⁺ cells was confirmed in the hearts of myosin-treated PD-1KO mice. Finally, the results of real-time PCR showed that myosin administration tended to increase gene expression of inflammatory cytokines and fibrosis markers in the hearts of PD-1KO mice.

It is expected that this model will be used to develop new prophylactic and therapeutic agents for ICI-associated myocarditis.

Tendinopathy is a degenerative disease characterized by rupture, pain or loss of strength at tendon tissues. Multiple intrinsic and extrinsic factors, such as aging and fluoroquinolones, are involved in the development of tendinopathy. However, despite much work, the exact pathophysiological mechanism remains unclear. In this study, we analyzed databases of self-reported adverse events and IBM MarketScan insurance claims database to explore a coexisting drug that reduced the incidence of tendinopathy, and found that dexamethasone prevented fluoroquinolone-induced tendinopathy. In experimental validation of the hypothesis, chronic treatment of pefloxacin to rats caused mechanical fragility and histological changes in tendon, which were both mitigated by the cotreatment of dexamethasone. For its molecular mechanism, in vitro studies revealed that oxidative stress was increased in pefloxacin-treated tenocytes, which was suppressed with the cotreatment of dexamethasone. Also, the increase in the gene expression level of glutathione peroxidase 3 (GPX3) was observed in dexamethasone-treated tenocytes. In fact, the overexpression of GPX3 mitigated pefloxacin-induced oxidative stress in tenocytes. These results suggest that dexamethasone reduces risk of tendinopathy by suppressing oxidative stress through the upregulation of GPX3. This data-driven approach based on clinical evidence will pave the way for the identification of therapeutic target for tendinopathy with high clinical predictability.

Subjective well-being (SWB) has been shown to correlate with longevity and healthy state. Several studies suggested that factors influencing SWB were at least physical activity, cognitive function, and social resource.

The fact that SWB, which is an emotional issue, is influenced by physical activity and cognitive activity, and vice versa SWB affects physical activity and cognitive activity, suggests that the locomotor system and cognitive function are closely related to mental health. However, the molecular basis of these interactions has not been clarified. We hypothesized that some molecules responsible for these interactions circulate the brain and the locomotor system. This clinical study aimed to find molecules responsible for controlling SWB from the blood circulation.

Subjects were healthy elderly people over 65 years old who have no functional troubles in daily life. Evaluation items were SWB, lifestyle, cognitive function (CF), motor function (MF) and daily activity (DA). To elucidate features of elder people with high SWB, subjects were divided by their SWB scores into 4 groups. High SWB was associated with high CF, MF and DA. Comprehensive analysis of responsible molecules in plasma for controlling high SWB are under investigation.

There has been no molecular explanation of why physical activity, cognitive activity, and social activity affect SWB. The present study has the potential to answer the question and to provide a new perspective for health and longevity research with significant impact for medical, psychological, and social sciences.

The epithelial cell adhesion molecule (EpCAM) is a cell surface glycoprotein, which is highly expressed on carcinoma cells. Because EpCAM is involved in cell adhesion, proliferation, survival, stemness, and tumorigenesis, it is thought to be a promising target for cancer diagnosis and therapy. Herein, we developed anti-EpCAM monoclonal antibodies (mAbs) using the Cell-Based Immunization and Screening (CBIS) method. One of the established anti-EpCAM mAb, EpMab-37 (mouse IgG₁, kappa), reacted with EpCAM-overexpressed Chinese hamster ovary-K1 cells (CHO/EpCAM) or a colorectal carcinoma cell line (Caco-2) in flow cytometry. In contrast, EpMab-37 did not react with EpCAM-knocked out Caco-2 (BINDS-16) cells in both flow cytometry and Western blot analysis. EpMab-37 could stain formalin-fixed paraffin-embedded colorectal carcinoma tissues by immunohistochemistry. Furthermore, we converted the subclass of EpMab-37 from mouse IgG₁ into IgG_2a (named as EpMab-37-mG_2a), and further produced a defucosylated version (EpMab-37-mG_2a-f), using FUT8-deficient ExpiCHO-S (BINDS-09) cells. The EpMab-37-mG_2a-f administration significantly suppressed the development of Caco-2 xenograft tumors in mice compared with the control IgG. In contrast, EpMab-37-mG_2a-f did not suppress the development of BINDS-16 xenograft tumors. These results indicated that EpMab-37 is useful for detecting EpCAM in tumors, and EpMab-37-mG_2a-f could contribute to the antibody therapy for EpCAM-positive tumors.

【Background & Aim】 We previously showed that prostaglandin D₂ (PGD₂) promotes allergic reaction by increasing antigen-specific IgE production. In the present study, we investigated the mechanism underlying the promotion of IgE production by PGD₂focusing on the role of its receptor, chemoattractant receptor-homologous molecule on Th2 cells (CRTH2).

【Methods & Results】 We intradermally sensitized wild type (WT) and CRTH2 deficient mice (Crth2^-/-) with ovalbumin (OVA). The serum OVA specific IgE level and the allergic reaction against OVA stimulation were lower in Crth2^-/- than those of WT. Immunostaining of lymph nodes showed that dendritic cells (DCs) expressed PGD₂ synthase. Consistently, bone marrow derived DCs released PGD₂ in response to OVA stimulation in vitro. The OVA-sensitization increased immune cell number in lymph node and Th2 cytokine productions from lymphocytes in WT. CRTH2 deficiency significantly decreased the immune cell number and cytokine productions. We finally revealed that intravenous transplantation of WT DCs but not T cells or B cells restored the serum levels of OVA specific IgE production and allergic reaction in Crth2^-/-.

【Conclusion】 In summary, antigen invasion stimulates PGD₂ production from DCs which promotes Th2 cytokine production in lymph node through CRTH2 signaling. These phenomena result in promoting antigen specific IgE production.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) and stimulator of interferon genes (STING) composes signal pathway which initiates innate immunity. 2'3'-cyclic GMP-AMP (cGAMP) is enzymatically produced by cGAS and activates several signaling pathway through binding to STING. Accumulating evidence indicates that cGAS-STING signaling play an important role in cancer, inflammatory disease, and senescence. Advanced glycation end products (AGEs) are biologically reactive compounds produced by prolonged exposure of proteins to carbonyl compounds. Chemical and physiological properties of AGEs depend on type of carbonyl compound. Accumulation of AGEs are observed in organs and tissues according to aging and leads to induction of proinflammatory effect. Therefore, AGEs are associated with the development of inflammatory and age-related diseases. However, relationship between cGAS-STING signal and AGEs remains unclear. In the present study, we investigate the effect of different types of AGEs on STING signal in macrophage. In THP-1 cells which is a human monocytic leukemia cell line, cGAMP transfection increased phosphorylation of TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3), resulting in upregulation of IFNβ and CXCL10. Glycolaldehyde-derived AGEs dose-dependently suppressed cGAMP-induced the phosphorylation of TBK1 and IRF3. In contrast, ribose-derived AGEs enhanced the phosphorylation of TBK1 and IRF3. These results may suggest that different types of AGEs contribute to the regulation of STING signal in macrophage.

