Atopic dermatitis (AD), a chronic inflammatory skin condition characterized by pruritus, can affect the patient’s quality of life. The pathogenesis of AD involves epidermal barrier dysfunction and inflammatory cytokines, with the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway playing a role in cytokine signaling. Although topical corticosteroids remain the mainstay of AD treatment, novel therapeutic agents, such as biologics and molecular-targeted drugs targeting cytokines and JAKs have been developed recently. AD has a complex multifactorial pathogenesis combining genetic and environmental factors. However, recent reports indicate that monogenic mutations may cause some atopic diseases. Recently, we encountered a father-son pair with severe AD, alopecia, and growth impairment, showing no response to conventional treatments. Genetic analysis revealed a JAK1 variant suspected of gain-of-function mutation. Subsequent treatment with upadacitinib, a selective JAK1 inhibitor, led to remarkable improvements in eczema and alopecia in both patients. This case highlights the potential of targeted therapies focusing on proteins derived from specific genes in cases caused by monogenic abnormalities, where molecular-targeted drugs could dramatically improve the symptoms. Thus, in severe AD patients exhibiting poor responses to conventional treatments or having growth impairment and atypical symptoms, considering the possibility of monogenic mutations is crucial for their diagnosis and management.

Allergic diseases are recognised as "common diseases” caused by multiple factors including genetic predisposition and environmental factors. However, in some patients with allergic diseases, comprehensive genomic analyses such as whole-exome sequencing (WES) analysis have recently revealed a causative single gene, which might be a good target molecule for drug discovery. In our WES analysis, a diagnosis was made in 42% of patients with some allergic symptoms, suggesting that allergic diseases, which are “common diseases”, include monogenic diseases.

In this symposium, we will present a severe case with multiple allergic symptoms in which a novel causative gene, STAT6 was identified by WES analysis. The proband showed multiple allergic symptoms including severe atopic dermatitis, food allergy, asthma, and eosinophilic gastroenteritis. In vitro experiments demonstrated that the STAT6 variant acted in a constitutively active manner. The variant knocked-in mice tended to spontaneously develop dermatitis similar to human atopic dermatitis. We propose that an exaggerated STAT6 response is a novel mechanism of allergic symptoms, which is a good therapeutic target. This mechanism has been shown in subsequent papers and is being established as a new entity of allergic diseases. We will also discuss recent findings surrounding STAT6.

Activity of various molecular network pathways are altered in cancer and infectious diseases. Analysis of molecular network pathways including epithelial-mesenchymal transition (EMT) and tumor microenvironment pathway are crucial to reveal mechanism in acquisition of treatment resistance in cancer. In the meantime, the coronavirus pathogenesis pathway is activated in coronavirus infection, where the role of reactive oxygen species (ROS) is unclear. To reveal the precise mechanism of cancer treatment-resistant networks and coronavirus pathogenesis pathway, public gene expression data were analyzed in Ingenuity Pathway Analysis (IPA). Among more than 100,000 analyses and datasets, 23,100 analyses were identified as being involved in EMT by growth factors pathway, and 106 analyses and 106 datasets were related to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Analysis on pancreatic ductal adenocarcinoma treated with palbociclib, a cyclin-dependent kinase, post 24 hours culture medium compared to the culture medium post 24 hours paclitaxel showed high activation of the regulation of the EMT by growth factors pathway. The 49 analyses were involved in SARS-CoV-2 and human, which comprise of 27 analyses including 9 analyses on tissue “skin” GSE156754 and 22 analyses on lung adenocarcinoma. FOS and JUN in the coronavirus pathogenesis pathway were activated in SARS-CoV-2 infected lung adenocarcinoma. Coronavirus pathogenesis pathway was activated in SARS-CoV-2 infected iPS cell-derived cardiomyocytes. Telmisartan, acetaminophen and arsenic trioxide were found to interact with the coronavirus pathogenesis pathway. Several microRNAs were identified to interact with the regulation of EMT by growth factors pathway and coronavirus pathogenesis pathway. The network interactions identified in molecular network pathway analysis have potential to identify the therapeutic mechanisms.

Environmental diseases are illness developed directly by environmental factors such as diet, radiation, and toxic chemicals. Understanding the molecular mechanisms behind environmental diseases not only enables the identification of potential intervention targets but also facilitates the development of specific biomarkers for early detection and accurate diagnosis. Quantitative mass spectrometry coupled with stable isotope-labeling techniques has played pivotal roles in the identification of biomarkers and elucidation of the molecular mechanisms associated with diseases. Deuterium oxide (D2O) emerges as an attractive stable isotope-labeling precursor for metabolic labeling due to its cost-effectiveness, simplicity of administration, and ability to label a wide range of biomolecules and biological systems. This presentation describes the unique characteristics of D2O as an isotope-labeling precursor and explore the biological pathways and target sites associated with D2O labeling for different types of biomolecules. Furthermore, practical applications of metabolic D2O labeling in diverse quantitative omics experiments are demonstrated with previous studies that employed this technique for metabolic flux analysis, biological turnover rate measurement, and relative quantification.

The alarming global increase of neuron-related diseases such as neurodegeneration and obesity suggest an urgent need for new therapeutic strategies. Current anti-obesity drugs are limited by off-target actions, and further research is needed to identify new targets and effective therapeutic approaches to regulate metabolism. Targeting brain lipid metabolism is a promising strategy to regulate energy balance and fight metabolic diseases tightly controlled by brain cells. The development of stable platforms for selective delivery of drugs, particularly to the hypothalamus, is a challenge but a possible solution for these diseases. Attenuating fatty acid oxidation in the hypothalamus via carnitine palmitoyltransferase 1A (CPT1A) inhibition leads to satiety, but this target is difficult to reach in vivo with the current drugs. We propose using an advanced crosslinked polymeric micelle-type nanomedicine that can stably load a CPT1A inhibitor for in vivo control of energy balance, leading to a rapid attenuation of food intake and body weight in mice via regulation of appetite-related neuropeptides and neuronal activation driving changes in the liver and adipose tissue. This investigation might contribute to the development and validation of a new generation of nanomedicine-based approaches targeting brain cells.

Physiological changes caused by environmental stress responses can result in environmental disease in wild animals, including humans. Previous studies have been reported that physiological changes by stress response induced the disturbance of neuroendocrine system via the hypothalamus-pituitary-adrenocortical (HPA) axis. Hereby, we hypothesize that acute- and long-term stress responses may influence physiological endocrine function, and finally lead to behavior- and reproductive disorder in wild fish. Indeed, through the impacts caused by exposure to various environmental chemicals, we confirmed that physiological dysfunction by stress response was associated with an increased risk of physical diseases induced by exposure concentration of chemicals. Therefore, we suggest that there is a strong interaction between environmental diseases and physiological dysfunction by long-term exposure to environmental stressors, such as hazardous chemicals. However, to clearly define the acceleration of physiological dysfunction by stress response, the scientific evidences regarding the impacts of different stressors, stress severity, and times of stress (i.e., acute- and chronic stress) on the physiological metabolic responses in the body are need. Moreover, the risk and durability of environmental disease in age, gender, and health status should be demonstrated.

Cognitive biases play a central role in shaping perceptions, attitudes, and decisions. These inherent biases often find their most striking manifestations in the realm of gender, particularly in fields traditionally seen as male-dominated, such as the sciences. In Japan, this problem is exacerbated by a notable lack of female university students in scientific disciplines. Historically, Japanese society has been characterized by defined gender roles, where societal expectations and traditions have often limited women's participation in certain fields, including science. While modern society is progressive in many ways, remnants of these ancient beliefs persist and subtly influence perceptions and decisions. The cognitive biases, which may be unconscious, play a central role in perpetuating the gender gap in academic choices. Such biases can limit opportunities and encouragement for young women to pursue scientific careers. But addressing this issue isn't just about recognizing and challenging these biases. It's also about cultivating an educational environment that actively supports and encourages women's participation in science. This symposium aims to dissect these biases, understand their origins, and propose actionable solutions. By shedding light on this issue, we hope to pave the way for a more equitable scientific community by breaking the chains of historical biases and cultural expectations.