Myeloid-derived suppressor cells (MDSC) are heterogeneous population of immature myeloid cells, that support tumor growth, by reducing T cell activity. Therefore, drugs which can inhibit MDSC are new predictive immunotherapeutic medicines. We have previously shown that Kampo medicines, Ninjinyoeito (NYT) and Juzentaihoto (JTT), suppress the differentiation of MDSC. In the tumor-bearing state, MDSC migrate to the tumor microenvironment (TME). In the present study, we have investigated the effects of NYT and JTT on the migration to TME. MDSC were isolated from C57BL/6J mice and differentiated into MDSC by the treatment with IL-6 and GM-CSF, after which the migration activity was assessed with transwell assay. The migration of MDSC was stimulated by treatment of 4T1 cancer cells or 4T1-conditioned media, NYT and JTT significantly inhibited the migration. Inaddition, NYT inhibits the phosphorylation of ERK1/2 in MDSC induced by 4T1-conditioned media. Furthermore, NYT considerably suppressed the expression of CCR2 in MDSC. These data indicated that, NYT and JTT suppress not only differentiation, but also the migration of MDSC to TME. This multi-step approach in cancer treatment may be important in the immunomodulatory effects of these Kampo medicines.

Recent advances in single cell technology now allow researchers to simultaneously profile the transcriptional program and chromatin accessibility from individual cells, providing access to both the characterization of cell types and states, and the exploration of gene regulatory programs at the same time and in the same cells.

Here, we present an analysis of single cell transcriptomes and chromatin accessibility profiles in PBMCs from a group of SARS-COV-2 infected subjects with a range of disease severities. We were able to identify several immune cell types at a coarse level based on transcriptomic profiles. By additional subclustering, we found 4 clusters of CD8T cells which show differential distribution across COVID-19 severities. Analysis of DEGs revealed higher expression of genes associated with CD8 T cell terminal differentiation and effector functions in the cluster enriched in mild patients. Chromatin accessibility analysis of the selected DEGs in CD8T cells confirms higher accessibility in patients with mild disease vs severe patients. Interestingly, the transcripion factor ZEB2 was identified as one of the top markers of the mild-severity cluster. Further motif analysis will clarify the importance of this TF in CD8T cell differentiation and outcome in SARS-COV-2 infected subjects.

【Background and Purpose】Feline mammary tumor is known to be the third most common tumors in cats and high grade tumors but more effective treatment has not been established. The culture method of organoids is also familiar with a method which can maintain the epithelial tissue structures and the characteristic like stem cell by three-dimensional culture using a special culture medium. In previous study, we created the feline mammary tumor organoids from tissue extracted patients and revealed the suitable culture condition. Then, we compared original tissue and organoid, and clarified the relation with the pathological characteristics or the expressions of hormone receptor, the differences of anti-cancer drug sensitivity in each case. In this study, we compared the gene expression of feline mammary tumor organoids with normal mammary organoids and searched for new therapeutic targets.

【Method】RNA was extracted from organoids produced from normal mammary glands 3 cases and feline mammary tumor 6 cases, and RNA sequencing was performed using next generation sequencer.

【Result】In RNA sequencing, there were 112 significant differentially genes. 81 genes were markedly upregulated in feline mammary tumor, and 31 genes were in normal mammary glands. In addition, the expression of X genes related with estrogen receptor was significantly upregulated in feline mammary tumor. In GESA analysis, the pathways related to apoptosis, epithelial-mesenchymal transition, and estrogen were activated.

【Conclusion】In feline mammary tumor and normal mammary glands, genetic differences  was cleared and a strong association with estrogen was observed. We will plan to analysis the relationship between the gene identified in this study and feline mammary tumor, and the metabolite activity by metabolome analysis using feline serum, search for biomarkers lead to more earlier diagnosis.

Oxytocin is a peptide hormone known for its strong central actions regulating a variety of behaviors including parenthood and social bonding to others, in addition to the long-known peripheral actions. Despite increasing realization of its importance, dynamics and sites of action of oxytocin in the brain are poorly understood due to a lack of appropriate probe; it is too small to be tagged with bulky fluorophores for visualization. Therefore, to overcome the current technical limitation and to facilitate the understandings of oxytocin's action in the brain, we tried to develop and apply a new probe. To this end, we conjugated oxytocin with "alkyne-tag" via a widely applicable simple coupling reaction. The alkyne-tag is far smaller than oxytocin, so it is expected that its tagging will be possible without significantly changing the original properties of oxytocin molecule. After incubation with the living brain tissues where alkyne-oxytocin behaves similarly to endogenous oxytocin, the tagged-oxytocin can be specifically visualized by a click chemistry reaction. Using this probe, we conducted various experiments to characterize the spatiotemporal dynamics of oxytocin in brain tissues. Here, I will introduce our novel strategy and findings brought by this probe including the region-specific binding sites and dynamics of oxytocin.

【Background】

Idiopathic Basal Ganglia Calcification (IBGC) is a progressive neurodegenerative disease characterized by calcification of the basal ganglia and cerebellar dentate nucleus. Several gene mutations of IBGC are reported, including SLC20A2 which are involved in phosphate transport. IBGC is thought to be caused by abnormal phosphate transport. However, its mechanism is not yet clear in central nervous system. Therefore, we elucidate the regulation of phosphate transport in neuronal cells, focusing on PDGF-BB, which is one of the causative gene of IBGC and activates phosphate transport in vascular smooth muscle cells.

【Methods】

The effect of PDGF-BB on phosphate uptake was evaluated with SLC20A2-KD (knockdown) SH-SY5Y cells, which is the human neuroblastoma cell type. To determine whether the effect of PDGF-BB is mediated by SLC20A1 or SLC20A2, knockdown assays were conducted. To investigate the mechanism of PDGF-BB on the phosphate uptake, the expression and membrane translocation of phosphate transporters, PiT1 and PiT2 (encoded by SLC20A1 or SLC20A2) was evaluated when PDGF-BB was treated.

【Results】

PDGF-BB enhanced the phosphate uptake by SLC20A1, not SLC20A2. Interestingly, the activation of phosphate uptake by PDGF-BB was not due to increase in the expression of phosphate transporters but to activation of membrane translocation. The activation of membrane translocation by PDGF-BB was canceled when Akt inhibitor was treated.

【Conclusion】

PDGF-BB enhanced the phosphate uptake by the membrane translocation of PiT1, the mechanism of which is thought to be Akt signaling.

We propose that L-DOPA by itself is a neurotransmitter. Recently, GPR143 was identified as an L-DOPA receptor. We previously showed non-effective dose of L-DOPA potentiates behavioral response to quinpirole, a dopamine D2 receptor (D2R) agonist. However, it remains undetermined whether and how GPR143 regulates D2R-mediated behaviors. We analyzed behavioral responses to several D2R ligands using Gpr143 gene-deficient (GPR143-KO) mice. We found that haloperidol, a D2R antagonist (0.5mg/kg)-induced catalepsy was attenuated in GPR143-KO mice compared to wild-type (WT) mice. To clarify which neural circuits that are responsible for this phenotype, we investigated haloperidol-induced catalepsy using conditional KO mice that expressing cre recombinase in D2R-, adenosine A2A receptor (indirect pathway)-, and in choline acetyltransferase (cholinergic interneuron)-positive neurons. Haloperidol-induced catalepsy was attenuated in D2R-cre (+); Gpr143^flox/y and ChAT-cre; Gpr143^flox/y mice. Furthermore, we found that a synthetic peptide, which inhibited the interaction between GPR143 and D2R, attenuated haloperidol-induced catalepsy. These results suggest that GPR143 expressed in the striatal cholinergic interneurons modulates haloperidol-induced extrapyramidal symptoms through coupling GPR143 and D2R.