SARS-CoV-2, the causative agent of COVID-19, emerged at the end of 2019. During its global spread over the past 3 years, SARS-CoV-2 has been highly diversified, and these SARS-CoV-2 variants have been considered to be the potential threats to the human society. In order to elucidate the virological characteristics of newly emerging SARS-CoV-2 variants in real-time, I launched a consortium called “The Genotype to Phenotype Japan (G2P-Japan)” in January 2021. With the colleagues who joined the G2P-Japan consortium, we have revealed the virological characteristics of SARS-CoV-2 variants. In May 2023, WHO declared the end of the Public Health Emergency of International Concern (PHEIC) for COVID-19. However, “the next pandemic” will come in the future, and we need to gather our wisdom learned from the COVID-19 pandemic for the preparedness of future pandemic. Here, I will talk about our findings on SARS-CoV-2 variants and future perspectives to combat the outbreaks and pandemic that will happen in the future.

The COVID-19 pandemic has changed how we live, work, learn, and interact in less than a year since the virus emerged. This also changed the way of virus research more translational. One of major contributors is air-liquid interface (ALI) cultured airway epithelium, ready-made or user-friendly construction kits. There are a lot of evidence of the limitation of the virus work using cancer cell lines, submerged monolayer culture or human respiratory virus semi-permissive animals. ALI airway epithelium, which closely mimic human airway epithelium morphologically or physiologically, are infected well with human respiratory viruses including influenza and coronavirus. Antiviral test of new compounds in this model would provide the best proof-of-concept (PoC) and help to build confidence for further clinical development. However, this system is more useful for industry than we thought. This system can be used to determine dosing regimen (prophylaxis, therapeutic etc) due to a long shelf life, clinical dose prediction, effective clinical parameter prediction, dosing route decision, power calculation for clinical trials, PK-PD relationship, prediction of inhaled toxicity and to test in disease population. Most importantly, as the use of system consequently reduces animal study, this contributes to 3Rs concept, which is very important for future drug discovery. Thus, ALI airway epithelium is not only a biology system simply to evaluate virus replication or a PoC, but also can be used for preclinical drug development broadly for industry.

以下の症例について、フロアーの参加者とともに考察します。

【症例】 79歳、男性。164cm、58.5kg。耐糖能障害、B型肝炎の既往（HBs抗原:陰性、HBs抗体:陽性）あり。

当院でX-3年より、高血圧、脂質異常症、胃潰瘍に対してアムロジピン（5mg）1T、ランソプラゾール（15mg）1T、アトルバスタチン（5mg）1T/日を処方されていた。

X-1年：降圧不十分（BP:156／84mmHg）にてトリクロルメチアジド（2mg）1T/日追加された。

X-5ヶ月：腰痛、下肢痛（脊椎間狭窄症疑い）に対しプレガバリン（75mg）2T（朝・夕）/日、また妻に対して易怒性、不眠とのことでペロスピロン（4mg）1T/日を追加された。この際の検査値は以下のとおり肝腎機能に異常は認めなかった。WBC:7400/μL、RBC:447万/μL、Hb:13.7g/dL、血小板:22.8万/μL、血糖（食後）:161mg/dL、HbA1c:6.6%、AST:19U/L、ALT:17、γ-GTP:34、LDH:187、BUN:20.1mg/dL、Cr:0.82、eGFR:69.1ml/min/1.73m2、Na:142mEq/L、K:3.5、Cl:97。追加薬剤はある程度の効果を認めていた。

X-2ヶ月：下肢のしびれ、疼痛の改善不十分にて他院を受診し、芍薬甘草湯を処方され１～３包（2.5～7.5ｇ）/日を服用していた。

X:脱力のため階段が上れなくなった。また2週間くらい前より、口渇感を感じるようになり当院受診した。外来担当医より、高血糖（BS:338mg/dl）と、肝障害（AST:545、ALT:155、LDH:1397）を認めたということで、診察依頼を受けた。

身体所見では、BP:158/70ｍｍHg、PR:76/分、浮腫なし、眼瞼結膜に貧血なし、胸部、腹部には特に異常所見なし。四肢に軽度筋力低下（MMT:上肢4＋/5、下肢4/5）あるが、他に神経学的異常は認めない。

【設問】以下の有害事象［１）脱力、２）高血糖、３）AST、ALT、LDH上昇（肝障害疑い）］各々に関与した可能性のある薬物はどれか？またその機序は？

[Background] Macrophage (Mφ) is one of the innate immune cells and related to several chronic inflammatory diseases. Ionotropic purinergic P2X7 receptor plays a key role in the regulation of Mφ functions. Caveolin-1 (Cav-1) is known to modulate the activities of ion channels. In this study, the functional coupling between Cav-1 and P2X7 receptor was examined using Cav-1 knockout (Cav-1 KO) mice.

[Methods] Murine bone marrow-derived Mφ (BMDM) was used in this study. Molecular interaction between Cav-1 and P2X7 receptor was analyzed by proximal ligation assay (PLA). Intracellular [Ca²⁺] and [K⁺] were measured by confocal microscopy with each specific fluorescent indicator. Lytic cell death was analyzed by LDH assay.

[Results] In BMDMs, Cav-1 expression was increased by lipopolysaccharide. PLA revealed the molecular interaction between Cav-1 and P2X7 receptor. The treatment with 1 mM ATP evoked sustained Ca²⁺ influx and K⁺ efflux in BMDMs from wild-type mice. The response was enhanced in BMDMs from Cav-1 KO mice. ATP stimulation induced lytic cell death through P2X7 receptor activation, and this cell death was facilitated in Cav-1 KO mice.

[Conclusion] Cav-1 negatively regulates the activation of P2X7 receptors and results in cell death in Mφ. This study may lead to the development of novel drugs of chronic inflammatory diseases.

Obesity is known to be triggered by various factors and is a recognized as a risk factor for metabolic diseases. Leptin is an anti-obesity hormone, which can inhibit food intake and increase energy expenditure. It is suggested that leptin resistance is one of the underlying mechanisms of obesity. Our research group is studying the impact of endoplasmic reticulum (ER) stress on development of leptin resistance. We previously found that 4-hydroxynonenal, which contains aldehyde, induced ER stress and inhibited leptin-induced STAT3 phosphorylation. In the present study, we identified several food additives, which contains aldehydes that could potentially cause leptin resistance through screening using STAT3 luciferase assay at neuronal cells expressing Ob-Rb leptin receptor. We also evaluated the downstream of leptin signal by analyzing STAT3 signal by Western blotting. We found that the several food additive can inhibit leptin-induced STAT3 signal. We are now analyzing the effect of food additives on ObR leptin receptor function by BRET technique. Overall, our results suggests that some food additives, which contain aldehydes, may potentially lead to obesity through formation of leptin resistance.

Amyotrophic lateral sclerosis (ALS) is a rare but devastating disease characterized by progressive neurodegeneration of motor neurons, leading to skeletal muscle denervation. Neuromuscular junction (NMJ) degeneration is an early pathological change in ALS. Nonetheless, the exact regulatory mechanisms and potential therapeutic interventions underlying this process remain largely unknown. Through our preliminary experiment using heterochronic parabiosis, a surgical technique enabling blood circulation sharing between organisms, we coupled ALS mice (SOD1^G93A) with WT mice. Surprisingly, this led to NMJ shedding in the WT mice, a phenomenon not typically observed, thus hinting at the presence of NMJ-degenerating molecules in ALS mouse plasma. Next, we conducted an extensive analysis of cytokine levels in the plasma of SOD1^G93A mice and WT mice and found markedly elevated levels of cytokine X in the ALS mice plasma, with abundant production originating from intestinal vascular endothelial cells. Treating SOD1^G93A mice with neutralizing cytokine X antibodies significantly suppressed NMJ degeneration and improved sciatic compound muscle action potential (CMAP). Similarly, a comparable positive outcome was also observed with the treatment of lipid nanoparticles containing cytokine X receptor siRNA formulations in the skeletal muscle tissue of SOD1^G93A mice. In addition to exploring the correlation between nervous system cell dysfunction within the lesion, our study unveiled a crucial role of systemically expressed cytokine X in ALS pathogenesis, offering valuable insights into a potential therapeutic biomarker for the disease.

Nicotinamide adenine dinucleotide (NAD⁺) is an essential coenzyme involved in many cellular functions including energetics, circadian rhythm, transcription, and epigenetics etc. Imbalanced NAD⁺ levels lead to age-related pathologies. NAD⁺ is biosynthesized by de novo, Preiss-Handler, and salvage pathways. It has been shown that the salvage pathway is more vital in maintaining NAD⁺ levels in skeletal muscle. Whereas evidence proving the role of de novo and Preiss-Handler pathways remains elusive. NAD synthetase (NADS) is the last enzyme shared in de novo and Preiss-Handler pathways. In this study, we generated NADS KO mice and investigated the role of de novo and Preiss-Handler pathways in skeletal muscle. The skeletal muscle in NADS KO mice showed depleted NAD⁺ levels at a young age resulting in poor skeletal muscle performance assessed by grip strength and endurance on the treadmill. Histology of these mice showed decreased cross-sectional area resembling sarcopenia. Interestingly, old NADS KO mice restored NAD⁺ levels but these mice didn’t recover muscle strength and endurance suggesting that reduced NAD⁺ levels in young age influence exercise performance in old age. Further, RNA sequencing of skeletal muscle revealed circadian genes are altered in NADS KO mice. In addition, we found that NAD+ precursor replenishment rescued the NAD⁺ levels and muscle weakness in NADS KO mice. This study implies that maintaining NAD⁺ levels in young age is crucial in preventing skeletal muscle aging.

The vulnerability of pancreatic β-cells to endoplasmic reticulum stress (ER), is a common cause of β-cell apoptosis and dysfunction involving decreased insulin secretory response and a reduction in β-cell mass in type 2 diabetes mellitus. The possible role of naturally occurring polyphenols –known as flavonoids- in treating type 2 diabetes mellitus is a current area of focus. However, there is a dearth of information about the effect of the trihydroxyflavone, apigenin, on pancreatic β-cell functions.

We evaluated the effect of apigenin on glucose-induced insulin secretion (GSIS) and β-cell apoptosis, studying the mechanism underlying its antidiabetic effects, using the INS-ID β-cell line. The results showed that apigenin dose-dependently stimulated GSIS at all concentrations (1, 10, 30 and 100 μM), with a significant peak effect at 30 μM concentration. The downstream ER stress signaling proteins, CHOP and cleaved caspase-3, which were elevated by thapsigargin-induced apoptosis of INS-1 cells, were concentration-dependently and strongly attenuated by apigenin treatment at all concentrations, with peak suppression at concentrations of 30 and 100 μM. These results strongly correlated with the flow cytometric analysis of Annexin V/PI staining and the DNA fragmentation analysis, which is indicative of apoptosis, as determined by DNA laddering. The thapsigargin-induced increase in TXNIP expression was also dose-dependently suppressed by treatment with apigenin. Apigenin also inhibited high concentration of glucose-induced apoptotic increases in cleaved caspase-3 and CHOP expressions.

These results suggest that apigenin is an attractive candidate with remarkable and potent insulinotropic and anti-apoptotic effects on β-cells and that its anti-diabetic effect may be mediated by increasing GSIS and preventing ER stress-mediated β-cell apoptosis mediated by CHOP, cleaved caspase-3 and TXNIP, hence promoting β-cells survival and function.

We previously reported that peripheral sympathetic nervous activation enhanced glucose uptake in standard laboratory chow- (SCF) and high-fat-fed (HFF) rats, and that the uptake might be mediated by PGC-1α in the soleus muscle in the SCF rats. However, blood glucose (BG) reduction was only temporal. In the present study, we investigated the effects of peripheral sympathetic activation on glycemic control. We detected sympathetic signal with a microelectrode in the unilateral sciatic nerve under anesthetic condition in the SCF and HFF rats, and conducted electrical microstimulation (MS) via the electrode for 60 min. The MS resulted in no significant change or slight elevation of BG in the SCF or HFF rats, respectively without no change in plasma insulin level. In the SCF rats, the MS elevated G6Pase mRNA expression as well as glycogen content in the liver, and reduced triglyceride (TG) content in the white adipose tissue. Conversely, the MS had no effects on these parameters in the HFF rats. These results suggest that peripheral sympathetic activation enhances glucose uptake independently of insulin in the skeletal muscle while BG level might be compensated by hepatic glucose production, and that the TG reduction in white adipose tissue may lead to glycerol supply and gluconeogenesis in the liver in the SCF but not in HFF rats.

Connexin43 (Cx43) is highly expressed in astrocytes (AS), and its expression is reduced in the prefrontal cortex of depressive patients. We have previously demonstrated amitriptyline (AMI), a tricyclic antidepressant, increased the expression of brain-derived neurotrophic factor (BDNF) mediated by lysophosphatidic acid (LPA) receptors. Present study examined the detailed mechanism involved in these responses. Primary cultured AS were prepared from the cerebral cortex of neonatal Wistar rats. The expression of Cx43 was downregulated with RNA interference. The expression of mRNA and protein were measured by real-time PCR and western blotting, respectively. Protein kinase C (PKC) activity was measured by using the assay kit. The enhancement of BDNF expression in Cx43-knockdown (KD) AS was suppressed by inhibition of extracellular signal-regulated kinase (ERK) and PKC which were known as the downstream of LPA receptors. Moreover, ERK phosphorylation and PKC activity were increased by treatment with AMI, which were also enhanced in Cx43-KD AS. Present study revealed reduced Cx43 expression potentiated the AMI-induced BDNF expression by enhancement of intracellular signaling in cortical AS. These results indicate the downregulation of Cx43 observed in the depressive patients might contribute to therapeutic effect of antidepressants.

The proper development of neural circuits is essential for normal brain function and is believed to be governed by the Hebbian rule a well-known hypothesis in neural circuit research, which suggests that neurons that fire together wire together. However, despite its long-standing history as a hypothesis, direct evidence supporting this notion and its influence on brain function has remained elusive. Our research investigated the Hebbian rule by experimentally inducing synchronous firing during the development of neural circuits in the mouse visual cortex. This brain region is known for its close relationship between neural circuit structure and brain function. To achieve this, we utilized a non-invasive transcranial optogenetic stimulation method, which allowed us to induce synchronous firing in ChR2-positive neurons during a critical period. We revealed a higher connection probability between synchronized neurons compared to ChR2-negative neurons or the control group without photostimulation, and this increase in connection probability was prevented by chronic treatment with MK801, an NMDAR antagonist. We used two-photon calcium imaging to examine the orientation selectivity of the stimulated neurons and found that synchronized neurons responded more similarly than the others. The increases in synaptic connectivity and similarity of orientation selectivity were not observed in randomly fired conditions. These outcomes suggest that developmental synaptic connection and its function depend not on the firing rate but on synchronicity.

Neurotrophin-3 (NT-3) is a type of neurotrophic factor and regulates neural differentiation, survival, and plasticity in both peripheral and central nervous systems. NT-3 expresses in the adult hippocampal dentate gyrus (DG) and has been reported to be upregulated by stress. However, the detailed function of NT3 in the hippocampal DG is unknown. Therefore, we tried to clarify the function of NT3 by overexpressing NT-3 in the hippocampal DG. We generated NT-3 overexpressing mice in the hippocampus by administering adeno-associated virus carrying NT-3 gene. Expression of NT-3 mRNA in the hippocampus was higher more than 35-fold than in the control group. The expression of calbindin, a mature neuronal marker, and the number of FosB positive cells were increased in mature neurons by NT3 overexpression. Moreover, the number of proliferating cells and immature neurons in the hippocampal dentate gyrus was decreased by NT-3 overexpression. In addition, the expression of Vegfd, a factor regulating neurogenesis, was decreased. These results showed that high NT-3 levels in the hippocampus regulate the activation of mature DG neurons and suppress the early phase of neurogenic processes.