Expansion of CAG and CTG (CWG) triplet repeats is causal in a number of inherited neurological diseases. The CWG triplet repeat diseases can be broadly classified into coding and non-coding types based on their location in the genome, such as Huntington's disease (HD) by expanded CAG repeat in a coding region and myotonic dystrophy type-1 (DM1) by expanded CTG repeat in a 3'-untranslated region. The CWG repeat diseases are thought to induce complex pathogenic mechanisms through expanded CWG repeat-derived RNAs and polypeptides. Here we show a CWG repeat DNA targeting compound, cyclic Pyrrole-Imidazole Polyamide (CWG-cPIP) suppresses the pathogenesis of coding and non-coding CWG repeat diseases. CWG-cPIP binds to hairpin form of the mismatched CWG DNA, interfering with transcriptional elongation of RNA polymerase in a repeat length-dependent manner. CWG-cPIP inhibits pathogenic mRNA transcripts from expanded CWG repeat, result in reduction of CUG RNA foci and polyglutamine accumulations in DM1 and HD patient cells and mouse models, respectively. Treatment with CWG-cPIP also ameliorates learning and memory deficits and synaptic dysfunction in DM1 and HD mouse models with less off-target effects. Taken together, we present a novel candidate compound that targets expanded CWG repeat DNA independent of genomic location, demonstrating the concept of reducing the levels of pathogenic RNAs and proteins.

Spinal cord injury (SCI) is a devastating trauma that results in a severe disability and irreversible motor and sensory dysfunction, whereas efficient therapies have not been fully developed. After SCI, astrocytes are the predominant cellular component that proliferates around the lesion core and contributes to the glial scar formation, which has long been considered one of the primary causes of spinal cord regeneration failure. However, the molecular mechanisms underlying the proliferation of astrocytes in response to central nervous system (CNS) injury remain unclear. In this study, we found that heterogeneous nuclear ribonucleoprotein U (Hnrnpu), a DNA/RNA binding protein, regulated astrocyte proliferation after SCI. siRNA-mediated knockdown of Hnrnpu suppressed the primary astrocyte proliferation without affecting the cell viability in vitro. Moreover, in vivo, inhibition of Hnrnpu expression by intraspinal injection of AAV5-Hnrnpu shRNA under the control of the astrocytic glial fibrillary acidic protein (GFAP) promoter inhibited astrocyte proliferation, increased lesion size, and suppressed functional recovery of mice after SCI. Taken together, Hnrnpu exerts a crucial role in astrocyte proliferation, where its changes would be regarded as a hallmark of CNS diseases and injuries in which astrocytes are involved.

Microglia arise from embryonic yolk sac and colonize the brain parenchyma, followed by the maturation of microglia-specific gene expression. Single-cell transcriptional analysis has revealed that microglia are transcriptionally heterogeneous. However, the environmental factors that induce microglial heterogeneity are largely unknown. Here we found that developmental intracerebral hemorrhage induces microglial heterogeneity. In neonatal mice, we found that a portion of microglia phagocytose the red blood cells (RBCs) and expressed Hmox1, which encodes Heme Oxygenase 1, significantly higher than non-RBC-phagocytic microglia. To examine the effect of RBC phagocytosis on the transcriptional property of microglia, we labeled Hmox1-expressing microglia with red fluorescent protein by developing a transgenic mouse line, finding that microglia that underwent RBC phagocytosis expressed genes typical for yolk sac microglia in the second postnatal week. Thus, this study reveals that neonatal environmental factors induce microglial heterogeneity.

In behavioral addiction, addicts repeat certain behaviors, such as internet gaming and gambling, despite negative consequences. No cure has been established due to the lack of animal models to explore its neuronal mechanisms. Here, by focusing on voluntary wheel-running in rodents, we have developed a novel operant conditioning task as a model of behavioral addiction, in which male C57BL/6J mice (> 7 weeks old) were allowed for wheel-running after certain numbers of nose pokes. In this task, the magnitude of motivation for wheel-running is quantifiable by evaluating the number of nose pokes.

We first measured dopamine (DA) release using fiber photometry with GRAB-DA sensors and found that DA was increased in the nucleus accumbens (NAc) immediately after nose-poking. Systemic administration of antagonists for DA D1 receptor (SCH23390; 0.025–0.1 mg/kg), D2 receptor (raclopride; 0.1–0.6 mg/kg), or an agonist for adenosine A2A receptor (CGS21680; 0.05-0.1 mg/kg) dose-dependently decreased the number of nose pokes. Intra-NAc infusion of these drugs (SCH23390; 0.05 µg/side, raclopride; 0.3 µg/side, CGS21680; 1 ng/side) also reduced the number of nose pokes. Additionally, systemic administration of serotonin (5-HT)2A receptor antagonist (volinanserin; 0.01–0.1 mg/kg) or 5-HT2C receptor antagonist (SB242084; 0.3–1.0 mg/kg) dose-dependently decreased the number of nose pokes. These results suggest that neurotransmission via D1, D2, and A2A receptors in the NAc and 5-HT2A and 2C receptors are involved in the motivation for wheel-running.

Alexander disease (AxD) is an intractable neurodegenerative disorder caused by GFAP mutations. AxD astrocytes show several abnormal phenotypes. Our previous study has shown that astrocytes in AxD model mice show aberrant Ca²⁺ signal that was a cause of etiology of AxD. In addition, using 2 photon imaging and Iba1-GCaMP6-60TM, an AxD model for microglial Ca²⁺ imaging, we recently found that microglial Ca²⁺ signals were also dramatically enhanced in the AxD model with more frequent Ca²⁺ signals in both the processes and cell bodies. Such increases in Ca²⁺ signals were inhibited by P2Y₁₂R antagonist but not by TTX, suggesting that these enhancement should be independent of neuronal activity, but dependent on extracellular ATP-mediated signals. Thus, we hypothesized that these microglial abnormal Ca²⁺ signals would be caused by increase of ATP amount released from astrocytes. Our analysis data of scRNAseq suggested that some astrocyte subclusters unique to the AxD model exhibit the lower expression level of the gene of astrocyte-specific ectonucleotidase subtype. In in situ ATP imaging using in vivo injection of AAV GfaABC1D ATP1.0, the signals of locally puffed ATP persisted longer in acute slice in AxD model than control WT mice, indicating a delay of ATP degradation in AxD brain that could cause the hyperactive Ca²⁺ signals in microglia. To study if these P2Y₁₂R-mediated Ca²⁺ signals in AxD microglia play any significant roles in the mechanism of pathology, P2Y₁₂R antagonist was administered. AxD model with treatment of P2Y₁₂R antagonist showed an exacerbation of pathological markers. This suggested that microglia play a protective role in AxD pathology via P2Y₁₂R. Our findings hold promise for the future development of therapies based on microglial manipulation.