Copy number variants in the ARHGAP10 gene are associated with schizophrenia (SCZ). We have previously demonstrated that Rho-kinase (ROCK) inhibitor, fasudil, ameliorates the decreased spine density in the medial prefrontal cortex (mPFC) of Arhgap10 S490P/NHEJ mice carrying the variants that mimic the ARHGAP10 variants found in a Japanese SCZ patient. Accordingly, we have proposed that ROCK is a potentially novel therapeutic target in SCZ. It is well known that there are two subtypes of ROCK, ROCK1 and ROCK2, and that fasudil inhibits both subtypes. Since ROCK2 is highly expressed in the brain, here we evaluated the effect of a selective ROCK2 inhibitor, belumosudil (KD025), on spine density in Arhgap10 S490P/NHEJ mice. We measured the spine density of pyramidal neurons in layer 2/3 of the mPFC in Arhgap10 S490P/NHEJ mice following daily oral administration of KD025 for one week. Moreover, we evaluated the general behaviors in an open field and systolic blood pressure after KD025 treatment. KD025 ameliorated decreased spine density of cortical neurons in the mPFC of Arhgap10 S490P/NHEJ mice, but had little effects on general behaviors and systolic blood pressure induced by fasudil. These observations suggest that ROCK2 is a more appropriate therapeutic target in SCZ, with little inducibility of hypotension.

Selecting an appropriate behavior is critical for survival in conflict situations during which animals face both appetitive and aversive stimuli. Animals in conflict situations exhibit several processes. First, they remain still in a safe place (suspension); once they decide to take action, they assess risks at a place where danger may occur (risk assessment) and finally reach the reward. However, most studies have not addressed these conflict processes. We developed a new experimental paradigm – the three-compartment conflict task – to quantitatively evaluate conflict processes. Our apparatus consisted of start, flat, and grid compartments. Mice were required to explore the grid compartment where they might receive foot shocks while trying to obtain sucrose. We found that applying foot shocks increased sucrose acquisition latency in the subsequent trials, reflecting an elevated conflict level throughout the trials. Time spent in the start compartment and the number of retreats were measured as parameters for conflict levels in suspension and risk assessment, respectively. Both parameters were increased by foot shocks. Treatment with diazepam decreased these parameters. Our new paradigm is a valuable tool for quantitatively evaluating conflict processes and contributes to the development of effective treatments for psychiatric disorders associated with maladaptive behaviors in conflict situations.

Although animal models of depression are produced by loading chronic stress, inducing neuroinflammation, or administering drugs that induce depression, the results obtained in these models have poor reproducibility. Therefore, it is necessary to develop animal models that exhibit definitive symptoms of depression for studies on potential therapeutics. This study aimed to investigate depressive symptoms and their pathogenesis in lipopolysaccharide (LPS)-inflamed mice treated with dexamethasone (DEX). Male ICR mice were injected with LPS, followed by injection with DEX at day later once daily for 6 days. Mice in the LPS+DEX group had significantly longer immobility time in the tail-suspension and forced swim tests than did those in the LPS or DEX only and control groups at 7 days post-LPS administration. In immunohistochemical analysis, significantly lower number of the immature neuronal marker doublecortin-positive cells were observed in the hippocampal dentate gyrus of mice in the LPS+DEX group compared with those of mice in the LPS or DEX only and control groups at 7 days after LPS administration. These results suggest that repeated DEX administration to LPS-inflamed mice may induce definitive symptoms of depression by decreasing the number of immature neurons in the hippocampal dentate gyrus.

[Background and Purpose] A mouse aggressive response meter (ARM) is a device that measures aggressive biting behavior (ABB) toward a metal rod presented in front of a mouse. With this device, we have previously found that kamishoyosan, a Japanese traditional herbal medicine, reduces this aggressive behavior (Igarashi et al., Brain Res. 2021). Although, in the previous study, it was suggested that males and females have different effects on GABAA receptors, the details have not been clarified. In this study, we investigated the effects of diazepam, an agonist for GABAA receptors.

[Experimental methods and results] Male and female ddY mice were isolation-reared from 3 to 10 to 11 weeks after birth. Isolation-reared male mice showed a 51% decrease in ABB 1 hour after i.p. administration with 2 mg/kg diazepam (p<0.05). On the other hand, no significant changes were observed in female mice treated with 2mg/kg diazepam. ABB was reduced by approximately 63% in male and 53% in female mice treated with 4 mg/kg diazepam (p<0.05). Next, we examined the expression level of potassium-chloride ion transporter (KCC2) mRNA in the prefrontal cortex by real-time PCR, and we found that it was decreased to about 8% in isolation-reared female mice compared to group-reared female mice (p<0.05). No significant difference was found between isolated and group-housed male mice.

[Discussion] KCC2 contributes inhibitory neural transmission by exporting intracellular chloride ion (Cl-). In this study, KCC2 expression in the PFC was decreased only in isolated female mice, which may disturb inhibitory neural transmission, consequently make it difficult for diazepam to reduce aggression.

Obesity is said to be one of the exacerbating factors of chronic pain, however its mechanism is unclear. Recently, fatty acid binding protein 3 (FABP3) functions as not only an intracellular chaperone to transport fatty acids, but also the signal transduction and gene transcription. There are some recent reports that FABP3 is induced in response to an increased dietary fat load as well as obesity, inflammation and pain. Here we tested whether FABP3 involve in the mechanism for obesity-induced exacerbation of postoperative pain using FABP3 deficit (FABP3KO) mice. Male ddY and C57BL6J wild-type (WT) mice were used by experiment. WT and FABP3 KO were fed on control diet or high fat diet (HFD) for 8 weeks. Postoperative pain was induced by paw incision. Mechanical allodynia was evaluated by von Frey test. Mice with paw incision showed mechanical allodynia. Repeated intracerebroventricully injection of FABP-IN-1, a FABP inhibitor for FABP3, 5 and 7, suppressed paw incision-induced mechanical allodynia. The mice fed HF diet exacerbated paw incision-induced mechanical allodynia compared to those in control diet fed WT mice. On the other hand, FABP3KO mice fed HF diet suppressed paw incision-induced mechanical allodynia. Our findings suggest that FABP3 might at least in part involve in obesity-induced exacerbation of postoperative pain.

In Japan, 10% of the population suffer from hearing loss, which impairs daily life; it is an urgent issue to identify the causative factors and develop the innovative therapies. Irreversible hearing loss stems primarily from disorders of the cochlea of the inner ear. The cochlea consists of three tubular structures. Two tubules contain perilymph, which exhibits an ionic composition similar to plasma. Another tubule is filled with endolymph, which shows a highly positive potential of +100 mV. This unique environment sensitizes hearing. Because the endolymph is packed in the narrow space and its quantity is small of ~1 µL in a mouse cochlea, the protein content remains unclear. To address this challenge, we developed a sophisticated approach to collect the endolymph from the cochlea of live mice. We fabricated a micropipette filled with a conductive organic solvent and inserted it into the cochlea, while monitoring the potential. As detecting the high potential of the endolymph, we aspirated the fluid into the pipette. The sample was then analyzed by LC-MS/MS. When comparing the result to the profile of the perilymph, we found that a few proteins including molecules involving lipid metabolism were enriched in the endolymph. The protein data described here may be useful for understanding of the mechanisms underlying hearing loss.