Fluoxetine is a selective serotonin reuptake inhibitor (SSRI) and exerts antidepressant effects, which has been considered to be mediated by increasing serotonin concentration in the inter-synaptic cleft. However, the monoamine hypothesis has not been fully supported as there is a time lag between transient increases in brain serotonin levels after SSRI treatment and the expression of antidepressant effects. The temporal inconsistency may be partly reconciled by memory mechanisms, especially in the hippocampus. A theory of memory suggests that learned memory needs to be consolidated into neuronal circuits by repeated reactivation of memory-encoding neuronal activity. Our previous study demonstrated that the inhibition of the ventral hippocampus after stress experiences inhibits subsequent depression-like behavior in mice. Especially, we found that sharp wave ripples (SWRs), which represent synchronized neuronal spikes in the ventral hippocampus, are a primary neuronal activity pattern to mediate this effect. Here, we tested whether fluoxetine affects SWRs in the ventral hippocampus in resting and depression model mice and found that fluoxetine administration significantly reduced ventral hippocampal SWRs in both resting mice and mice that received social defeat stress. These results demonstrate that fluoxetine inhibits SWR-induced neuronal reactivation, suggesting an antidepressant effect mediated by suppressing memory consolidation.

We have recently reported that excitatory neurons in the claustrum mediate anxiety responses to acute psychological stressors that induce negative emotional states. However, it is unclear how claustral neurons represent information related to anxiety responses to a stressor. To address this question, here we performed calcium imaging of GCaMP6f-expressing claustral neurons in freely moving mice during three behavioral tests; the elevated plus maze, the open field test, and a second open field test after an exposure to a ten-minute single social defeat stress. Prior to exposure to a stressor, we found that a subset of claustral neurons displayed an increase in calcium levels upon transitioning to areas associated with increased anxiety in the elevated plus maze and the open field. In the open field test after exposure to social defeat stress, a different subset of neurons, including neurons that were activated by of stress, exhibited sustained high levels of calcium when entering and exiting the less anxiogenic corner zones of the open field. These results suggest that stress-related anxiety information is represented in a claustral neuronal population that is different from the population representing anxiety under non-stressed conditions.

Hypertensive disorder of pregnancy (HDP) affects about 10% of pregnant women, which may be caused by dysfunction of placental trophoblasts following impaired uterine spiral arterioles. Our previous analysis revealed that endoplasmic reticulum (ER) stress signaling may be altered in HDP, compared to normal pregnancy. In addition, the expression of high-temperature requirement A1 (HTRA1), a serine protease was decreased in the HDP placenta. However, a role of ER stress and HTRAs in HDP pathophysiology remains unknown. The relationship between of ER stress and HTRAs was explored in vitro human trophoblast cells. Treatment with ER stress inducers thapsigargin or tunicamycin increased the expression of HTRA1 and its subtype HTRA3, but did not alter HTRA2 and HTRA4 in trophoblasts. In vitro invasion assay revealed that either thapsigargin or tunicamycin treatment and the knockdown of HTRA1 or HTRA3 inhibited trophoblast invasion. In addition, ER stress inducers or knockdown of HTRA1 altered the ratio of soluble fms-like tyrosine kinase-1 (sFLT1) to placental growth factor (PGF), a severity index of HDP. The expression of HTRA1 was lower in HDP placenta compared to the normal placenta tissues. These results suggest that ER stress may regulate trophoblast invasion partly via HTRA1 and HTRA3 and is involved in the pathogenic mechanism of HDP. It might be possible to develop a therapeutic means that targets HTRA1 to improve pregnancy complications such as HDP.

Antidepressants are known to cause hyponatremia, but conflicting evidence exists regarding specific antidepressants. To identify antidepressants less likely to cause hyponatremia, we conducted a triangulation study integrating retrospective cohort, disproportionality, and pharmacodynamic studies. In the retrospective cohort study, a significant decrease in serum sodium levels was observed for selective serotonin reuptake inhibitors (SSRIs) and serotonin-noradrenaline reuptake inhibitors (SNRIs), whereas no decrease was found for a noradrenergic and specific serotonergic antidepressant (mirtazapine). Within-class comparison revealed no decrease in serum sodium levels for fluvoxamine among SSRIs and milnacipran among SNRIs. In the disproportionality analysis, a significant increase in hyponatremia reports was observed for SSRIs and SNRIs, but not for mirtazapine, fluvoxamine, and milnacipran. Finally, pharmacoepidemiological–pharmacodynamic analysis revealed a significant correlation between the decrease in serum sodium levels and binding affinity for serotonin transporter (SERT), suggesting that lower binding affinity of mirtazapine, fluvoxamine, and milnacipran against SERT is responsible for the above difference. These data suggest that mirtazapine, fluvoxamine, and milnacipran are less likely to cause hyponatremia.

Opioid analgesics are widely used to manage moderate to severe pain in cancer patients. The μ opioid receptor (MOR) is expressed in not only the brain and spinal cord, but also a wide range of peripheral sites, including the gastrointestinal tract and immune cells. It has been considered that the central µ-opioidergic system is closely associated with analgesia and euphoric effects, whereas the peripheral µ-opioidergic system is responsible for side effects such as constipation and nausea. However, little is known about the role of the endogenous µ-opioidergic system in the control of the innate immune response. In this study, we investigated the functional role of central and peripheral µ-opioidergic systems in tumor progression using a pharmacological and genetics approach. First, we found that treatment with peripheral MOR antagonists significantly decreased Lewis lung carcinoma (LLC)-graft compared to that in control mice. On the other hand, activation of the hypothalamic µ-opioidergic system using the Gq-DREADD technique significantly decreased the tumor volume compared to that in control mice. Taken together, these findings suggest that the central and peripheral µ-opioidergic systems may play a bidirectional role in the control of tumor progression.

Pain transmission in the spinal dorsal horn (SDH) is regulated by descending neuronal pathways from the brain, such as noradrenergic (NAergic) neurons from the locus coeruleus. While it is known that spinal NA produces an antinociceptive effect, we have recently shown that NA has an ability to produce pain hypersensitivity via Hes5-expressing SDH astrocytes. However, the mechanism underlying the bidirectional effect of spinal NA remains unknown. In this study, we showed that while intrathecal injection of NA at a low dose (NA^low) induced pain hypersensitivity via α_1A-adrenergic receptors (α_1A-ARs) in Hes5⁺ astrocytes, the hypersensitivity was not observed by intrathecal high-dose NA (NA^high). The effect of NA^high was also mediated by activation of inhibitory interneurons via α_1A-ARs. We found that NA^high also activated β₁-ARs in astrocytes that suppressed the astrocytic α_1A-ARs-mediated effect. However, if α_1A-ARs are expressed in inhibitory interneurons, why does NA^low produce pain hypersensitivity? We further found that activation of astrocytic α_1A-ARs increased release of adenosine, a factor that suppresses inhibitory interneurons. Therefore, our findings indicate that NA bidirectionally modulates pain transmission via astrocyte-neuron interactions in a concentration-dependent manner.