Finerenone, a nonsteroidal mineralocorticoid-receptor blocker, has been shown in clinical trials to have considerable cardioprotective benefits. However, its precise mechanism is not clear. Here, we aimed to test the hypothesis that cardioprotective effects of finerenone are linked to a decrease in salt buildup and, as a result, a lower macrophage chemotaxis and/or a change in its phenotype in heart tissues. First, effects of finerenone (10 mg/kg body weight by oral gavage) on myocardial injury and sodium accumulation were examined in uninephrectomized (UNx) Sprague-Dawley rats with chronic aldosterone infusion (0.75 μg/hr) and salt-loading through drinking water (1% NaCl) for 4 weeks. Echocardiography and gene expression analyses revealed an adverse cardiac remodeling as well as diastolic dysfunction with preserved ejection fraction. Notably, finerenone treatment completely prevented the cardiac dysfunction with the improved cardiac remodeling in these rats. Furthermore, sodium content in left ventricular tissues were markedly elevated in salt-loaded aldosterone-infused UNx rats, but significantly reduced in rats with concomitant finerenone treatment. Moreover, gene expression of F4/80 (a macrophage marker) was significantly reduced by finerenone treatment. Apart from that, finerenone dramatically reduced the salt-induced elevation in M1 markers (TNF-alpha and iNOS) in RAW264.7 cells, whereas M2 markers remained unaltered. These data indicate that finerenone has the potential to mitigate cardiac dysfunction in salt-loaded and aldosterone-infused rats by suppressing sodium accumulation in left ventricular tissues. These effects of finerenone may attenuate the subsequent inflammation by macrophages and adverse cardiovascular remodeling.

Aortic aneurysm and dissection (AAD) are common arterial diseases with high mortality. Recently, AAD have been reported as adverse events associated with fluoroquinolones. Previous studies showed fluoroquinolones could induce AAD through arterial media disorder, but the influence of fluoroquinolones to vascular endothelium is unknown. The aim of current study is to evaluate the effect of fluoroquinolone to vascular endothelium on the development of AAD. We analyzed the association in clinical situation between fluoroquinolones and AAD using VigiBase, the World Health Organization’s global database. We evaluated whether levofloxacin (LVFX) affects the AAD development using human umbilical vein endothelial cells (HUVECs) and aortic dissection model mice which induced by three drugs; nitric oxide synthase inhibitor, angiotensin II, and lysyl oxidase inhibitor. VigiBase analysis showed that the fluoroquinolones had the association of high reporting rate of AAD. Although LVFX did not significantly increase the incidence of AAD in mice, LVFX treatment increased VCAM-1 expression in HUVECs. Our results suggested that the endothelial dysfunction might be one candidate mechanism of fluoroquinolone-associated AAD.

Purpose: Calcitonin gene-related peptide (CGRP) regulates inflammation through receptor activity-modifying protein 1 (RAMP1), a subunit of CGRP receptor complex in immune cells. Acute respiratory distress syndrome (ARDS) is a severe respiratory dysfunction induced by cytokine storm that leads to alveolar epithelial damage and increased pulmonary vascular permeability. This study investigated the role of RAMP1 signaling in the pathology of ARDS.

Methods and Results: ARDS was created by an intratracheal administration of lipopolysaccharide (LPS) to male RAMP1-knockout (RAMP1-/-) mice and wild-type (WT) mice. As compared with WT, RAMP1-/- exhibited increases in fatality rates, infiltration of inflammatory cells and hemorrhage in the lung tissues, and levels of total protein, inflammatory cytokines (TNFα, IL-1β and, IL-6), and chemokine (CXCL12) in bronchoalveolar lavage fluid (BALF). RAMP1 was expressed in alveolar macrophages (AMs), and CGRP levels in BALF were increased in WT and RAMP1-/- at 72 h. Removal of AMs with clodronate liposome (CL) enhanced lung injury and total protein levels, and reduced cytokines (TNFα and IL-1β) in both genotypes at 6 h, but increased the cytokines and CXCL2 together with neutrophil accumulation at 72 h. Cultured AMs from RAMP1-/- showed higher levels of cytokines and chemokines than those in WT.

Conclusion: These results suggested that deletion of RAMP1 signaling in AMs aggravated LPS-induced acute lung injury by increasing vascular permeability, inflammatory cytokines production, and neutrophil accumulation.

The development of acute respiratory syndrome (ARDS) during sepsis clinically doubles the mortality rate of patients. ARDS is essentially non-cardiogenic pulmonary edema, based on increasing permeability of vascular endothelial and pulmonary epithelial cells due to their injury and cell death caused by excessive inflammation in lung. It has been also known that the expression of AQP5 in lung tissues is considerably reduced in LPS induced ARDS mouse model. Therefore, it is possible that the decrease in AQP5 expression contributes to the pathogenesis of ARDS. We have established a transgenic (Tg) mouse in which AQP5 is highly expressed specifically in the pulmonary epithelial cells. In this study, we have examined the significance of changes in AQP5 expression in ARDS, using this Tg mice. In wild type (WT) mice, intraperitoneal treatment of LPS decreased survival rate and caused pulmonary edema evaluated by lung wet/dry weight ratio. In AQP5-Tg mice, the decrease in survival rate and pulmonary edema caused by LPS was considerably improved. In addition, TUNEL stained lung tissue revealed that the apoptotic cells in AQP5-Tg mice was significantly less than that in WT mice. These results indicated that the decrease in AQP5 expression in ARDS enhances the apoptosis of pulmonary epithelial cells and exacerbate pulmonary edema.

Background & Aims

Liver fibrosis could lead to fatal secondary diseases such as cirrhosis and hepatocellular carcinoma, including osteoporosis. However, there are no effective treatments for liver fibrosis and subsequent osteoporosis, necessitating new therapeutic targets. Recently, fibroblast growth factor 23 (FGF23) has garnered attention as a potential fibrosis-promoting factor. FGF23 also controls the phosphorus level in the body; excess FGF23 level causes phosphorus deficiency, resulting in impaired bone microstructure. In this study, we hypothesized that the FGF23 level increases with liver injury, which in turn induces liver fibrosis and osteoporosis.

Results

We found that carbon tetrachloride-induced liver injury increased the serum FGF23 level. RNA sequencing analysis using FGF23-treated hepatic stellate cells showed that FGF23 promotes the production of Matrisomes, which helps form the extracellular matrix. The FGF receptor antagonist pemigatinib alleviated carbon tetrachloride-induced liver fibrosis and dysfunction. Moreover, pemigatinib suppressed the deleterious alterations in bone density and microstructure.

Conclusion

We found that the serum FGF23 level increased with liver injury, FGF23 promoted liver fibrosis, and inhibition of FGF23–FGFR signaling alleviated liver fibrosis and subsequent osteoporosis. These findings suggest that FGF23–FGFR signaling may be a new therapeutic target for liver fibrosis and subsequent osteoporosis.

Objective: Acetaminophen (APAP) overdose causes severe acute liver failure. Impaired liver repair and regeneration after APAP hepatotoxicity leads to failed recovery and mortality. Accumulating evidence indicates that macrophages play a critical role in liver repair after APAP-induced liver injury; however, underlying mechanisms of involvement of macrophages remain unknown. Here, we examined the role of endogenous thromboxane A₂ (TXA₂) in macrophages in liver repair after APAP-induced liver injury.

Methods and Results: APAP (300 mg/kg, ip) was administered to macrophage-specific thromboxane prostanoid receptor (TP) deficient mice (mTPKO) and control mice (Cont). Compared with Cont, mTPKO exhibited severe liver injury as indicated by increased levels of ALT, necrotic area, and pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and decreased expression of PCNA, a marker of hepatocyte proliferation at 48 h after APAP treatment. CD68-positive macrophages less accumulated in the livers from mTPKO, accompanied by reduced expressions of chemokines. Flow cytometry analysis revealed that the numbers of M1 macrophages in mTPKO were higher than control, while the numbers of M2 macrophages in mTPKO were lower than control. In cultured bone marrow-derived macrophages from mTPKO, M1-related gene expressions were increased and M2-related gene expressions were decreased.

Conclusions: TP receptor signaling in macrophages promoted liver repair after APAP-induced liver injury by accumulating M2 macrophages in the livers.

Macrophage recruitment and polarization play pivotal roles in the initiation and progression of liver fibrosis. Our previous study has demonstrated that Sphingosine kinase 1 (SphK1) has distinct roles in the activation of Kupffer cells (KCs) and hepatic stellate cells (HSCs) in liver fibrosis. However, the role of SphK1 in hepatic macrophage recruitment and polarization remains unclear. In this study, Single-cell transcriptomics illustrated that SphK1 is highly expressed in monocytes/macrophages and upregulated during both stages of macrophage M1 and M2 polarization. Consistently, SphK1 expression was elevated and positively correlated with macrophage M1 and M2 polarization in human fibrotic livers. SphK1 deletion reduced the recruitment of hepatic macrophages and inhibited M1 and M2 polarization in CCl₄-induced mice. SphK1 deficiency in endogenous liver cells attenuated macrophage recruitment via CCL2. SphK1 in macrophage activated the ASK1-JNK1/2-p38 signaling pathway to promote M1 polarization. Furthermore, macrophage SphK1 downregulated small ubiquitin-like modifier (SUMO) specific peptidase1 (SENP1) so as to decrease de-SUMOylation of Kruppel-like factor 4 (KLF4) to promote M2 polarization. Together, our findings highlighting that SphK1 aggravated liver fibrosis by promoting macrophage recruitment and polarization and might serve as a potential drug target for the treatment of liver fibrosis.

【Background】Cholesterol-induced cholelithiasis is the most popular diseases in all of cholelithiasis. Cholesterol gall stones are generally formed caused by various factor following unbalance cholesterol secretion. Recently, it has been reported that there are some functional changes of gall bladder like mucus secretion or motility as one of the factors for cholelithiasis, but there has not been established the mechanism related to the functional changes of gall bladder mucosa. Additionally, organoid is often used for the pathological evaluation because it can reproduce cell composition, characteristics, and function, but it has not been established organoid model for cholelithiasis.

【Objectives】We established gall bladder organoid derived from cholesterol-induced cholelithiasis model mouse and evaluated the functional disorder mechanism for cholesterol-induced cholelithiasis.

【Methods】We feeded inducing diet to 4 weeks or 8 weeks mice, and produced cholesterol-induces cholelithiasis model mice. Using extracted tissue, we evaluated the pathological characteristics about gall stones formation and gall bladder structure. And also, we cultured gall bladder organoid derived from extracted tissue and compared its structure or mucus production with original tissue. Additionally, the gene expression difference was analyzed by RNA sequence.

【Results】It was observed the gall stones formation in 4 weeks feeding mouse. Organoid size in 4 weeks feeding mouse was significantly bigger than control group. In 8 weeks feeding mouse, organoid size was also increased significantly and gallbladder inflammation or gallbladder wall hypertrophy were recognized in gallbladder tissue. Furthermore, in RNA sequence, there were genetic expression differences between control and feeding group.

【Conclusion】It was suggested that gall bladder organoid derived from cholesterol-induced cholelithiasis model mouse can reproduce pathological characteristics of cholelithiasis. We will plan to do more functional analysis based on RNA sequence.  

Immune checkpoint inhibitor discovery represents a turning point in cancer treatment. However, the response rates of solid tumors remain approximately 10%–30%; consequently, prognostic and immune-related adverse event (irAE) predictors are being explored. The programmed cell death protein 1 (PD-1) receptor occupancy (RO) of PD-1 inhibitors depends on the number of peripheral blood lymphocytes and their PD-1 expression levels, suggesting that the RO may be related to efficacy and adverse events. As PD-1 inhibition affects each T-cell subset differently, the RO of each cell population must be characterized. However, relevant data have not been reported, and the prognostic relevance of this parameter is not known. In this study, we aimed to clarify the association between the nivolumab RO in each T-cell population and patient prognosis and reveal the development of irAEs in nivolumab-treated patients. Thirty-two patients were included in the study, and the mean follow-up period was 364 days. The nivolumab RO on effector regulatory T cells (eTregs) was significantly lower in the group that presented clinical benefits, and a significant negative association was observed between PD-1 occupancy on eTregs and all-cause mortality. The results suggest that the nivolumab RO on eTregs may be a prognostic factor in PD-1 inhibitor therapy, implying that the inhibition of PD-1/PD-ligand 1 (PD-L1) signaling on eTregs may attenuate antitumor effects.

Lung fibrosis is developed in severe asthmatic patients, whereas the mechanisms are unclear. We have established a murine model of steroid-insensitive asthma, which shows lung fibrosis. RNA-seq analyses revealed genes encoding CCR5 and its ligands were upregulated in the lung. However, roles of CCR5 in the development of fibrosis were unclear. In this study, we analyzed whether a CCR5 antagonist, maraviroc improves the fibrosis and increase in one of profibrotic cell types, monocyte-derived alveolar macrophages (MoAMs). OVA-sensitized BALB/c mice were intratracheally challenged with OVA. Dexamethasone (DEX, 1 mg/kg i.p.) or maraviroc (50 mg/kg p.o.) was administered during the challenges. The development of lung fibrosis and the number of MoAMs (CD45⁺ CD64⁺ Ly-6C⁺ Ly-6G^-/low Siglec-F^-) in the lung were analyzed. Maraviroc improved the development of lung fibrosis, whereas DEX did not affect it. Increase in MoAMs was suppressed by neither DEX nor maraviroc. Yet, interestingly, the number of CCR5⁺ cells were decreased in the maraviroc-treated mice. The decreased CCR5⁺ cells expressed CD45, CD11b, Ly-6C and Ly-6G, which displayed phenotype of monocyte-derived suppressor cells (MDSC). It was suggested that CCR5 possibly contributes to the increase in CCR5⁺ MDSC-like profibrotic cells, leading to the development of lung fibrosis.

Myeloid-Derived Suppressor Cells (MDSCs) are generated during tumor-bearing condition, and inhibit T-cell activity to promote cancer growth. Therefore, drugs which can inhibit MDSCs are new predictive immunotherapeutic medicines. On the other hand, AMP-activated protein kinase (AMPK) and mTOR signaling plays not only important role in energy homeostasis, but also recently revealed to plays a central role in an anti-tumor response. In this study, we examined the effect of metformin, an AMPK activator, and rapamycin, an inhibitor of mTOR on MDSCs differentiation. Bone marrow cells were isolated from of C57BL/6J mice and differentiated into MDSCs by the treatment with IL-6 and GM-CSF. Both metformin and rapamycin dose-dependently decreased differentiated MDSCs. These drugs also decreased the suppressing ability of MDSCs on T cell proliferation. Interestingly, subtype analysis of MDSC has shown that, these drugs decreased the ratio of monocytic MDSCs (M-MDSCs), whereas that of granulocytic MDSCs (G-MDSCs) was increased. Taken together, we assume that activation of AMPK and following inhibition of mTOR selectively inhibits M-MDSCs differentiation, and their immunosuppressive property. We expect that this study will provide new insights into the pharmacological regulation tumor immunology.

Ovarian cancer is a gynecologic malignancy with a high mortality rate. Eribulin, a non-taxane microtubule inhibitor approved for breast cancer and sarcoma, exerts antitumor efficacy in ovarian cancer cells (author et al, BPB. 2022:45). Ferroptosis, an iron-dependent cell death resulting from lipid peroxidation, is triggered by an accumulation of intracellular iron leading to oxidative stress. Reactive oxygen species (ROS) are a　cause of oxidative stress, and crucial for mitochondrial homeostasis. We explored the involvement of　ferroptosis and its mechanism in the antitumor activity of eribulin in ovarian cancer cells (RMG-1). Eribulin-induced cell death was mitigated by deferoxamine, an iron chelator. Eribulin elevated the levels of　intracellular iron, lipid peroxides, ROS, and mitochondrial membrane potential. Eribulin downregulated NRF2, heme oxygenase-1 (HO-1) and dihydroorotate dehydrogenase (DHODH), whereas glutathione peroxidase (GPX4) protein level remained unaffected. Combining eribulin with ML210, a GPX4-inhibiting ferroptosis inducer, enhanced eribulin-induced cell death. Taken together, eribulin triggers ferroptosis characterized by increased intracellular iron, lipid peroxidation, and ROS in ovarian cancer cells. The ferroptosis-inducing effect may be orchestrated through suppression of the NRF2/HO-1 signaling pathway and lipid peroxidation inhibition by DHODH. These findings illuminate the potential of eribulin-induced ferroptosis as a therapeutic strategy in ovarian cancer treatment.