Fatty acid-binding protein (FABP) regulates polyunsaturated fatty acids intracellular trafficking and functions as a signal transduction via modulation of gene expression. We have demonstrated that FABP3 protein was observed in microglia of the median eminence (ME) of hypothalamus and this protein was increased in the ME of pain model mice. These changes were correlated with the increment of hypothalamic docosahexaenoic acid (DHA) levels. Here, we assessed the effect of DHA on FABP3 expression using MG6 cell, a microglia cell line. Also, we tested the effect of FABP inhibitor on the mechanical allodynia in postoperative pain model mice. MG6 cells were cultured in Dulbecco's modified eagle medium with or without 10% fetal bovine serum (FBS) as a cell stress. FABP3 was measured by qPCR. Mechanical allodynia was assessed by von Frey test. FABP3 mRNA was expressed on the MG6 cell. Under the condition of serum-free media, FABP3 mRNA was also significantly increased compared to the media with 10% FBS. This increment was suppressed by DHA (300 μM). Repeated intraventricular injection of FABP inhibitor was significantly suppressed mechanical allodynia in postoperative pain mice. These results indicated that DHA might be involved in the regulation of microglial FABP3, and brain FABP might work as a regulator of pain.

Parkinson's disease (PD) is one of the most prevalent neurodegenerative diseases and disease-modifying treatment is required to inhibit the disease progression. We have previously reported that 8-OH-DPAT, serotonin (5-HT)1A full agonist, induced expression of antioxidant metallothionein (MT) in astrocyte and exhibited protective effects against 6-OHDA-induced neurodegeneration of nigrostriatal dopamine (DA) neuron. In this study, we investigated neuroprotective effect of anti-depressant mirtazapine, as an indirect 5-HT1A agonist, against dopaminergic neuronal death. Mirtazapine administration to 6-OHDA-injected hemi-parkinsonian mice significantly increased MT expression in striatal astrocytes and inhibited the reduction of nigrostriatal DA neuron. These effects were cancelled by simultaneous administration of 5-HT1A antagonist. To explore the precise neuroprotective mechanism, we examined effects of mirtazapine using primary cultured mesencephalic neurons and striatal astrocytes from rat fetus. Neuroprotection by mirtazapine was observed only in neuron-astrocyte co-cultured condition. Furthermore, MT expression in astrocyte was significantly increased when astrocytes were treated with mirtazapine-pretreated neuronal conditioned medium (Mir-NCM). Treatment with medium from Mir-NCM-treated astrocytes (Mir-NCM-ACM) showed dopaminergic protection against 6-OHDA. These effects were cancelled when astrocytes were treated Mir-NCM and 5-HT1A antagonist. Moreover, MT antibody completely cancelled the neuroprotective effects of Mir-NCM-ACM. These results suggested that mirtazapine protected DA neurons by inducing expression and secretion in/from astrocytes via indirect stimulation on astrocytic 5-HT1A receptor.

Osteoarthritis (OA) is a common joint disease characterized by the breakdown of subchondral bone and cartilage damage, most often affecting middle-aged and elderly people. Although the etiology of OA is still unknown, some reports suggest that inflammatory factors such as interleukin (IL)-1β mediate the progression of OA. In order to investigate the effect of IL-1β and the possibility of treatment for OA, we used 2-carba-cyclic phosphatidic acid (2ccPA) and its derivatives on human chondrocytes. 2ccPA is a synthesized phospholipid based from a bioactive phospholipid mediator: cyclic phosphatidic acid (cPA). It is previously reported that 2ccPA exhibits anti-inflammatory and chondroprotective effects on an OA animal model. 2ccPA and its ring-opened body (ROB) derivative significantly suppressed IL-1β-induced upregulation of IL-6, matrix metalloproteinase-13, and cyclooxygenase-2, as well as the degradation of type II collagen and aggrecan. However, the other two derivatives, the deacylated body and the ring-opened deacylated body showed little effect on IL-1β-exposed human chondrosarcoma cell-line. These data suggest that acyl chain of 2ccPA and ROB is essential for anti-inflammatory effect on OA. Taken together, this study provides evidence that 2ccPA and ROB would be a novel therapeutic agent for OA.

Cannabidiol (CBD) is the second major cannabinoid which is often said to improveanxiety and sleep with no psychoactive effect. It is said that CBD can promotethe production of wake-related neurotransmitters such as dopamine. Further,co-injection of CBD and THC increased sleeping time in rats. However, no studyhas been done for the in-vivo effect of CBD to circadian clock. Present studyaimed to elucidate whether CBD can modify peripheral circadian rhythm.

PER2::LUC knock-in female mice were used to determine the effect of CBD to theperipheral clock. Mice were divided into six groups: CBD isolate (99%crystalline) in MCT oil, CBD isolate in 5% EtOH/5% cremophor/water, andwater-soluble CBD nanopowder in water, with respective vehicle controls. Eachdrug was orally administered at ZT4 or ZT16 for three days, then PER2 geneexpression in the liver, kidney, and submandibular gland is observed by in-vivoimaging.

We found that phase advance in the liver and the submandibular gland only happenswhen MCT or CBD in MCT (CBD/MCT) was administered at ZT4. However, the phaseadvance did not differ between the MCT and CBD/MCT groups. Furthermore, neitherthe vehicle nor the CBD affected the peripheral clock when administered atZT16. 

This study suggested that not CBD but rather MCT oil affect the circadian clockin mice. As MCT oil is commonly used as a base for CBD products, we propose thatMCT might be the possible factor that affects the circadian clock and causerhythm improvement. Therefore further detailed studies on the effect of CBDproducts will be needed.

L-Theanine (LTE) is a derivative of glutamic acid, which is abundant in tea leaves and contributes to the umami and sweetness of tea. In recent years, its effects on the brain, such as relaxation, have been attracting attention. In this study, we used imaging mass spectrometry (IMS) to visually analyze the changes in the catecholamine system in the brain after the administration of LTE. In IMS, we applied derivatization reagent to improve detection limit. Simultaneous imaging of catecholamines, LTE and g-aminobutyric acid (GABA) is particularly useful to understand a metabolic pathway. We investigated whether symptom of depression is improved or not by free drinking of LTE water. The mice that are freely drinking of theanine (Group 1), symptoms of depression was milder than that of drinking of water (Group 2). IMS showed dopamine (DA) marginally produced from caudate putamen from Group 2, but DA was produced from the Group 1. Interestingly, GABA increased at hypothalamus nucleus paraventricularis (PVN), which controls eating amount, from Group 1 compared with Group 2. We hypothesized an increasing of GABA at PVN works an appetite stimulation. Practically, an appetite of mouse in Group 1 was not decreased compared with that in Group 2. IMS visually gives us the pathway of catechol amine and improvement of depression by theanine.

【Background】

Skeletal muscles are composed of slow-twitch fiber and fast-twitch fiber. Transcriptional factor Tead induces muscle differentiation with its cofactor Taz. However, whether Tead controls slow- and fast-twitch fiber is unknown. Thus, we focused on Vgll3, another cofactor of Tead, and investigated its role in muscle differentiation. 

【Result】

We established Vgll3 expressing C2C12 cells and differentiated these cells. Vgll3 expressing cells showed upregulation of muscle differentiation markers and an increase in slow-twitch fiber marker Myh7 with a decrease in fast-twitch fiber marker Myh4. These results suggest that Vgll3 increases slow-twitch fiber.