Many types of cancer cells have increased demand for specific amino acids, depending on either exogenous supply or upregulated de novo synthesis. Intracellular accumulation of cysteine (Cys) is often observed in cancer cells, which is thought to contribute to the elimination of oxidative stress associated with rapid cell proliferation and/or exposure to anticancer drugs. However, the mechanism of metabolic reprogramming of cysteine during the oncogenesis is not fully understood yet. In this study, we found that the expression levels of genes responsible for cysteine synthesis were downregulated in the hepatocarcinoma-formed tumor tissues implanted in mice. On the other hand, the expression levels of cystine uptake transporter, xCT, were increased in hepatic tumor tissues as compared with healthy liver. The expression of DNA methyltransferase was also increased in hepatocarcinoma tumor tissues and caused DNA methylation of cysteine synthesis genes thereby repressing their expression. Pharmacological inhibition of cysteine synthesis resulted in a temporal decrease in intracellular cysteine contents and upregulation of xCT expression. Therefore, reduction of intracellular cysteine levels appeared to repulsively increase Cys uptake via promoting xCT expression. These findings suggest that the accumulation of Cys in hepatocarcinoma tumor cells results from enhancement of exogenous supply. This metabolic reprogramming may be required for the survival ability of oncogenic transformed cells.

Experimental animals including mice interacts with others and exhibits variety of behaviors. However, conventional behavioral tests mostly focused on single mouse behavior since visual tracking for multiple mice is practically impossible. Here, we aimed to develop the tracking tool for multiple mice using deep learning methods for image recognition. Behaviors of two to four C57BL/6 mice were recorded with handy camera in an open field arena. First, we manually labeled the mouse contours for hundreds of frame images and trained a deep learning model with labeled images. Next, mouse counters in all frames were predicted by the trained model, and assigned IDs by calculating similarities of every pair of contours between frames. Finally, we tracked the geometric center of contours that has the same IDs and semi-automatically corrected predictive errors to improve the performance. The established system could accurately track two to four mice under light conditions. In addition, we confirmed that this system accurately predicted the videos with bedding in the arena and could evaluate the videos recorded with infrared lights. This technology provides a new approach to evaluate mouse behaviors in pharmacological research.

Monoamine oxidase-B (MAOB) is a crucial enzyme not only as a therapeutic target for Parkinson's disease but also as a binding target for PET tracers that image reactive astrogliosis. We have developed a PET tracer named [¹⁸F]SMBT-1 designed for in vivo MAOB imaging. This was achieved by altering the chemical structure of tau PET tracer THK5351, which had high affinity for MAOB. To better understand the interaction of MAOB with these PET tracers, we determined the atomic structure of MAOB-SMBT-1 and MAOB-THK5351 complexes by X-ray crystallography with soaking method. Initially, we confirmed the high binding affinity of [¹⁸F]SMBT-1 against purified MAOB (K_D = 4.4, B_max = 2.8 nmol/mg protein), which was consistent with previously determined values against commercially available microsomal MAOB (K_D = 3.7 nM, B_max = 0.11 nmol/mg protein). In vitro competitive binding and biochemical assays demonstrated that SMBT-1 and THK5351 possess higher binding affinity and enzymatic inhibitory activity against MAOB than clinically used MAOB inhibitors Rasagiline and Safinamide. The crystal structures of MAOB in complexes with SMBT-1 and THK5351 (determined at 2.25 - 2.6Å) showed that they bind in the active site cavity of the protein in front of the flavin adenine dinucleotide cofactor. The findings achieved in this study would provide a potentially practical guide to avoid off-target binding to MAOB or to develop higher potency radiopharmaceuticals.

Metformin is a drug for type 2 diabetes used as the first-line mainly in North America and Europe. Its mechanism of action is believed to lower blood glucose levels by inhibiting gluconeogenesis in the liver and increasing glucose consumption in peripheral tissues. However, there are still controversies regarding the target molecules and the biochemical networks that induce pharmacological actions downstream of the target molecules. We applied the methodology of trans-omics analysis, which elucidates metabolic regulatory mechanisms as a large-scale molecular network, to characterizing the mechanism of action of metformin in the liver. We will show comprehensive identification of target proteins and reconstruction of metabolic regulatory networks that induce pharmacological actions of metformin.

The number of nerve fibers in the epidermis is increased during skin inflammation, which causes inflammatory pain. Sialyl glycosphingolipid, ganglioside, regulates axonal elongation and maintains axon morphology. Our previous study showed that Arthrobacter ureafaciens sialidase that degrades sialyl conjugates attenuated the number of epidermal nerve fibers and inflammatory pain. Moreover, F-11 cells derived from the dorsal root ganglion neuron treated with sialidase showed reduced neurite length and enhanced calcitonin gene-related peptide (CGRP)-immunoreactivity.

Thus, we investigated the effects of olcegepant, a CGRP receptor antagonist, on the inhibition of epidermal nerve fibers and inflammatory pain when treated with sialidase. One day after intraplantar injection of complete Freund’s adjuvant into the mouse hind paw to initiate skin inflammation, olcegepant and sialidase were injected into inflamed skin. The number of nerve fibers in the epidermis was counted using immunofluorescent staining with anti-PGP 9.5 antibody. Olcegepant attenuated the analgesic effects and epidermal nerve fiber collapse by sialidase. These results suggested that sialidase degenerated epidermal nerve fibers in inflamed skin via CGRP receptor activation, resulting in analgesia.

Neuropathic pain is a pathological pain state caused by a lesion or disease affecting the somatosensory system. Because existing analgesics often do not work, the development of new drugs for neuropathic pain is needed. A mouse model of neuropathic pain in which the fourth lumbar spinal nerve is transected (SpNT: spinal nerve transection) shows pain behavior that is resolved spontaneously. Recently, we found that a CD11c+ microglia subset emerged in the spinal cord after SpNT is necessary for the pain resolution. However, the role of CD11c+ microglia in other neuropathic pain models remains to be determined, especially in spared nerve injury (SNI) model that does not exhibit the spontaneous resolution of pain behavior. In this study, we found the number of CD11c+ microglia in the spinal cord was lower in the SNI model than the SpNT model, suggesting that prolonged behavioral pain hypersensitivity in the SNI model may be related to an impaired emergence of CD11c+ microglia. In addition, increasing the number of CD11c+ microglia by a cytokine administrated exogenously facilitated the resolution of pain behavior in both models. The alleviating effect was abolished by depletion of spinal CD11c+ microglia. Thus, increasing CD11c+ microglia or augmenting their function could be a new therapeutic strategy for neuropathic pain.