Recent studies reported Taz activation induces Vgll3 expression. Thus, we used the Lats1/2 inhibitor, which induces Taz activation. Lats1/2 inhibitor induces Vgll3 expression and upregulation of Myh7 and Myh4. This result suggests that Lats1/2 inhibition induces Taz activation to increase fast-twitch fiber and Vgll3 expression to increase slow-twitch fiber simultaneously.

【Conclusion】

Our results suggested that Vgll3 increases slow-twitch fiber, and Lats1/2 inhibition increase slow- and fast-twitch fiber via activation of Vgll3 and Taz. Aging significantly decreases fast-twitch fiber and causes older adults injury. Thus, Tead and its cofactors are important for aging treatment.

【Background, Purpose】

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, irreversible, and ultimately fatal lung disease. IPF occurs due to TGF-β1/Smad signaling activation.

Previously, we observed that eliglustat, glucosylceramide synthase inhibitor exhibited anti-fibrotic effects against nomal human lung fibroblast. So, we checked whether eliglustat exhibits anti-fibrotic effects also against IPF patient-derived cells (IPF cells), and tried to elucidate its mechanism.

【Results, Discussion】

First, we tested the anti-fibrotic effects of eliglustat in IPF cells. Treatment of IPF cells with eliglustat similarly suppressed the up-regulation of fibrotic proteins such as α-SMA and collagen by TGF-β1. Eliglustat had no effects on phosphorylation and translocation to the nucleus of Smad. The knockdown of glucosylceramide synthase did not inhibit the up-regulation of α-SMA by TGF-β1. These results suggest that eliglustat inhibits fibrotic protein transcription by Smad independently of its inhibitory effect against glucosylceramide synthase. Next, we focused on sterol regulatory element-binding protein2 (SREBP2) to elucidate the anti-fibrotic mechanism of eliglustat. SREBP2 regulates intracellular cholesterol levels and is known to inhibit the transcript activity of Smad. Treatment of IPF cells with eliglustat induced translocation of SREBP2 to the nucleus and up-regulation of downstream genes of SREBP2. Inhibition of SREBP2 attenuated the eliglustat-induced down-regulation of α-SMA expression. These results suggest that eliglustat exhibits anti-fibrotic effects through activation of SREBP2.

P2Y1 receptor is upregulated in astrocytes in many neurological diseases. We have previously shown that elevated P2Y1 receptor expression in astrocytes causes neuronal hyperexcitability by enhancing neuron-astrocyte communication in the hippocampal CA1 region. However, contribution of microglia to the astrocytic P2Y1 receptor-mediated neuron-astrocyte communication is not known, despite the fact that microglia are also activated in such pathological conditions. To this end, we attempted to investigate the role of microglia in astrocyte P2Y1 receptor signaling by depleting microglia with a CSF1 receptor antagonist, PLX5622. The results are summarized in the following two points: 1) Microglia depletion increased P2ry1 gene expression in astrocytes and enhanced Ca²⁺ signal via P2Y1 receptor, indicating that microglia would have a role to inhibit P2Y1 receptor expression in astrocytes. 2) Microglia depletion prolonged the time required for degradation of exogenously applied ATP. Because microglia highly express an ATP degrading enzyme CD39, they would play a central role in shutting-off of P2Y1 receptor signals by metabolizing ATP. Taken together, it is suggested that microglia would also play an important role in neuron-astrocyte communication via 2 different modes, i.e., inhibition of P2Y1 receptor expression and degradation of extracellular ATP.

Because the expression of up to 10% of genes is under the control of the circadian machinery consisting of clock genes, it should not come as a surprise that the disfunction of clock gene affects the onset and/or state of various disease. Diurnal variations in pain hypersensitivity are common in chronic pain disorders, but pathological relevance of clock genes in neuropathic pain hypersensitivity remains unknown. In this study, we investigated the threshold of mechanical pain hypersensitivity in peripheral sciatic nerve-ligated (PSL) animals and found that clock gene deficient mice (Per2^m/m mice) failed to develop the neuropathic pain hypersensitivity. As observed in wild-type mice, PSL- Per2^m/m mice also activation of glial cells in the dorsal horn of the spinal cord, as well as increased expression of pain-related molecules. On the other hand, the descending pain suppressor system and endocannabinoid system were upregulated in Per2^m/m mice, suggesting that the suppression mechanism against neuropathic pain is enhanced by dysfunction of clock gene. Therefore, Per2^m/m mice are less likely to develop pain hypersensitivity even when peripheral nerves are injured. These findings indicate that endogenous pain suppression system are under the control of circadian clock. Identification of circadian clock controlled pain suppressor molecule would be a therapeutic target for treatment of neuropathic pain.

Pain information transmission/processing in the spinal dorsal horn (SDH) is strongly controlled by descending neurons from the brain. One of the major neurotransmitters of descending pathways is noradrenaline (NA). Descending NAergic neurons from the locus coeruleus (LC) is known to produce analgesic effects via activation of inhibitory interneurons in the SDH. However, the identity of the inhibitory interneuron subset in the SDH is poorly understood. Recently, we have found a subset of the SDH inhibitory interneurons captured by adeno-associated viral (AAV) vectors incorporating a neuropeptide Y promoter (AAV-NpyP⁺) that is crucial for neuropathic allodynia. Here, we showed that this neuronal subset is a major target of spinal NA to inhibit pain information transmission/processing. Whole-cell patch-clamp recordings using spinal cord slices revealed that NA predominantly depolarizes AAV-NpyP⁺ neurons. This effect was suppressed by a pharmacological blockade and genetic knockdown of α_1B-adrenoceptor (AR) in AAV-NpyP⁺ neurons in the SDH. Furthermore, we found that the analgesic effect of duloxetine on neuropathic pain which is associated with an increase in the spinal NA level by inhibiting NA reuptake into presynaptic terminals is reduced by AAV-NpyP⁺ neuron-selective knockdown of α_1B-ARs. These results indicate that α_1B-ARs expressed in AAV-NpyP⁺ neurons would be a target of spinal NA presumably from descending LC neurons and contribute to the analgesic effect of duloxetine. Thus, spinal α_1B-ARs could be a new therapeutic target.

Purpose: Paclitaxel (PTX) is a typical anticancer drug that induces peripheral neuropathy and significantly reduces patients' quality of life. Previous our study showed gabapentinoids, such as mirogabalin, attenuate PTX-induced peripheral neuropathic pain by acting on the spinal dorsal horn. PTX-induced peripheral neuropathy has so far focused on peripheral inflammation, but it is possible that changes in the spinal dorsal horn may contribute to PTX-induced peripheral neuropathy. In this study, we investigated the mechanism of synaptic plasticity in the spinal dorsal horn by using electrophysiological and immunohistochemical analysis.

Methods: We administered a single intraperitoneal dose of PTX 5 mg/kg to C57BL/6NCr mice.　We analyzed the frequency of spontaneous and von Frey filament (vFF; 0.69 mN) evoked firing in spinal dorsal horn neurons by using in vivo extracellular recording. Immunohistochemical staining was performed on spinal cord (L4-6) slices.

Results: Electrophsiological data showed the frequency of spontaneous and vFF evoked firing in spinal dorsal horn neurons were significantly enhanced in PTX model mice.

The levels of the neuronal activation marker c-fos were increased with mechanical allodynia formation in PTX model mice. Glia-associated makers Iba1 and GFAP also showed chronological changes after PTX-treatment.　

Conclusion: The present study suggests that synaptic plastic changes occur not only in the periphery but also in the spinal dorsal horn in the PTX-induced peripheral neuropathic pain model.

Osteoarthritis (OA) is characterized by pain caused by inflammation and degradation of cartilage matrix in joint. Although the number of patients is expected to increase due to the aging of the population, the use of anti-inflammatory drugs, which is the main therapeutics, may not be effective in pathology of OA. REV-ERBs are one of nuclear receptors involved in a wide range of physiological functions. We have previously shown that REV-ERBs were expressed in primary cultured chondrocytes, and REV-ERB agonist suppresses the upregulation of proinflammatory cytokines and matrix degradation enzymes in these cells under inflammatory conditions. However, the role of REV-ERBs in pathogenesis of OA is not clear. Thus, we investigated the effect of REV-ERB agonist on nociceptive hypersensitivity in monoiodoacetate (MIA)-induced OA model. SR9009, a REV-ERB agonist, was administered intraarticularly twice a week, starting 3 days after MIA administration. Mechanical thresholds were measured by the von Frey test. MIA induced mechanical hypersensitivity from day 3 after administration, which persisted at least until day 28. Administration of SR9009 significantly ameliorated mechanical hypersensitivity from day 14 after MIA administration. These results suggest that activation of REV-ERB might induce an analgesic effect on OA pain.

Monitoring of plasma drug concentrations is required for effective pharmacotherapy. Repetitive collection of whole blood followed by analysis of plasma samples with conventional methods delays representation of crucial results. Skin is an easily accessible organ; a portion of systemically circulating drug molecules is diffused to the dermal interstitial fluid. Thus, the compound's pharmacokinetics (PK) in the fluid mirrors the plasma PK. To approach such local dermal space, here we describe a microsensing system with a needle-type boron-doped diamond (BDD) electrode, which detects chemical compounds by redox reaction. As a test analyte we chose an anticancer drug, doxorubicin. In an in vitro experiment with a BDD microsensor, doxorubicin elicited a current in response to applied negative potential. Calibration curve covered the therapeutic window (10−100 nM). The sensor's performance was also tested in the collected interstitial fluids. Finally, the sensor was inserted into the dermis layer in anesthetized live rats; after doxorubicin was intravenously injected, the local PK was tracked for >1 hour with the C_max and T_max 3.1 ± 1.4 nM and 33.6 ± 20.6 mins, respectively (n = 7). By combining a formula linking the local measurements to plasma data, this microsensing system may be applicable to real-time monitoring of systemic PK.

P-glycoprotein (P-gp) is mainly found in the cell membrane of the small intestine and blood-brain barrier in vivo, and is responsible for the extracellular transport of cytotoxic hydrophobic compounds. P-gp is known to transport many pharmaceutical compounds as substrates. If we can understand the substrate recognition mechanism of P-gp, it will be possible to design pharmaceutical compounds that are not recognized by P-gp. Recently, the complex structures of human P-gp with substrates and inhibitors have been reported by single-particle analysis using Cryo-EM, and the differences in the binding pockets of substrates and inhibitors have been clarified. However, a detailed understanding of how P-gp can identify compounds as substrates or inhibitors has not been achieved. In this study, we aim to elucidate the detailed substrate recognition mechanism by elucidating and comparing multiple complex structures of P-gp and compounds. First, we established a system for expression and purification of human P-gp. Further, we have established a simple system for reconstitution into Nanodisc. Recently, we succeeded to obtain the 3D structure at the highest resolution (2.93 Å) as human P-gp. In this presentation, we will introduce the expression, purification, and Nanodisc reconstruction systems of P-gp and the obtained 3D structures.

[Background] Macrophage (Mφ) plays crucial roles in immunity and its dysfunction leads to the chronic inflammatory diseases such as arteriosclerosis. Several Mφ functions are modulated by the activation of ionotropic purinergic P2X7 receptor. Caveolin-1 (Cav-1) enables effective intracellular Ca²⁺ signaling by accumulating ion channels within caveolae domain. In this study, we analyzed the functional coupling between Cav-1 and P2X7 receptor using Cav-1 knockout (Cav-1 KO) mice.

[Methods] In murine bone marrow-derived Mφ (BMDM), the expression of Cav-1 was analyzed by real-time PCR and Western Blotting. Interaction of Cav-1 and P2X7 receptor was analyzed by proximal ligation assay. Ca²⁺ influx, K⁺ efflux and reactive oxygen species (ROS) production were measured with confocal microscopy. Cell death was analyzed by LDH assay.

[Results] The expression of Cav-1 was increased by LPS (lipopolysaccharide)-induced inflammatory stimulation in BMDM. Cav-1 was interacted with P2X7 receptor. Thereafter, ATP-evoked Ca²⁺ influx and K⁺ efflux were increased in Cav-1 KO BMDM. ROS production and cell death evoked by ATP were also enhanced in Cav-1 KO BMDM.

[Conclusion] Cav-1 suppresses the activation of P2X7 receptor and modulates immune responses in Mφ. This study may lead to the development of novel drugs for chronic inflammatory diseases.

A drug that blocks the cardiac myocyte voltage-gated K⁺ channels encoded by the hERG carries a potential risk of long QT syndrome and life-threatening cardiac arrhythmia. Interestingly, certain hERG blockers can also facilitate hERG activation to increase hERG currents, which may reduce proarrhythmic potential. However, the molecular mechanism remains unclear. The hallmark feature of the facilitation effect by hERG blockers is that a depolarizing preconditioning pulse shifts voltage-dependence of hERG activation to more negative voltages. Here we utilize a D540K hERG mutant to study the mechanism of the facilitation effect. D540K hERG is activated by not only depolarization but also hyperpolarization. With D540K hERG, we find that nifekalant, a hERG blocker and Class III antiarrhythmic agent, blocks and facilitates not only current activation by depolarization but also current activation by hyperpolarization, suggesting a shared gating process upon depolarization and hyperpolarization. Moreover, in response to hyperpolarizing conditionings, nifekalant facilitates D540K hERG currents but not wild-type currents. Our results indicate that induction of facilitation is coupled to pore opening, not voltage per se. We propose that gated access to the hERG central cavity underlies the voltage-dependence of induction of facilitation.

A group of TRPCs, TRPC3, TRPC6, and TRPC7, form Ca²⁺-permeable channels directly activated by diacylglycerol (DAG) and play important roles in regulating neuronal survival and dendritic growth, cardiovascular fibrosis in vitro and in vivo  through regulation of Ca²⁺signaling. Various compounds targeting these TRPC channels have been developed for the treatment of serious diseases such as sudden pulmonary fibrosis and chronic nephropathy. However, none of these compounds have yet reached clinical application, and therefore development of new TRPC3/C6/C7 inhibitors has been much-needed. Here, we have developed a piperazine derivative targeting TRPC3/C6 channels. This compound suppressed receptor-activated Ca²⁺influx in a dose-dependent manner in human embryonic kidney cells 293 expressed with human TRPC3 or TRPC6 (TRPC3, IC₅₀ = 0.086: TRPC6, IC₅₀ = 0.034µM). This drug showed no significant inhibitory or stimulatory effect on other TRPs including TRPC7. Interestingly, during isolation of human TRPC7, we obtained a new splice variant of human TRPC7; we are in the process to characterize biophysical and pharmacological properties of the variant that has a deletion in one of the functionally critical domains.