Central post-stroke pain (CPSP) is a type of central neuropathic pain, and its mechanisms remain unknown. Recently, we identified a significant increase of lipocalin 2 (LCN2) in the spinal cord of bilateral carotid artery occlusion (BCAO)-induced CPSP model mice using DNA microarray analysis. Generally, LCN2 is synthesized and secreted from activated glial cells. Also, glial cells derived LCN2 play a crucial role in the pathogenesis of neuropathic pain and stroke. In this study, we evaluated whether spinal glial cell-derived LCN2 is involved in the development of central post-stroke pain. Male ddY mice were subjected to 30 min of BCAO. Mechanical hypersensitivity was assessed by the von Frey test. LCN2 protein and its mRNA were evaluated by immunofluorescence stain and real-time PCR, respectively. We tested the expression of LCN2 in lipopolysaccharide (LPS)-activated MG6 microglial cells. BCAO mice showed hypersensitivity against mechanical stimuli and the activation of microglia and astrocyte in the spinal cord 3 days after BCAO. Spinal LCN2 protein was significantly increased and observed in the superficial dorsal horn of BCAO mice. LPS-activated microglial cells significantly and dose-dependently increased LCN2 mRNA expression. These results indicate that LCN2 in the spinal glial cells may involve in the development of CPSP.

Mammalian sperm, including human, must undergo several physiological and biochemical changes, collectively called capacitation, to be fertilization-competent. Capacitated sperm actively generate reactive oxygen species (ROS). A low level of ROS facilitates capacitation whereas an excessive ROS impairs capacitation. Hypotaurine (HT) is a precursor of taurine, and is abundant in the oviduct. HT is known to mitigate oxidative stress in hamster sperm, and is transported by taurine transporter (TauT) in a Na⁺- and Cl^-- dependent manner. However, how HT protect sperm from oxidative stress remains unknown. This study aimed to elucidate the antioxidant mechanisms by HT in hamster sperm, focusing on the involvement of TauT.

We first examined the effects of HT on sperm motility, intra- and extra- cellular ROS levels. HT was shown to be necessary to maintain motility. HT lowered intracellular ROS levels, but had no effect on extracellular ROS levels at the concentration tested, although HT itself has an antioxidative capacity at higher concentrations. Incorporation and enrichment of HT in sperm were confirmed by LC-MS/MS analysis. Next, the involvement of TauT was investigated. TauT was present in hamster sperm. β-alanine, a blocker of TauT, inhibited HT transport, increased intracellular ROS levels and impaired sperm motility. Moreover, the elimination of Na⁺ and Cl^- inhibited HT transport, and increased intracellular ROS levels.

In conclusions, the results indicate that hamster sperm incorporate and concentrate HT via TauT to protect themselves from ROS.

GPCR signalling utilizes an allosteric coupling between the extracellular facing ligand-binding pocket and the cytoplasmic domain of the receptor that interacts with the signalling proteins. GPCR ligands impart different level of activation or deactivation of signalling proteins via GPCRs that are selectively and specifically regulated, in phenomena called ligand efficacy. The ligand efficacy remarkably affects the therapeutic properties of the ligand. Therefore, it is important to understand the molecular mechanism that determines the ligand efficacy in drug discovery research. Recently, we have attempted to use FTIR spectroscopy to study the conformational changes in muscarinic receptor (M₂R) induced by ligand binding. The systematic ligand binding-induced difference FTIR spectroscopy on ligands with four different efficacies (agonist, partial agonist, antagonist, and inverse agonist) demonstrate the novel direct method for the quantitative evaluation of ligand efficacy on M₂R. Notably, FTIR signals strongly correlates with the results of G-protein activity in cells. Thus, this approach emphasizes that the FTIR signal can serve as a probe to distinguish the ligand efficacy of M₂R, and how FTIR spectroscopy can efficiently contribute to elucidate the underlying mechanism of ligand engagement and action towards the receptors.

G protein-coupled receptor 3 (GPR3) is highly expressed in various neurons and can constitutively activate the Gαs protein in the absence of ligands, thereby elevating the basal intracellular cAMP levels. We have shown that GPR3 is upregulated during neuronal differentiation and contributes to neurite outgrowth and neuronal survival. Meanwhile, GPR3 is rapidly induced in neurons, T cells, and mast cells upon stimuli; however, the potential mechanisms related to the rapid GPR3 induction remain elusive. In this study, we investigated the regulatory mechanism underlying GPR3 expression and its effect on downstream gene expression during neuronal differentiation in PC12 cells. PC12 cells stimulated using the cAMP activator forskolin strongly upregulated GPR3 mRNA as early as 1–2 h, declining thereafter. In addition, the GPR3 expression induced by forskolin was significantly augmented with the increased intracellular Ca²⁺ level produced by ionomycin. In addition, a luciferase-based promoter assay revealed that the cAMP response element in the 5'-flanking region of the rat GPR3 genome was associated with GPR3 transcription. Moreover, the upregulated GPR3 expression resulted in increased NR4A1 gene expression, further upregulating the expression of synapsin1, a downstream target of NR4A1. These results suggest that immediate early GPR3 upregulation by cAMP and Ca²⁺ stimuli may further enhance cAMP signaling, thereby modulating downstream gene expressions.

Na⁺/ Ca²⁺ exchangers (NCXs) are predominantly expressed in neuronal plasma membranes and consist of three mammalian NCX isoforms (NCX1, NCX2, and NCX3). However, the biological function of NCX in the brain still remains unknown. Here, we examined behavioral changes, as well as underlying molecular properties in the NCX3 heterozygous (NCX3^+/-) mice. We found that hyperactivity and social deficits in NCX3^+/- mice, which are ameliorated by the treatment of methylphenidate. In addition, we have identified that NCX3 was localized in the dopaminergic neuron of the ventral tegmental area which was the source of the dopamine innervation of the prefrontal cortex (PFC). In the PFC, NCX3^+/- mice displayed decreased extracellular levels of dopamine triggered by social stimuli and persistent elevation of basal dopamine levels relative to WT mice. In concordance with the increase of extracellular dopamine levels in the PFC, NCX3^+/- mice exhibited the activation of dopamine D1 receptor signaling pathways including PKA and DARPP-32 relative to WT mice in the PFC. Thus, the decreased expression of NCX3 leads to impair dopaminergic neurotransmission in the PFC, which likely accounts for the hyperactivity and social dysfunction in NCX3^+/- mice.

The central serotonergic system is involved in various functions such as instinct, emotion, and cognitive functions in mammals. However, it is not clear how much molecular and functional conservation the central serotonergic system has in vertebrates. It has become clear that birds have advanced cognitive functions comparable to mammals. Therefore, we attempted to elucidate the molecular and anatomical structure of the central serotonergic system using the chicken (Gallus gallus domesticus) as an avian model.

First, we determined the location of the dorsal raphe (DR) nucleus and median raphe (MR) nucleus in the chicken using the expression distribution of the chicken orthologs of serotonin transporter (SERT) and tryptophan hydroxylase 2 (TPH2) in the brainstem. Next, we clarified the serotonin receptors (5-HT receptors, 5-HTRs) expressed in serotonergic neurons contained in the DR and MR (Fujita, et al., 2022a). Our data indicate that the molecular properties of serotonergic neurons are evolutionarily well conserved between birds and mammals. In addition, we comprehensively elucidated the expression regions of almost all 5-HTR genes in the chicken telencephalon (Fujita, et al., 2020; 2022b). Our data has comprehensively revealed the brain regions and receptors modulated by serotonin in the avian telencephalon, making it possible to access the understanding of the neural circuits that govern cognition and emotion modulated by the serotonergic system in birds.

Gain-of-function mutations in RyR2 are known to cause lethal arrhythmias such as catecholaminergic ventricular tachycardia (CPVT). In CPVT, reduction of RyR2 activity is thought to suppress arrhythmias, but there are no clinically available antiarrhythmic drugs with RyR2-specific inhibitory action. We developed a high-affinity (IC50 of ~15nM) and selective RyR2 inhibitor, TMDJ-035, based on a hit compound identified in a high-throughput screening. TMDJ-035 effectively suppressed arrhythmias in CPVT mouse models harboring mutant RyR2s. Unlike conventional anti-arrhythmic drugs, i.e., Na channel inhibitors, Ca channel inhibitors, ß-blockers, TMDJ-035 did not affect ECG parameters or cardiac contractile function at the effective doses. Our results demonstrate that the specific suppression of RyR2 activity is highly effective in preventing and treating arrhythmias caused by RyR2 hyperactivation.