In vascular smooth muscles, the activity of Ca²⁺-activated Cl^- (Cl_Ca) channels regulates the membrane excitability and myogenic tone. TMEM16A channels are predominantly form Cl_Ca currents in vascular smooth muscles including portal vein smooth muscles (PVSMs). Caveola is a cholesterol-rich membrane invaginations and structurally contributes to effective and efficient signal transduction. Caveolin 1 (Cav1) is accumulated in the caveolin and plays a key role in forming the functional complex among enzymes, receptors, and ion channels. In this study, the functional roles of Cav1 on the expression and activity of TMEM16A Cl_Ca channels were examined in portal vein smooth muscle cells (PVSMCs) from wild-type (WT) and Cav1-knockout (KO) mice. Spontaneous contractions of PVSMs were recorded using an isotonic transducer. TMEM16A-mediated Cl_Ca currents were recorded by whole-cell patch-clamp configurations. The expression of TMEM16A channels was quantitatively analyzed by real-time PCR. The amplitude of spontaneous contractions of PVSMs was larger in Cav1-KO mice than WT mice. Whole-cell Cl_Ca currents were also larger in Cav1-KO PVSMCs than WT PVSMCs. Importantly, Ani9 (a specific blocker for TMEM16A channels)-sensitive currents were increased in Cav1-KO PVSMCs compared to WT PVSMCs. The expression of TMEM16A channels was higher in Cav1-KO PVSMs than WT PVSMs. The present data strongly suggest that the caveola structure formed by Cav1 negatively regulates the expression and activity of TMEM16A-mediated Cl_Ca channels in vascular smooth muscle cells.

We propose that L-DOPA by itself is a neurotransmitter. Recently, a G-protein coupled receptor GPR143, a gene product of ocular albinism1, was identified as a receptor for L-DOPA. In this study, to identify the physiological role of GPR143, we performed phenotypic analysis using Gpr143-gene deficient (GPR143-KO) mice. To assess anxiety- and exploration-related behaviors, we employed zero-maze test, and found that time spent in open arms was decreased in GPR143-KO mice when compared to wild-type (WT) mice. The time spent in open arms was also decreased in striatal indirect pathway specific GPR143-KO mice. To investigate the involvement of endogenous L-DOPA, we examined the effect of alpha-methyl-para-tyrosine, a synthetic inhibitor of L-DOPA on mouse behavior. We found that administration of α-MPT at the dose of 3mg/kg (i.p.) decreased the release of L-DOPA without affecting that of dopamine from the dorsal striatum. The administration of α-MPT decreased the time spent in open arms in WT mice, while this effect was not observed in GPR143-KO mice. Furthermore, intraventricular administration of a synthetic peptide, which inhibited the interaction between GPR143 and dopamine D2 receptor (DRD2), increased anxiety-like behavior. These results suggest that L-DOPA regulates anxiety-like behavior through GPR143 and DRD2 coupling in the striatal indirect pathway.

Nutrient uptake is essential for maintaining the enhanced growth and proliferation of cancer cells. LAT1 (SLC7A5), which preferentially transports large neutral amino acids, is highly expressed in various cancers. LAT1 inhibitors are preclinically shown to suppress the cancer cell proliferation and tumor growth, and a representative compound JPH203 is under clinical evaluation. However, detailed pharmacological influence of LAT1 inhibition on the overall uptake of large neutral amino acids and the protein synthesis in cancer cells that are thought to be crucial for its anti-cancer effects have not been elucidated yet. Here, we showed that JPH203 dramatically inhibits the uptake of all the large neutral amino acids in multiple pancreatic cancer cell lines. We also found that JPH203 significantly inhibits the amino acid uptake even in cell culture media containing high concentrations of various amino acids. Analyses of the protein synthesis activity based on the binding state of mRNA with ribosomes (Polysome analysis) and the incorporation of puromycin into nascent polypeptides (SUnSET) revealed that JPH203 suppresses global translation. These results advance our understanding of pharmacological activities underlying the anti-cancer effects of LAT1 inhibitors, further supporting the adequacy of cancer treatments targeting LAT1.

Gastrointestinal symptoms, including nausea and vomiting, are common adverse effects of oral iron tablets, but the mechanism of iron-induced nausea and vomiting is not yet known. Studies have shown that there are close relationships between gastrointestinal motility and gastrointestinal symptoms such as nausea, vomiting, and diarrhea, with more than 90% of patients with delayed gastric emptying experiencing nausea and vomiting. However, the effect of iron on gastrointestinal motility has not yet been investigated. In the present study, we aimed to elucidate the effects of iron on gastrointestinal motility using sodium ferrous citrate (SFC),  the most commonly used iron tablets. Gastric emptying in mice was assessed by ¹³C-octanoic acid breath test to examine the effect of SFC (3-30 mg Fe/kg, p.o.) with or without the 5-HT₃ receptor antagonist, palonosetron hydrochloride (5 mg/kg. s.c.). Colon transit was also measured by the beads method. The results showed that SFC delayed the gastric emptying, which was ameliorated by administration of palonosetron hydrochloride. It was also confirmed that ingredients of the tablets had no effect on gastric emptying. SFC also had no effect on the colon transit in vivo. These results lead the possibility that the iron-induced delayed gastric emptying may be mediated through nausea and vomiting.

Cannabis is the most widely used addictive drug following alcohol and tobacco. However, the mechanisms involved in the mental effects and dependence formation are unclear. Δ9-tetrahydrocannabinol (THC), the main active substance in cannabis, binds and affects cannabinoid type 1 receptors (CB1R) in the brain. The mice were i.p. administered arachidonylcyclopropylamide (ACPA), a CB1R-selective agonist, and then two behavioral experiments were performed. Treatments of ACPA induced the anxiolytic-like behavior in the elevated plus maze test. ACPA increased place preference in the conditioned place preference test. The BLA of mice highly expresses CB1R in the GABAergic interneurons. We aimed to reveal the role of CB1R in BLA for ACPA-induced behaviors. AM251, a CB1R selective antagonist, was administered intra-BLA before i.p. administration of ACPA. Intra-BLA administration of AM251 inhibited ACPA-induced anxiolytic-like behavior and place preference. Furthermore, in vivo microdialysis was performed to measure basal GABA levels in the BLA. Acute administration of ACPA had significantly increased basal GABA levels. Chronic administration of ACPA did't affect basal GABA levels. These results suggest that CB1R in the BLA contributes to behavior disorders caused by the acute or chronic use of cannabis and these behaviors maight be through a complex control system involving GABA. This study suggests that CB1R in the BLA may lead to new therapeutic targets in the treatment of cannabis-induced adverse effects.